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Sample records for kinase scaffolding complex

  1. Conformational coupling between receptor and kinase binding sites through a conserved salt bridge in a signaling complex scaffold protein.

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    Davi R Ortega

    Full Text Available Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD, NMR spectroscopy, and circular dichroism (CD, we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.

  2. Disrupting the scaffold to improve focal adhesion kinase-targeted cancer therapeutics.

    Science.gov (United States)

    Cance, William G; Kurenova, Elena; Marlowe, Timothy; Golubovskaya, Vita

    2013-03-26

    Focal adhesion kinase (FAK) is emerging as a promising cancer target because it is highly expressed at both the transcriptional and translational level in cancer and is involved in many aspects of tumor growth, invasion, and metastasis. Existing FAK-based therapeutics focus on inhibiting the kinase's catalytic function and not the large scaffold it creates that includes many oncogenic receptor tyrosine kinases and tumor suppressor proteins. Targeting the FAK scaffold is a feasible and promising approach for developing highly specific therapeutics that disrupt FAK signaling pathways in cancer.

  3. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

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    Wilfried Jonkers

    2014-11-01

    Full Text Available Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this

  4. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

  5. The NDR kinase scaffold HYM1/MO25 is essential for MAK2 map kinase signaling in Neurospora crassa.

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    Anne Dettmann

    2012-09-01

    Full Text Available Cell communication is essential for eukaryotic development, but our knowledge of molecules and mechanisms required for intercellular communication is fragmentary. In particular, the connection between signal sensing and regulation of cell polarity is poorly understood. In the filamentous ascomycete Neurospora crassa, germinating spores mutually attract each other and subsequently fuse. During these tropic interactions, the two communicating cells rapidly alternate between two different physiological states, probably associated with signal delivery and response. The MAK2 MAP kinase cascade mediates cell-cell signaling. Here, we show that the conserved scaffolding protein HYM1/MO25 controls the cell shape-regulating NDR kinase module as well as the signal-receiving MAP kinase cascade. HYM1 functions as an integral part of the COT1 NDR kinase complex to regulate the interaction with its upstream kinase POD6 and thereby COT1 activity. In addition, HYM1 interacts with NRC1, MEK2, and MAK2, the three kinases of the MAK2 MAP kinase cascade, and co-localizes with MAK2 at the apex of growing cells. During cell fusion, the three kinases of the MAP kinase module as well as HYM1 are recruited to the point of cell-cell contact. hym-1 mutants phenocopy all defects observed for MAK2 pathway mutants by abolishing MAK2 activity. An NRC1-MEK2 fusion protein reconstitutes MAK2 signaling in hym-1, while constitutive activation of NRC1 and MEK2 does not. These data identify HYM1 as a novel regulator of the NRC1-MEK2-MAK2 pathway, which may coordinate NDR and MAP kinase signaling during cell polarity and intercellular communication.

  6. Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis.

    Science.gov (United States)

    Princz, Lissa N; Wild, Philipp; Bittmann, Julia; Aguado, F Javier; Blanco, Miguel G; Matos, Joao; Pfander, Boris

    2017-03-01

    DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution. © 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  7. Integrin-linked kinase: a Scaffold protein unique among its ilk.

    Science.gov (United States)

    Dagnino, Lina

    2011-06-01

    Integrin-linked kinase (ILK) is a scaffolding protein with central roles in tissue development and homeostasis. Much debate has focused on whether ILK is a bona fide or a pseudo- kinase. This aspect of ILK function has been complicated by the large volumes of conflicting observations obtained from a wide variety of experimental approaches, from in vitro models, to analyses in invertebrates and in mammals. Key findings in support or against the notion that ILK is catalytically active are summarized. The importance of ILK as an adaptor protein is well established, and defining its role as a signaling hub will be the next key step to understand its distinct biological roles across tissues and species.

  8. Coordinating ERK signaling via the molecular scaffold Kinase Suppressor of Ras [version 1; referees: 2 approved

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    Danielle Frodyma

    2017-08-01

    Full Text Available Many cancers, including those of the colon, lung, and pancreas, depend upon the signaling pathways induced by mutated and constitutively active Ras. The molecular scaffolds Kinase Suppressor of Ras 1 and 2 (KSR1 and KSR2 play potent roles in promoting Ras-mediated signaling through the Raf/MEK/ERK kinase cascade. Here we summarize the canonical role of KSR in cells, including its central role as a scaffold protein for the Raf/MEK/ERK kinase cascade, its regulation of various cellular pathways mediated through different binding partners, and the phenotypic consequences of KSR1 or KSR2 genetic inactivation. Mammalian KSR proteins have a demonstrated role in cellular and organismal energy balance with implications for cancer and obesity. Targeting KSR1 in cancer using small molecule inhibitors has potential for therapy with reduced toxicity to the patient. RNAi and small molecule screens using KSR1 as a reference standard have the potential to expose and target vulnerabilities in cancer. Interestingly, although KSR1 and KSR2 are similar in structure, KSR2 has a distinct physiological role in regulating energy balance. Although KSR proteins have been studied for two decades, additional analysis is required to elucidate both the regulation of these molecular scaffolds and their potent effect on the spatial and temporal control of ERK activation in health and disease.

  9. Polyelectrolyte-complex nanostructured fibrous scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Verma, Devendra; Katti, Kalpana S.; Katti, Dinesh R.

    2009-01-01

    In the current work, polyelectrolyte complex (PEC) fibrous scaffolds for tissue engineering have been synthesized and a mechanism of their formation has been investigated. The scaffolds are synthesized using polygalacturonic acid and chitosan using the freeze drying methodology. Highly interconnected pores of sizes in the range of 5-20 μm are observed in the scaffolds. The thickness of the fibers was found to be in the range of 1-2 μm. Individual fibers have a nanogranular structure as observed using AFM imaging. In these scaffolds, PEC nanoparticles assemble together at the interface of ice crystals during freeze drying process. Further investigation shows that the freezing temperature and concentration have a remarkable effect on structure of scaffolds. Biocompatibility studies show that scaffold containing chitosan, polygalacturonic acid and hydroxyapatite promotes cell adhesion and proliferation. On the other hand, cells on scaffolds fabricated without hydroxyapatite nanoparticles showed poor adhesion.

  10. Fungal communication requires the MAK-2 pathway elements STE-20 and RAS-2, the NRC-1 adapter STE-50 and the MAP kinase scaffold HAM-5.

    Science.gov (United States)

    Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

    2014-11-01

    Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell-cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell-cell communication in fungi and higher eukaryotes.

  11. p21-activated Kinase1(PAK1) can promote ERK activation in a kinase independent manner

    DEFF Research Database (Denmark)

    Wang, Zhipeng; Fu, Meng; Wang, Lifeng

    2013-01-01

    204) although phosphorylation of b-Raf (Ser445) and c-Raf (Ser 338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1 and Mek1, independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate...... MEK activity in a kinase independent manner, probably by serving as a scaffold to facilitate interaction of c-Raf....

  12. Scaffolding Proteins: Not Such Innocent Bystanders

    OpenAIRE

    Smith, F. Donelson; Scott, John D.

    2013-01-01

    Sequential transfer of information from one enzyme to the next within the confines of a protein kinase scaffold enhances signal transduction. Though frequently considered to be inert organizational elements, two recent reports implicate kinase-scaffolding proteins as active participants in signal relay.

  13. The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...

  14. Scaffolding proteins: not such innocent bystanders.

    Science.gov (United States)

    Smith, F Donelson; Scott, John D

    2013-06-17

    Sequential transfer of information from one enzyme to the next within the confines of a protein kinase scaffold enhances signal transduction. Though frequently considered to be inert organizational elements, two recent reports implicate kinase-scaffolding proteins as active participants in signal relay. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

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    Ines Teichert

    2014-09-01

    Full Text Available Mitogen-activated protein kinase (MAPK pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1. We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

  16. Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets.

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    Lai, Justine Y Q; Langston, Steven; Adams, Ruth; Beevers, Rebekah E; Boyce, Richard; Burckhardt, Svenja; Cobb, James; Ferguson, Yvonne; Figueroa, Eva; Grimster, Neil; Henry, Andrew H; Khan, Nawaz; Jenkins, Kerry; Jones, Mark W; Judkins, Robert; Major, Jeremy; Masood, Abid; Nally, James; Payne, Helen; Payne, Lloyd; Raphy, Gilles; Raynham, Tony; Reader, John; Reader, Valérie; Reid, Alison; Ruprah, Parminder; Shaw, Michael; Sore, Hannah; Stirling, Matthew; Talbot, Adam; Taylor, Jess; Thompson, Stephen; Wada, Hiroki; Walker, David

    2005-05-01

    This work describes the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS). These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. Several of these are based upon literature kinase templates, or adaptations of them to provide novelty. The routes to their preparation are outlined. A variety of automation techniques were used to prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to remove excess reagents and when necessary, preparative high performance liquid chromatography (HPLC) was used for purification. These compounds were screened against an 'in-house' kinase panel. The success rate in HTS was significantly higher than the corporate compound collection. Copyright (c) 2004 Wiley Periodicals, Inc.

  17. The secret life of kinases: functions beyond catalysis.

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

    Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  18. On Mineral Retrosynthesis of a Complex Biogenic Scaffold

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    Ashit Rao

    2017-03-01

    Full Text Available Synergistic relations between organic molecules and mineral precursors regulate biogenic mineralization. Given the remarkable material properties of the egg shell as a biogenic ceramic, it serves as an important model to elucidate biomineral growth. With established roles of complex anionic biopolymers and a heterogeneous organic scaffold in egg shell mineralization, the present study explores the regulation over mineralization attained by applying synthetic polymeric counterparts (polyethylene glycol, poly(acrylic acid, poly(aspartic acid and poly(4-styrenesulfonic acid-co-maleic acid as additives during remineralization of decalcified eggshell membranes. By applying Mg2+ ions as a co-additive species, mineral retrosynthesis is achieved in a manner that modulates the polymorph and structure of mineral products. Notable features of the mineralization process include distinct local wettability of the biogenic organic scaffold by mineral precursors and mineralization-induced membrane actuation. Overall, the form, structure and polymorph of the mineralization products are synergistically affected by the additive and the content of Mg2+ ions. We also revisit the physicochemical nature of the biomineral scaffold and demonstrate the distinct spatial distribution of anionic biomolecules associated with the scaffold-mineral interface, as well as highlight the hydrogel-like properties of mammillae-associated macromolecules.

  19. The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development

    Science.gov (United States)

    2011-01-01

    Background Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here. Results Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. Conclusions Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling. PMID:21401930

  20. The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development.

    Science.gov (United States)

    Mouchel-Vielh, Emmanuèle; Rougeot, Julien; Decoville, Martine; Peronnet, Frédérique

    2011-03-14

    Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here. Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling.

  1. The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development

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    Peronnet Frédérique

    2011-03-01

    Full Text Available Abstract Background Mitogen-activated protein kinase (MAPK cascades (p38, JNK, ERK pathways are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here. Results Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. Conclusions Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling.

  2. Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors.

    Science.gov (United States)

    Yang, Bin; Vasbinder, Melissa M; Hird, Alexander W; Su, Qibin; Wang, Haixia; Yu, Yan; Toader, Dorin; Lyne, Paul D; Read, Jon A; Breed, Jason; Ioannidis, Stephanos; Deng, Chun; Grondine, Michael; DeGrace, Nancy; Whitston, David; Brassil, Patrick; Janetka, James W

    2018-02-08

    Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

  3. Protein scaffolds and higher-order complexes in synthetic biology

    NARCIS (Netherlands)

    den Hamer, A.; Rosier, B.J.H.M.; Brunsveld, L.; de Greef, T.F.A.; Ryadnov, M.; Brunsveld, L.; Suga, H.

    2017-01-01

    Interactions between proteins control molecular functions such as signalling or metabolic activity. Assembly of proteins via scaffold proteins or in higher-order complexes is a key regulatory mechanism. Understanding and functionally applying this concept requires the construction, study, and

  4. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-26

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystallographic structures in the Protein Data Bank (PDB) by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of potential autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. We have identified 15 unique autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described in the relevant publications on the crystal structures (N-terminal juxtamembrane regions of CSF1R and EPHA2, activation loop tyrosines of LCK and IGF1R, and a serine in a nuclear localization signal region of CLK2. Mutation of residues in the autophosphorylation complex interface of LCK either severely impaired autophosphorylation or increased it. Taking the autophosphorylation complexes as a whole and comparing them with peptide-substrate/kinase complexes, we observe a number of important features among them. The novel and previously observed autophosphorylation sites are conserved in many kinases, indicating that by homology we can extend the relevance of these complexes to many other clinically relevant drug targets.

  5. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction

    Science.gov (United States)

    Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

    2015-01-01

    Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process. PMID:26393507

  6. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-01-01

    is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain

  7. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

    Science.gov (United States)

    Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

  8. Porous ceramic scaffolds with complex architectures

    Science.gov (United States)

    Munch, E.; Franco, J.; Deville, S.; Hunger, P.; Saiz, E.; Tomsia, A. P.

    2008-06-01

    This work compares two novel techniques for the fabrication of ceramic scaffolds for bone tissue engineering with complex porosity: robocasting and freeze casting. Both techniques are based on the preparation of concentrated ceramic suspensions with suitable properties for the process. In robocasting, the computer-guided deposition of the suspensions is used to build porous materials with designed three dimensional geometries and microstructures. Freeze casting uses ice crystals as a template to form porous lamellar ceramic materials. Preliminary results on the compressive strengths of the materials are also reported.

  9. Influence of processing parameters on pore structure of 3D porous chitosan-alginate polyelectrolyte complex scaffolds.

    Science.gov (United States)

    Florczyk, Stephen J; Kim, Dae-Joon; Wood, David L; Zhang, Miqin

    2011-09-15

    Fabrication of porous polymeric scaffolds with controlled structure can be challenging. In this study, we investigated the influence of key experimental parameters on the structures and mechanical properties of resultant porous chitosan-alginate (CA) polyelectrolyte complex (PEC) scaffolds, and on proliferation of MG-63 osteoblast-like cells, targeted at bone tissue engineering. We demonstrated that the porous structure is largely affected by the solution viscosity, which can be regulated by the acetic acid and alginate concentrations. We found that the CA PEC solutions with viscosity below 300 Pa.s yielded scaffolds of uniform pore structure and that more neutral pH promoted more complete complexation of chitosan and alginate, yielding stiffer scaffolds. CA PEC scaffolds produced from solutions with viscosities below 300 Pa.s also showed enhanced cell proliferation compared with other samples. By controlling the key experimental parameters identified in this study, CA PEC scaffolds of different structures can be made to suit various tissue engineering applications. Copyright © 2011 Wiley Periodicals, Inc.

  10. The fabrication and cell culture of three-dimensional rolled scaffolds with complex micro-architectures

    International Nuclear Information System (INIS)

    Liu Yaxiong; Li Xiao; Qu Xiaoli; Zhu Lin; He Jiankang; Zhao Qian; Wu Wanquan; Li Dichen

    2012-01-01

    Cell cultures for tissue engineering are traditionally prepared on two-dimensional or three-dimensional scaffolds with simple pores; however, this limits mass transportation, which is necessary for cell viability and function. In this paper, an innovative method is proposed for fabricating porous scaffolds with designed complex micro-architectures. Channels devised by computer-aided design were used to simulate features of blood vessels in native rat liver. Rapid prototyping and microreplication were used to produce a negative polydimethylsiloxane mold, and then a planar porous scaffold with predefined microchannel parameters was obtained by freeze-drying a silk fibroin/gelatin solution of an optimized concentration. After seeding with rat primary hepatocytes, the planar scaffold was rolled up to build spatial channels. By reconstructing the three-dimensional channel model in the scaffold in the form of micro-computed topography data and observing the cross-sections of the scroll, we confirmed that the bent channels were still interconnected, with restricted deviations. A comparison of the primary hepatocyte culture in the scaffolds with and without the devised channels proved that our design influenced cell organization and improved cell survival and proliferation. This method can be used for the construction of complex tissues for implantation and for culturing cells in vitro for biological tests and observations.

  11. HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

    Science.gov (United States)

    Diani, Erica; Avesani, Francesca; Bergamo, Elisa; Cremonese, Giorgia; Bertazzoni, Umberto; Romanelli, Maria Grazia

    2015-02-01

    The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Disrupting the Scaffold to Improve Focal Adhesion Kinase–Targeted Cancer Therapeutics

    Science.gov (United States)

    Cance, William G.; Kurenova, Elena; Marlowe, Timothy; Golubovskaya, Vita

    2013-01-01

    Focal adhesion kinase (FAK) is emerging as a promising cancer target because it is highly expressed at both the transcriptional and translational level in cancer and is involved in many aspects of tumor growth, invasion, and metastasis. Existing FAK-based therapeutics focus on inhibiting the kinase's catalytic function and not the large scaffold it creates that includes many oncogenic receptor tyrosine kinases and tumor suppressor proteins. Targeting the FAK scaffold is a feasible and promising approach for developing highly specific therapeutics that disrupt FAK signaling pathways in cancer. PMID:23532331

  13. Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex.

    Science.gov (United States)

    Ma, Jiong; Kelich, Joseph M; Junod, Samuel L; Yang, Weidong

    2017-04-01

    The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. © 2017. Published by The Company of Biologists Ltd.

  14. Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

    Science.gov (United States)

    Child, Matthew A.; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A.; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A.; Boothroyd, John C.; Reese, Michael L.

    2017-01-01

    ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. PMID:28246362

  15. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine

  16. PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

    Science.gov (United States)

    Deng, Youping; Xu, Hu; Riedel, Heimo

    2007-02-15

    The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

  17. Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold.

    Directory of Open Access Journals (Sweden)

    Rawin Poonperm

    Full Text Available Kinesin family member 4 (KIF4 and condensins I and II are essential chromosomal proteins for chromosome organization by locating primarily to the chromosome scaffold. However, the mechanism of how KIF4 and condensins localize to the chromosome scaffold is poorly understood. Here, we demonstrate a close relationship between the chromosome localization of KIF4 and condensin I, but not condensin II, and show that KIF4 and condensin I assist each other for stable scaffold formation by forming a stable complex. Moreover, phosphorylation of KIF4 and condensin I by Aurora B and polo-like kinase 1 (Plk1 is important for KIF4 and condensin I localization to the chromosome. Aurora B activity facilitates the targeting of KIF4 and condensin I to the chromosome, whereas Plk1 activity promotes the dissociation of these proteins from the chromosome. Thus, the interdependency between KIF4 and condensin I, and their phosphorylation states play important roles in chromosome scaffold organization during mitosis.

  18. Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold.

    Science.gov (United States)

    Poonperm, Rawin; Takata, Hideaki; Uchiyama, Susumu; Fukui, Kiichi

    2017-01-01

    Kinesin family member 4 (KIF4) and condensins I and II are essential chromosomal proteins for chromosome organization by locating primarily to the chromosome scaffold. However, the mechanism of how KIF4 and condensins localize to the chromosome scaffold is poorly understood. Here, we demonstrate a close relationship between the chromosome localization of KIF4 and condensin I, but not condensin II, and show that KIF4 and condensin I assist each other for stable scaffold formation by forming a stable complex. Moreover, phosphorylation of KIF4 and condensin I by Aurora B and polo-like kinase 1 (Plk1) is important for KIF4 and condensin I localization to the chromosome. Aurora B activity facilitates the targeting of KIF4 and condensin I to the chromosome, whereas Plk1 activity promotes the dissociation of these proteins from the chromosome. Thus, the interdependency between KIF4 and condensin I, and their phosphorylation states play important roles in chromosome scaffold organization during mitosis.

  19. Staurosporine scaffold-based rational discovery of the wild-type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog-sensitive kinase technology.

    Science.gov (United States)

    Song, Xiaoyun; Liu, Xingcai; Ding, Xi

    2017-04-01

    The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain

  20. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    Energy Technology Data Exchange (ETDEWEB)

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  1. MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics

    Directory of Open Access Journals (Sweden)

    Dhanendra Tomar

    2016-05-01

    Full Text Available Mitochondrial Ca2+ Uniporter (MCU-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1 have severely impaired [Ca2+]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metabolism.

  2. Experimental research of ZrO{sub 2}/BCP/PCL scaffold with complex pore pattern for bone tissue

    Energy Technology Data Exchange (ETDEWEB)

    Sa, Min Woo; Shin, Hae Ri; Kim, Jong Young [Dept. of Mechanical Engineering, Andong National University, Andong (Korea, Republic of)

    2015-11-15

    Recently, synthetic biopolymers and bioceramics such as poly (-caprolactone)(PCL), hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate(BCP), and zirconia have been used as substrates to generate various tissues or organs in tissue engineering. Thus, the purpose of this study was the characterization of ZrO{sub 2}/BCP/PCL(ZBP) scaffold for bone tissue regeneration. Based on the result of single-line test, blended 3D ZBP scaffolds with fully interconnected pores and new complex pore pattern of -type and staggered-type were successfully fabricated using a polymer deposition system. Furthermore, the effect of ZBP scaffold on mechanical property was analyzed. In addition, in vitro cell interaction of ZBP scaffold on MG63 cells was evaluated using a cell counting kit-8(CCK-8) assay.

  3. Chemical and morphological gradient scaffolds to mimic hierarchically complex tissues: From theoretical modeling to their fabrication.

    Science.gov (United States)

    Marrella, Alessandra; Aiello, Maurizio; Quarto, Rodolfo; Scaglione, Silvia

    2016-10-01

    Porous multiphase scaffolds have been proposed in different tissue engineering applications because of their potential to artificially recreate the heterogeneous structure of hierarchically complex tissues. Recently, graded scaffolds have been also realized, offering a continuum at the interface among different phases for an enhanced structural stability of the scaffold. However, their internal architecture is often obtained empirically and the architectural parameters rarely predetermined. The aim of this work is to offer a theoretical model as tool for the design and fabrication of functional and structural complex graded scaffolds with predicted morphological and chemical features, to overcome the time-consuming trial and error experimental method. This developed mathematical model uses laws of motions, Stokes equations, and viscosity laws to describe the dependence between centrifugation speed and fiber/particles sedimentation velocity over time, which finally affects the fiber packing, and thus the total porosity of the 3D scaffolds. The efficacy of the theoretical model was tested by realizing engineered graded grafts for osteochondral tissue engineering applications. The procedure, based on combined centrifugation and freeze-drying technique, was applied on both polycaprolactone (PCL) and collagen-type-I (COL) to test the versatility of the entire process. A functional gradient was combined to the morphological one by adding hydroxyapatite (HA) powders, to mimic the bone mineral phase. Results show that 3D bioactive morphologically and chemically graded grafts can be properly designed and realized in agreement with the theoretical model. Biotechnol. Bioeng. 2016;113: 2286-2297. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain

    Directory of Open Access Journals (Sweden)

    Pesti Szabolcs

    2012-11-01

    Full Text Available Abstract Background Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown. Results Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly. Conclusion Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.

  5. Protein kinase that phosphorylates light-harvesting complex is autophosphorylated and is associated with photosystem II

    International Nuclear Information System (INIS)

    Coughlan, S.J.; Hind, G.

    1987-01-01

    Thylakoid membranes were phosphorylated with [γ- 32 P]ATP and extracted with octyl glucoside and cholate. Among the radiolabeled phosphoproteins in the extract was a previously characterized protein kinase of 64-kDa apparent mass. The ability of this enzyme to undergo autophosphorylation in situ was used to monitor its distribution in the membrane. Fractionation studies showed that the kinase is confined to granal regions of the thylakoid, where it appears to be associated with the light-harvesting chlorophyll-protein complex of photosystem II. The kinetics of kinase autophosphorylation were investigated both in situ and in extracted, purified enzyme. In the membrane, autophosphorylation saturated within 20-30 min and was reversed with a half-time of 7-8 min upon removal of ATP or oxidative inactivation of the kinase; the accompanying dephosphorylation of light-harvesting complex was slower and kinetically complex. Fluoride (10 mM) inhibited these dephosphorylations. Autophosphorylation of the isolated kinase was independent of enzyme concentration, indicative of an intramolecular mechanism. A maximum of one serine residue per mole of kinase was esterified. Autophosphorylation was more rapid in the presence of histone IIIs, an exogenous substrate. Dephosphorylation of the isolated enzyme was not observed

  6. Small structural changes on a hydroquinone scaffold determine the complex I inhibition or uncoupling of tumoral oxidative phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Urra, Félix A., E-mail: felix.urra@qf.uchile.cl [Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Casilla 7, Santiago (Chile); Córdova-Delgado, Miguel [Departamento de Química Orgánica y Físico-Química, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1 (Chile); Lapier, Michel; Orellana-Manzano, Andrea [Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Casilla 7, Santiago (Chile); Acevedo-Arévalo, Luis; Pessoa-Mahana, Hernán; González-Vivanco, Jaime M. [Departamento de Química Orgánica y Físico-Química, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1 (Chile); Martínez-Cifuentes, Maximiliano [Instituto de Química de Recursos Naturales, Universidad de Talca, Casilla 747, Talca (Chile); and others

    2016-01-15

    Mitochondria participate in several distinctiveness of cancer cell, being a promising target for the design of anti-cancer compounds. Previously, we described that ortho-carbonyl hydroquinone scaffold 14 inhibits the complex I-dependent respiration with selective anti-proliferative effect on mouse mammary adenocarcinoma TA3/Ha cancer cells; however, the structural requirements of this hydroquinone scaffold to affect the oxidative phosphorylation (OXPHOS) of cancer cells have not been studied in detail. Here, we characterize the mitochondrial metabolism of TA3/Ha cancer cells, which exhibit a high oxidative metabolism, and evaluate the effect of small structural changes of the hydroquinone scaffold 14 on the respiration of this cell line. Our results indicate that these structural changes modify the effect on OXPHOS, obtaining compounds with three alternative actions: inhibitors of complex I-dependent respiration, uncoupler of OXPHOS and compounds with both actions. To confirm this, the effect of a bicyclic hydroquinone (9) was evaluated in isolated mitochondria. Hydroquinone 9 increased mitochondrial respiration in state 4o without effects on the ADP-stimulated respiration (state 3{sub ADP}), decreasing the complexes I and II-dependent respiratory control ratio. The effect on mitochondrial respiration was reversed by 6-ketocholestanol addition, indicating that this hydroquinone is a protonophoric uncoupling agent. In intact TA3/Ha cells, hydroquinone 9 caused mitochondrial depolarization, decreasing intracellular ATP and NAD(P)H levels and GSH/GSSG ratio, and slightly increasing the ROS levels. Moreover, it exhibited selective NAD(P)H availability-dependent anti-proliferative effect on cancer cells. Therefore, our results indicate that the ortho-carbonyl hydroquinone scaffold offers the possibility to design compounds with specific actions on OXPHOS of cancer cells. - Highlights: • Small changes on a hydroquinone scaffold modify the action on OXPHOS of cancer

  7. Complexes of γ-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

    International Nuclear Information System (INIS)

    Kukharskyy, Vitaliy; Sulimenko, Vadym; Macurek, Libor; Sulimenko, Tetyana; Draberova, Eduarda; Draber, Pavel

    2004-01-01

    Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that γ-tubulin (γ-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis

  8. Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

    DEFF Research Database (Denmark)

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

    2012-01-01

    , but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα......GDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest...

  9. Composite Scaffold of Poly(Vinyl Alcohol) and Interfacial Polyelectrolyte Complexation Fibers for Controlled Biomolecule Delivery

    Science.gov (United States)

    Cutiongco, Marie Francene A.; Choo, Royden K. T.; Shen, Nathaniel J. X.; Chua, Bryan M. X.; Sju, Ervi; Choo, Amanda W. L.; Le Visage, Catherine; Yim, Evelyn K. F.

    2015-01-01

    Controlled delivery of hydrophilic proteins is an important therapeutic strategy. However, widely used methods for protein delivery suffer from low incorporation efficiency and loss of bioactivity. The versatile interfacial polyelectrolyte complexation (IPC) fibers have the capacity for precise spatiotemporal release and protection of protein, growth factor, and cell bioactivity. Yet its weak mechanical properties limit its application and translation into a viable clinical solution. To overcome this limitation, IPC fibers can be incorporated into polymeric scaffolds such as the biocompatible poly(vinyl alcohol) hydrogel (PVA). Therefore, we explored the use of a composite scaffold of PVA and IPC fibers for controlled biomolecule release. We first observed that the permeability of biomolecules through PVA films were dependent on molecular weight. Next, IPC fibers were incorporated in between layers of PVA to produce PVA–IPC composite scaffolds with different IPC fiber orientation. The composite scaffold demonstrated excellent mechanical properties and efficient biomolecule incorporation. The rate of biomolecule release from PVA–IPC composite grafts exhibited dependence on molecular weight, with lysozyme showing near-linear release for 1 month. Angiogenic factors were also incorporated into the PVA–IPC grafts, as a potential biomedical application of the composite graft. While vascular endothelial growth factor only showed a maximum cumulative release of 3%, the smaller PEGylated-QK peptide showed maximum release of 33%. Notably, the released angiogenic biomolecules induced endothelial cell activity thus indicating retention of bioactivity. We also observed lack of significant macrophage response against PVA–IPC grafts in a rabbit model. Showing permeability, mechanical strength, precise temporal growth factor release, and bioinertness, PVA–IPC fibers composite scaffolds are excellent scaffolds for controlled biomolecule delivery in soft tissue

  10. Composite scaffold of poly(vinyl alcohol) and interfacial polyelectrolyte complexation fibers for controlled biomolecule delivery.

    Science.gov (United States)

    Cutiongco, Marie Francene A; Choo, Royden K T; Shen, Nathaniel J X; Chua, Bryan M X; Sju, Ervi; Choo, Amanda W L; Le Visage, Catherine; Yim, Evelyn K F

    2015-01-01

    Controlled delivery of hydrophilic proteins is an important therapeutic strategy. However, widely used methods for protein delivery suffer from low incorporation efficiency and loss of bioactivity. The versatile interfacial polyelectrolyte complexation (IPC) fibers have the capacity for precise spatiotemporal release and protection of protein, growth factor, and cell bioactivity. Yet its weak mechanical properties limit its application and translation into a viable clinical solution. To overcome this limitation, IPC fibers can be incorporated into polymeric scaffolds such as the biocompatible poly(vinyl alcohol) hydrogel (PVA). Therefore, we explored the use of a composite scaffold of PVA and IPC fibers for controlled biomolecule release. We first observed that the permeability of biomolecules through PVA films were dependent on molecular weight. Next, IPC fibers were incorporated in between layers of PVA to produce PVA-IPC composite scaffolds with different IPC fiber orientation. The composite scaffold demonstrated excellent mechanical properties and efficient biomolecule incorporation. The rate of biomolecule release from PVA-IPC composite grafts exhibited dependence on molecular weight, with lysozyme showing near-linear release for 1 month. Angiogenic factors were also incorporated into the PVA-IPC grafts, as a potential biomedical application of the composite graft. While vascular endothelial growth factor only showed a maximum cumulative release of 3%, the smaller PEGylated-QK peptide showed maximum release of 33%. Notably, the released angiogenic biomolecules induced endothelial cell activity thus indicating retention of bioactivity. We also observed lack of significant macrophage response against PVA-IPC grafts in a rabbit model. Showing permeability, mechanical strength, precise temporal growth factor release, and bioinertness, PVA-IPC fibers composite scaffolds are excellent scaffolds for controlled biomolecule delivery in soft tissue engineering.

  11. Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

    Science.gov (United States)

    Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

    2014-01-01

    Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

  12. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-05-01

    Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

  13. BESST--efficient scaffolding of large fragmented assemblies.

    Science.gov (United States)

    Sahlin, Kristoffer; Vezzi, Francesco; Nystedt, Björn; Lundeberg, Joakim; Arvestad, Lars

    2014-08-15

    The use of short reads from High Throughput Sequencing (HTS) techniques is now commonplace in de novo assembly. Yet, obtaining contiguous assemblies from short reads is challenging, thus making scaffolding an important step in the assembly pipeline. Different algorithms have been proposed but many of them use the number of read pairs supporting a linking of two contigs as an indicator of reliability. This reasoning is intuitive, but fails to account for variation in link count due to contig features.We have also noted that published scaffolders are only evaluated on small datasets using output from only one assembler. Two issues arise from this. Firstly, some of the available tools are not well suited for complex genomes. Secondly, these evaluations provide little support for inferring a software's general performance. We propose a new algorithm, implemented in a tool called BESST, which can scaffold genomes of all sizes and complexities and was used to scaffold the genome of P. abies (20 Gbp). We performed a comprehensive comparison of BESST against the most popular stand-alone scaffolders on a large variety of datasets. Our results confirm that some of the popular scaffolders are not practical to run on complex datasets. Furthermore, no single stand-alone scaffolder outperforms the others on all datasets. However, BESST fares favorably to the other tested scaffolders on GAGE datasets and, moreover, outperforms the other methods when library insert size distribution is wide. We conclude from our results that information sources other than the quantity of links, as is commonly used, can provide useful information about genome structure when scaffolding.

  14. Developmental Scaffolding

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio

    2015-01-01

    . Within the developmental hierarchy, each module yields an inter-level relationship that makes it possible for the scaffolding to mediate the production of selectable variations. Awide range of genetic, cellular and morphological mechanisms allows the scaffolding to integrate these modular variations...... to the complexity of sign recognition proper of a cellular community. In this semiotic perspective, the apparent goal directness of any developmental strategy should no longer be accounted for by a predetermined genetic program, but by the gradual definition of the relationships selected amongst the ones...

  15. The Discovery of Aurora Kinase Inhibitor by Multi-Docking-Based Virtual Screening

    Directory of Open Access Journals (Sweden)

    Jun-Tae Kim

    2014-11-01

    Full Text Available We report the discovery of aurora kinase inhibitor using the fragment-based virtual screening by multi-docking strategy. Among a number of fragments collected from eMololecules, we found four fragment molecules showing potent activity (>50% at 100 μM against aurora kinase. Based on the explored fragment scaffold, we selected two compounds in our synthesized library and validated the biological activity against Aurora kinase.

  16. Molecular evolution of a-kinase anchoring protein (AKAP-7: implications in comparative PKA compartmentalization

    Directory of Open Access Journals (Sweden)

    Johnson Keven R

    2012-07-01

    Full Text Available Abstract Background A-Kinase Anchoring Proteins (AKAPs are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA, directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18 are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport. Results Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels that promote the production of AKAP7γ instead of AKAP7δ. Conclusions This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.

  17. Magnesium Oxide Nanoparticles Reinforced Electrospun Alginate-Based Nanofibrous Scaffolds with Improved Physical Properties

    Directory of Open Access Journals (Sweden)

    R. T. De Silva

    2017-01-01

    Full Text Available Mechanically robust alginate-based nanofibrous scaffolds were successfully fabricated by electrospinning method to mimic the natural extracellular matrix structure which benefits development and regeneration of tissues. Alginate-based nanofibres were electrospun from an alginate/poly(vinyl alcohol (PVA polyelectrolyte complex. SEM images revealed the spinnability of the complex composite nanofibrous scaffolds, showing randomly oriented, ultrafine, and virtually defects-free alginate-based/MgO nanofibrous scaffolds. Here, it is shown that an alginate/PVA complex scaffold, blended with near-spherical MgO nanoparticles (⌀ 45 nm at a predetermined concentration (10% (w/w, is electrospinnable to produce a complex composite nanofibrous scaffold with enhanced mechanical stability. For the comparison purpose, chemically cross-linked electrospun alginate-based scaffolds were also fabricated. Tensile test to rupture revealed the significant differences in the tensile strength and elastic modulus among the alginate scaffolds, alginate/MgO scaffolds, and cross-linked alginate scaffolds (P<0.05. In contrast to cross-linked alginate scaffolds, alginate/MgO scaffolds yielded the highest tensile strength and elastic modulus while preserving the interfibre porosity of the scaffolds. According to the thermogravimetric analysis, MgO reinforced alginate nanofibrous scaffolds exhibited improved thermal stability. These novel alginate-based/MgO scaffolds are economical and versatile and may be further optimised for use as extracellular matrix substitutes for repair and regeneration of tissues.

  18. Direct 3D powder printing of biphasic calcium phosphate scaffolds for substitution of complex bone defects

    International Nuclear Information System (INIS)

    Castilho, Miguel; Pires, Inês; Moseke, Claus; Ewald, Andrea; Gbureck, Uwe; Groll, Jürgen; Teßmar, Jörg; Vorndran, Elke

    2014-01-01

    The 3D printing technique based on cement powders is an excellent method for the fabrication of individual and complex bone substitutes even in the case of large defects. The outstanding bone remodeling capacity of biphasic calcium phosphates (BCPs) containing hydroxyapatite (HA) as well as tricalcium phosphate (TCP) in varying ratios makes the adaption of powder systems resulting in BCP materials to this fabrication technique a desirable aim. This study presents the synthesis and characterization of a novel powder system for the 3D printing process, intended for the production of complexly shaped BCP scaffolds by a hydraulic setting reaction of calcium carbonate and TCP with phosphoric acid. The HA/TCP ratio in the specimens could be tailored by the calcium/phosphate ratio of the starting powder. The scaffolds could be fabricated with a dimensional accuracy of >96.5% and a minimal macro pore size of 300 µm. Independent of the phase composition the printed specimens showed a microporosity of approximately 68%, while the compressive strength strongly depended on the chemical composition and increased with rising TCP content in the scaffolds to a maximum of 1.81 MPa. Post-treatment of the scaffolds with a polylactic-co-glycolic acid-solution enhanced the mechanical properties by a factor of 8. In vitro studies showed that all BCP scaffolds were cytocompatible and enhanced the cell viability as well as the cell proliferation, as compared with pure TCP. Cell proliferation is even better on BCP when compared to HA and cell viability is in a similar range on these materials. (paper)

  19. Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors.

    Science.gov (United States)

    Kwiatkowski, Jacek; Liu, Boping; Tee, Doris Hui Ying; Chen, Guoying; Ahmad, Nur Huda Binte; Wong, Yun Xuan; Poh, Zhi Ying; Ang, Shi Hua; Tan, Eldwin Sum Wai; Ong, Esther Hq; Nurul Dinie; Poulsen, Anders; Pendharkar, Vishal; Sangthongpitag, Kanda; Lee, May Ann; Sepramaniam, Sugunavathi; Ho, Soo Yei; Cherian, Joseph; Hill, Jeffrey; Keller, Thomas H; Hung, Alvin W

    2018-05-24

    Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

  20. Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors

    Directory of Open Access Journals (Sweden)

    Nir Qvit

    2016-04-01

    Full Text Available Parasitic diseases cause ∼500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase and TRACK (Trypanosoma receptor for activated C-kinase. We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase, may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs. Keywords: Chagas disease, Leishmaniasis, Peptide, LACK, TRACK, Scaffold protein

  1. SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Sung Jun [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Ni, Lisheng [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Osinski, Adam [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Tomchick, Diana R. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Brautigam, Chad A. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States; Luo, Xuelian [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

    2017-10-24

    The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1–MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.

  2. Tunable signal processing in synthetic MAP kinase cascades.

    Science.gov (United States)

    O'Shaughnessy, Ellen C; Palani, Santhosh; Collins, James J; Sarkar, Casim A

    2011-01-07

    The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  4. The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

    Science.gov (United States)

    Gajiwala, Ketan S; Grodsky, Neil; Bolaños, Ben; Feng, Junli; Ferre, RoseAnn; Timofeevski, Sergei; Xu, Meirong; Murray, Brion W; Johnson, Ted W; Stewart, Al

    2017-09-22

    The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. How protein kinases co-ordinate mitosis in animal cells.

    Science.gov (United States)

    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

    Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

  6. The dual kinase complex FAK-Src as a promising therapeutic target in cancer

    Science.gov (United States)

    Bolós, Victoria; Gasent, Joan Manuel; López-Tarruella, Sara; Grande, Enrique

    2010-01-01

    Focal adhesion kinase (FAK) and steroid receptor coactivator (Src) are intracellular (nonreceptor) tyrosine kinases that physically and functionally interact to promote a variety of cellular responses. Plenty of reports have already suggested an additional central role for this complex in cancer through its ability to promote proliferation and anoikis resistance in tumor cells. An important role for the FAK/Src complex in tumor angiogenesis has also been established. Furthermore, FAK and Src have been associated with solid tumor metastasis through their ability to promote the epithelial mesenchymal transition. In fact, a strong correlation between increased FAK/Src expression/phosphorylation and the invasive phenotype in human tumors has been found. Additionally, an association for FAK/Src with resistances to the current anticancer therapies has already been established. Currently, novel anticancer agents that target FAK or Src are under development in a broad variety of solid tumors. In this article we will review the normal cellular functions of the FAK/Src complex as an effector of integrin and/or tyrosine kinase receptor signaling. We will also collect data about their role in cancer and we will summarize the most recent data from the FAK and Src inhibitors under clinical and preclinical development. Furthermore, the association of both these proteins with chemotherapy and hormonal therapy resistances, as a rationale for new combined therapeutic approaches with these novel agents, to abrogate treatment associated resistances, will also be reviewed. PMID:20616959

  7. Privileged scaffolds or promiscuous binders: a glance of pyrrolo[2,1-f][1,2,4]triazines and related bridgehead nitrogen heterocycles in medicinal chemistry.

    Science.gov (United States)

    Song, Yu'ning; Zhan, Peng; Zhang, Qingzhu; Liu, Xinyong

    2013-01-01

    Pyrrolo[2,1-f][1,2,4]triazine template, a unique bridgehead nitrogen heterocycle, certainly deserves the title of "privileged scaffold" in the drug discovery field because of the versatility and potential to yield derivatives with a wide range of biological activities, such as anti-anaplastic lymphoma kinase (ALK), Janus kinase 2 (JAK2), VEGFR-2, EGFR and/or HER2, Met kinase, p38α mitogen-activated protein (MAP) kinase and insulin-like growth factor receptor (IGF-1R) kinase activities, etc. These different biological properties of pyrrolo[2,1-f][1,2,4]triazine derivatives have motivated new studies in searching for novel derivatives with improved activity and also other applications in pharmaceutical field. However, no systematic review is available in the literature on the pyrrolo[2,1- f][1,2,4]triazine derivatives concerning the design of potent drug-like compounds. Owing to the importance of this heterocyclic system, the present paper is an attempt to the pharmacological activities, structural modifications and the structure-activity relationship (SAR) reported for bridgehead nitrogen heterocycles in the current literature, making an effort to highlight the importance and therapeutic potentials of the pyrrolo[2,1-f][1,2,4]triazine scaffold and its bridgehead nitrogen bioisosters as heterocyclic privileged medicinal scaffolds.

  8. Hsp27 regulates Akt activation and polymorphonuclear leukocyte apoptosis by scaffolding MK2 to Akt signal complex.

    Science.gov (United States)

    Wu, Rui; Kausar, Hina; Johnson, Paul; Montoya-Durango, Diego E; Merchant, Michael; Rane, Madhavi J

    2007-07-27

    We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117-128) on Akt as an Hsp27 binding region. Deletion of amino acids 117-128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate Akt(Delta117-128) mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2.

  9. A Review on Fabricating Tissue Scaffolds using Vat Photopolymerization.

    Science.gov (United States)

    Chartrain, Nicholas A; Williams, Christopher B; Whittington, Abby R

    2018-05-09

    Vat Photopolymerization (stereolithography, SLA), an Additive Manufacturing (AM) or 3D printing technology, holds particular promise for the fabrication of tissue scaffolds for use in regenerative medicine. Unlike traditional tissue scaffold fabrication techniques, SLA is capable of fabricating designed scaffolds through the selective photopolymerization of a photopolymer resin on the micron scale. SLA offers unprecedented control over scaffold porosity and permeability, as well as pore size, shape, and interconnectivity. Perhaps even more significantly, SLA can be used to fabricate vascular networks that may encourage angio and vasculogenesis. Fulfilling this potential requires the development of new photopolymers, the incorporation of biochemical factors into printed scaffolds, and an understanding of the effects scaffold geometry have on cell viability, proliferation, and differentiation. This review compares SLA to other scaffold fabrication techniques, highlights significant advances in the field, and offers a perspective on the field's challenges and future directions. Engineering de novo tissues continues to be challenging due, in part, to our inability to fabricate complex tissue scaffolds that can support cell proliferation and encourage the formation of developed tissue. The goal of this review is to first introduce the reader to traditional and Additive Manufacturing scaffold fabrication techniques. The bulk of this review will then focus on apprising the reader of current research and provide a perspective on the promising use of vat photopolymerization (stereolithography, SLA) for the fabrication of complex tissue scaffolds. Copyright © 2018. Published by Elsevier Ltd.

  10. Microchip Immunoaffinity Electrophoresis of Antibody-Thymidine Kinase 1 Complex

    Science.gov (United States)

    Pagaduan, Jayson V.; Ramsden, Madison; O’Neill, Kim; Woolley, Adam T.

    2015-01-01

    Thymidine kinase-1 (TK1) is an important cancer biomarker whose serum levels are elevated in early cancer development. We developed a microchip electrophoresis immunoaffinity assay to measure recombinant purified TK1 (pTK1) using an antibody that binds to human TK1. We fabricated poly(methyl methacrylate) microfluidic devices to test the feasibility of detecting antibody (Ab)-pTK1 immune complexes as a step towards TK1 analysis in clinical serum samples. We were able to separate immune complexes from unbound antibodies using 0.5X phosphate buffer saline (pH 7.4) containing 0.01% Tween-20, with 1% w/v methylcellulose that acts as a dynamic surface coating and sieving matrix. Separation of the antibody and Ab-pTK1 complex was observed within a 5 mm effective separation length. This method of detecting pTK1 is easy to perform, requires only a 10 μL sample volume, and takes just 1 minute for separation. PMID:25486911

  11. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

    Science.gov (United States)

    Frey, Stefan; Reschka, Eva J; Pöggeler, Stefanie

    2015-01-01

    The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

  12. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK Complex.

    Directory of Open Access Journals (Sweden)

    Stefan Frey

    Full Text Available The striatin-interacting phosphatase and kinase (STRIPAK complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP. In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

  13. Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

    Science.gov (United States)

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

    2010-01-01

    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

  14. Identification and Characterization of Amlexanox as a G Protein-Coupled Receptor Kinase 5 Inhibitor

    Directory of Open Access Journals (Sweden)

    Kristoff T. Homan

    2014-10-01

    Full Text Available G protein-coupled receptor kinases (GRKs have been implicated in human diseases ranging from heart failure to diabetes. Previous studies have identified several compounds that selectively inhibit GRK2, such as paroxetine and balanol. Far fewer selective inhibitors have been reported for GRK5, a target for the treatment of cardiac hypertrophy, and the mechanism of action of reported compounds is unknown. To identify novel scaffolds that selectively inhibit GRK5, a differential scanning fluorometry screen was used to probe a library of 4480 compounds. The best hit was amlexanox, an FDA-approved anti-inflammatory, anti-allergic immunomodulator. The crystal structure of amlexanox in complex with GRK1 demonstrates that its tricyclic aromatic ring system forms ATP-like interactions with the hinge of the kinase domain, which is likely similar to how this drug binds to IκB kinase ε (IKKε, another kinase known to be inhibited by this compound. Amlexanox was also able to inhibit myocyte enhancer factor 2 transcriptional activity in neonatal rat ventricular myocytes in a manner consistent with GRK5 inhibition. The GRK1 amlexanox structure thus serves as a springboard for the rational design of inhibitors with improved potency and selectivity for GRK5 and IKKε.

  15. The drug-eluting resorbable magnesium vascular scaffold in complex coronary bifurcations: insights from an in vivo multimodality imaging study.

    Science.gov (United States)

    Bennett, Johan; Vanhaverbeke, Maarten; Vanden Driessche, Nina; Hiltrop, Nick; Adriaenssens, Tom; Desmet, Walter; Sinnaeve, Peter; Dubois, Christophe

    2018-04-20

    This acute in vivo study sought to provide insights regarding the feasibility of performing complex bifurcation stenting with Magmaris magnesium alloy bioresorbable scaffolds (Biotronik, Bulach, Switzerland). Twenty-five New Zealand White rabbits underwent stenting of non-diseased aorto-iliac bifurcations with the Magmaris using provisional (PS; n=5), culotte (n=6), modified T (n=6), or T and protrusion (TAP, n=8) stenting techniques. Angiography, optical coherence tomography and micro-computed tomography were performed. Angiographic results were good without evidence of side branch (SB) compromise. In 9/25 procedures, strut fractures were identified with minimal luminal compromise in two cases. PS opened the SB optimally without evidence of scaffold compromise. Culotte resulted in complete bifurcation coverage and good scaffold expansion; single strut fractures were present in three out of six and double fractures in one out of six procedures. Modified T and TAP resulted in complete bifurcation coverage, minimal neocarina double-strut layers and good expansion. In two out of six modified T procedures, strut fractures were present with SB scaffold deformity present in an additional two out of six procedures. In three out of eight TAP procedures, strut fractures were present without compromising overall scaffold integrity. Bifurcation stenting using Magmaris appears feasible. PS with additional TAP whenever needed seems a reasonable approach. Whenever a two-stent technique is planned, TAP appears most favourable whilst modified T and culotte stenting should probably be avoided.

  16. AJUBA LIM Proteins Limit Hippo Activity in Proliferating Cells by Sequestering the Hippo Core Kinase Complex in the Cytosol.

    Science.gov (United States)

    Jagannathan, Radhika; Schimizzi, Gregory V; Zhang, Kun; Loza, Andrew J; Yabuta, Norikazu; Nojima, Hitoshi; Longmore, Gregory D

    2016-10-15

    The Hippo pathway controls organ growth and is implicated in cancer development. Whether and how Hippo pathway activity is limited to sustain or initiate cell growth when needed is not understood. The members of the AJUBA family of LIM proteins are negative regulators of the Hippo pathway. In mammalian epithelial cells, we found that AJUBA LIM proteins limit Hippo regulation of YAP, in proliferating cells only, by sequestering a cytosolic Hippo kinase complex in which LATS kinase is inhibited. At the plasma membranes of growth-arrested cells, AJUBA LIM proteins do not inhibit or associate with the Hippo kinase complex. The ability of AJUBA LIM proteins to inhibit YAP regulation by Hippo and to associate with the kinase complex directly correlate with their capacity to limit Hippo signaling during Drosophila wing development. AJUBA LIM proteins did not influence YAP activity in response to cell-extrinsic or cell-intrinsic mechanical signals. Thus, AJUBA LIM proteins limit Hippo pathway activity in contexts where cell proliferation is needed. Copyright © 2016 Jagannathan et al.

  17. An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

    Science.gov (United States)

    Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

    2013-01-01

    Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

  18. Image-based characterization of foamed polymeric tissue scaffolds

    International Nuclear Information System (INIS)

    Mather, Melissa L; Morgan, Stephen P; Crowe, John A; White, Lisa J; Shakesheff, Kevin M; Tai, Hongyun; Howdle, Steven M; Kockenberger, Walter

    2008-01-01

    Tissue scaffolds are integral to many regenerative medicine therapies, providing suitable environments for tissue regeneration. In order to assess their suitability, methods to routinely and reproducibly characterize scaffolds are needed. Scaffold structures are typically complex, and thus their characterization is far from trivial. The work presented in this paper is centred on the application of the principles of scaffold characterization outlined in guidelines developed by ASTM International. Specifically, this work demonstrates the capabilities of different imaging modalities and analysis techniques used to characterize scaffolds fabricated from poly(lactic-co-glycolic acid) using supercritical carbon dioxide. Three structurally different scaffolds were used. The scaffolds were imaged using: scanning electron microscopy, micro x-ray computed tomography, magnetic resonance imaging and terahertz pulsed imaging. In each case two-dimensional images were obtained from which scaffold properties were determined using image processing. The findings of this work highlight how the chosen imaging modality and image-processing technique can influence the results of scaffold characterization. It is concluded that in order to obtain useful results from image-based scaffold characterization, an imaging methodology providing sufficient contrast and resolution must be used along with robust image segmentation methods to allow intercomparison of results

  19. Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1.

    Science.gov (United States)

    Yoo, Byung-Chun; Lee, Jung-Youn; Lucas, William J

    2002-05-03

    In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. In this study, the complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Supporting evidence was obtained in that only five putative protein kinases (three calcium-independent and two calcium-dependent protein kinases) were detected within the phloem sap extracted from stem tissues. Biochemical methods were used to purify one such calcium-dependent protein kinase. The gene for this C. maxima calmodulin-like domain protein kinase 1 (CmCPK1), was cloned using peptide microsequences. A combination of mass spectrometry, peptide fingerprinting, and amino-terminal sequencing established that, in the phloem sap, CmCPK1 exists as an amino-terminally cleaved protein. A second highly homologous isoform, CmCPK2, was identified, but although transcripts could be detected in the companion cells, peptide fingerprint analysis suggested that CmCPK2 does not enter the phloem sap. Potential substrates for CmCPK1, within the phloem sap, were also detected using an on-membrane phosphorylation assay. Entry of CmCPK1 into sieve elements via plasmodesmata and the potential roles played by these phloem protein kinases are discussed.

  20. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

    Science.gov (United States)

    Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

    2017-09-19

    Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress

    Directory of Open Access Journals (Sweden)

    Shashi Kant

    2017-09-01

    Full Text Available Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA activation of a non-receptor tyrosine kinase (SRC-dependent cJun NH2-terminal kinase (JNK signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway.

  2. Applying ligands profiling using multiple extended electron distribution based field templates and feature trees similarity searching in the discovery of new generation of urea-based antineoplastic kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Eman M Dokla

    Full Text Available This study provides a comprehensive computational procedure for the discovery of novel urea-based antineoplastic kinase inhibitors while focusing on diversification of both chemotype and selectivity pattern. It presents a systematic structural analysis of the different binding motifs of urea-based kinase inhibitors and the corresponding configurations of the kinase enzymes. The computational model depends on simultaneous application of two protocols. The first protocol applies multiple consecutive validated virtual screening filters including SMARTS, support vector-machine model (ROC = 0.98, Bayesian model (ROC = 0.86 and structure-based pharmacophore filters based on urea-based kinase inhibitors complexes retrieved from literature. This is followed by hits profiling against different extended electron distribution (XED based field templates representing different kinase targets. The second protocol enables cancericidal activity verification by using the algorithm of feature trees (Ftrees similarity searching against NCI database. Being a proof-of-concept study, this combined procedure was experimentally validated by its utilization in developing a novel series of urea-based derivatives of strong anticancer activity. This new series is based on 3-benzylbenzo[d]thiazol-2(3H-one scaffold which has interesting chemical feasibility and wide diversification capability. Antineoplastic activity of this series was assayed in vitro against NCI 60 tumor-cell lines showing very strong inhibition of GI(50 as low as 0.9 uM. Additionally, its mechanism was unleashed using KINEX™ protein kinase microarray-based small molecule inhibitor profiling platform and cell cycle analysis showing a peculiar selectivity pattern against Zap70, c-src, Mink1, csk and MeKK2 kinases. Interestingly, it showed activity on syk kinase confirming the recent studies finding of the high activity of diphenyl urea containing compounds against this kinase. Allover, the new series

  3. CK2 phospho-dependent binding of R2TP complex to TEL2 is essential for mTOR and SMG1 stability.

    Science.gov (United States)

    Horejsí, Zuzana; Takai, Hiroyuki; Adelman, Carrie A; Collis, Spencer J; Flynn, Helen; Maslen, Sarah; Skehel, J Mark; de Lange, Titia; Boulton, Simon J

    2010-09-24

    TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Customized biomimetic scaffolds created by indirect three-dimensional printing for tissue engineering

    International Nuclear Information System (INIS)

    Lee, Ju-Yeon; Choi, Bogyu; Wu, Benjamin; Lee, Min

    2013-01-01

    Three-dimensional printing (3DP) is a rapid prototyping technique that can create complex 3D structures by inkjet printing of a liquid binder onto powder biomaterials for tissue engineering scaffolds. Direct fabrication of scaffolds from 3DP, however, imposes a limitation on material choices by manufacturing processes. In this study, we report an indirect 3DP approach wherein a positive replica of desired shapes was printed using gelatin particles, and the final scaffold was directly produced from the printed mold. To create patient-specific scaffolds that match precisely to a patient's external contours, we integrated our indirect 3DP technique with imaging technologies and successfully created custom scaffolds mimicking human mandibular condyle using polycaprolactone and chitosan for potential osteochondral tissue engineering. To test the ability of the technique to precisely control the internal morphology of the scaffolds, we created orthogonal interconnected channels within the scaffolds using computer-aided-design models. Because very few biomaterials are truly osteoinductive, we modified inert 3D printed materials with bioactive apatite coating. The feasibility of these scaffolds to support cell growth was investigated using bone marrow stromal cells (BMSC). The BMSCs showed good viability in the scaffolds, and the apatite coating further enhanced cellular spreading and proliferation. This technique may be valuable for complex scaffold fabrication. (paper)

  5. Tubular inverse opal scaffolds for biomimetic vessels

    Science.gov (United States)

    Zhao, Ze; Wang, Jie; Lu, Jie; Yu, Yunru; Fu, Fanfan; Wang, Huan; Liu, Yuxiao; Zhao, Yuanjin; Gu, Zhongze

    2016-07-01

    There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially oriented elliptical pattern microstructures on their surfaces. It is demonstrated that these tailored tubular scaffolds can effectively make endothelial cells to form an integrated hollow tubular structure on their inner surface and induce smooth muscle cells to form a circumferential orientation on their outer surface. These features of our tubular scaffolds make them highly promising for the construction of biomimetic blood vessels.There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially

  6. Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development

    Directory of Open Access Journals (Sweden)

    Mohna Bandyopadhyay

    2016-12-01

    Full Text Available CK2 is a Ser/Thr protein kinase that is highly conserved amongst all eukaryotes. It is a well-known oncogenic kinase that regulates vital cell autonomous functions and animal development. Genetic studies in the fruit fly Drosophila are providing unique insights into the roles of CK2 in cell signaling, embryogenesis, organogenesis, neurogenesis, and the circadian clock, and are revealing hitherto unknown complexities in CK2 functions and regulation. Here, we review Drosophila CK2 with respect to its structure, subunit diversity, potential mechanisms of regulation, developmental abnormalities linked to mutations in the gene encoding CK2 subunits, and emerging roles in multiple aspects of eye development. We examine the Drosophila CK2 “interaction map” and the eye-specific “transcriptome” databases, which raise the prospect that this protein kinase has many additional targets in the developing eye. We discuss the possibility that CK2 functions during early retinal neurogenesis in Drosophila and mammals bear greater similarity than has been recognized, and that this conservation may extend to other developmental programs. Together, these studies underscore the immense power of the Drosophila model organism to provide new insights and avenues to further investigate developmentally relevant targets of this protein kinase.

  7. Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

    Science.gov (United States)

    Valentine, Cathleen D.; Haggie, Peter M.

    2011-01-01

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

  8. Confinement of β(1)- and β(2)-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae.

    Science.gov (United States)

    Valentine, Cathleen D; Haggie, Peter M

    2011-08-15

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β(1)- and β(2)AR, are structurally similar but mediate distinct signaling responses. Scaffold protein-mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β(1)- and β(2)AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)-domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β(2)AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β(2)AR confinement. For both β(1)- and β(2)AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β(1)- or β(2)AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.

  9. The effect of MRN complex and ATM kinase inhibitors on Zebrafish embryonic development

    Science.gov (United States)

    Kumaran, Malina; Fazry, Shazrul

    2018-04-01

    Zebrafish is an ideal animal model to study developmental biology due to its transparent embryos and rapid development stages of embryogenesis. Here we investigate the role of DNA damage proteins, specifically Mre11/Rad50/NBN (MRN) complex and ataxia-telangiectasia mutated (ATM) kinase during embryogenesis by inhibiting its function using specific MRN complex (Mirin) and ATM Kinase inhibitors (Ku60019 and Ku55933). Zebrafish embryos at midblastula transition (MBT) stage are treated with Mirin, Ku60019 and Ku55933. The embryonic development of the embryos was monitored at 24 hours-post fertilisation (hpf), 48 hpf and 72 hpf. We observed that at the lowest concentrations (3 µM of Mirin, 1.5 nM of Ku60019 and 3 nM of Ku55933), the inhibitors treated embryos have 100% survivability. However, with increasing inhibitor concentration, the survivability drops. Control or mock treatment of all embryos shows 100 % survivability rate. This study suggests that DNA damage repair proteins may be crucial for normal zebrafish embryo development and survival.

  10. Bone tissue engineering scaffolding: computer-aided scaffolding techniques.

    Science.gov (United States)

    Thavornyutikarn, Boonlom; Chantarapanich, Nattapon; Sitthiseripratip, Kriskrai; Thouas, George A; Chen, Qizhi

    Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).

  11. β-Catenin destruction complex-independent regulation of Hippo–YAP signaling by APC in intestinal tumorigenesis

    Science.gov (United States)

    Cai, Jing; Maitra, Anirban; Anders, Robert A.; Taketo, Makoto M.; Pan, Duojia

    2015-01-01

    Mutations in Adenomatous polyposis coli (APC) underlie familial adenomatous polyposis (FAP), an inherited cancer syndrome characterized by the widespread development of colorectal polyps. APC is best known as a scaffold protein in the β-catenin destruction complex, whose activity is antagonized by canonical Wnt signaling. Whether other effector pathways mediate APC's tumor suppressor function is less clear. Here we report that activation of YAP, the downstream effector of the Hippo signaling pathway, is a general hallmark of tubular adenomas from FAP patients. We show that APC functions as a scaffold protein that facilitates the Hippo kinase cascade by interacting with Sav1 and Lats1. Consistent with the molecular link between APC and the Hippo signaling pathway, genetic analysis reveals that YAP is absolutely required for the development of APC-deficient adenomas. These findings establish Hippo–YAP signaling as a critical effector pathway downstream from APC, independent from its involvement in the β-catenin destruction complex. PMID:26193883

  12. Heparin-Functionalized chitosan/ κ-carrageenan complexes as potential scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Dofeliz, Joni L.; Rojas, Nina Rosario L.

    2015-01-01

    Cell-based approaches to tissue regeneration are playing an increasingly important role in bone and cartilage repair. This is made possible through the use of growth factors, which are signaling molecules that induce a number of effects such as cell proliferation, migration and differentiation. But problems arise as these growth factors tend to be expensive, short-lived and slow moving through the extracellular matrix, making them very inefficient in their current form of introduction. One such growth factor is basic fibroblast growth factor (bFGF). The general objective of this study is to construct heparin-functionalized chitosan/κ-carrageenan scaffolds, which when bound to basic fibroblast growth factor (bFGF), may be used in the culture of mesenchymal stem cells for differentiation into cartilage. In this study, gels of semi-interpenetrating networks (semi-IPN) of chitosan and κ-carrageenan (2:4:1 blend ration) were prepared using calcium chloride as a cross-linker. The gels were then functionalized with heparin, which is known for its growth factor binding ability, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as cross-linker. The functionalized gels were then reshaped into scaffold on the wells of 48-well plates through freeze-dry method. The scaffolds were found to be realatively porous with pore sizes larger than 100 μm which satisfies the requirements for cell culture. Subsequent thermogravimetric analysis shows that the scaffolds were relatively stable with a degradation temperature of 277°C. Additionally, there was no observable separation of the individual degradation temperatures of chitosan and κ-carrageenan, confirming that a single miscible phase in the form of a semi-IPN structure was created. Results of scanning electron microscopy also showed that the scaffolds were stable under 2 hours of UV sterilization. The FTIR spectra of the heparinized PEC showed characteristic absorption bands (S=O asymetric stretch) in the area of 1160

  13. Structures of the inactive and active states of RIP2 kinase inform on the mechanism of activation.

    Directory of Open Access Journals (Sweden)

    Erika Pellegrini

    Full Text Available Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated. Here we analyse RIP2K activation by combining crystal structures of the active and inactive states with mass spectrometric characterization of their phosphorylation profiles. The active state has Helix αC inwardly displaced and the phosphorylated Activation Segment (AS disordered, whilst in the inactive state Helix αC is outwardly displaced and packed against the helical, non-phosphorylated AS. Biophysical measurements show that the active state is a stable dimer whilst the inactive kinase is in a monomer-dimer equilibrium, consistent with the observed structural differences at the dimer interface. We conclude that RIP2 kinase auto-phosphorylation is intimately coupled to dimerization, similar to the case of BRAF. Our results will help drug design efforts targeting RIP2 as a potential treatment for NOD2-RIP2 related inflammatory diseases.

  14. Overlapping bio-absorbable scaffolds: Aim for D2D technique?

    Science.gov (United States)

    Khan, Asaad A; Dangas, George D

    2018-06-01

    The results of overlapping metallic stents have been concerning but this practice is often unavoidable in the setting of long or tortuous lesions, diameter discrepancy of proximal and distal vessel, and for residual dissections. Theoretically, bio-absorbable scaffolds may carry an advantage over metallic stents due to the progressive resorption of the scaffold theoretically rendering the overlap a non-issue; this has not been clinically evident. Since stent/scaffold overlap cannot be entirely avoided, improved stent delivery/deployment and scaffold design modification may reduce complications in this complex patient subset. © 2018 Wiley Periodicals, Inc.

  15. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-01-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

  16. Cell-derived matrix coatings for polymeric scaffolds.

    Science.gov (United States)

    Decaris, Martin L; Binder, Bernard Y; Soicher, Matthew A; Bhat, Archana; Leach, J Kent

    2012-10-01

    Cells in culture deposit a complex extracellular matrix that remains intact following decellularization and possesses the capacity to modulate cell phenotype. The direct application of such decellularized matrices (DMs) to 3D substrates is problematic, as transport issues influence the homogeneous deposition, decellularization, and modification of DM surface coatings. In an attempt to address this shortcoming, we hypothesized that DMs deposited by human mesenchymal stem cells (MSCs) could be transferred to the surface of polymeric scaffolds while maintaining their capacity to direct cell fate. The ability of the transferred DM (tDM)-coated scaffolds to enhance the osteogenic differentiation of undifferentiated and osteogenically induced MSCs under osteogenic conditions in vitro was confirmed. tDM-coated scaffolds increased MSC expression of osteogenic marker genes (BGLAP, IBSP) and intracellular alkaline phosphatase production. In addition, undifferentiated MSCs deposited significantly more calcium when seeded onto tDM-coated scaffolds compared with control scaffolds. MSC-seeded tDM-coated scaffolds subcutaneously implanted in nude rats displayed significantly higher blood vessel density after 2 weeks compared with cells on uncoated scaffolds, but we did not observe significant differences in mineral deposition after 8 weeks. These data demonstrate that DM-coatings produced in 2D culture can be successfully transferred to 3D substrates and retain their capacity to modulate cell phenotype.

  17. Large branched self-assembled DNA complexes

    International Nuclear Information System (INIS)

    Tosch, Paul; Waelti, Christoph; Middelberg, Anton P J; Davies, A Giles

    2007-01-01

    Many biological molecules have been demonstrated to self-assemble into complex structures and networks by using their very efficient and selective molecular recognition processes. The use of biological molecules as scaffolds for the construction of functional devices by self-assembling nanoscale complexes onto the scaffolds has recently attracted significant attention and many different applications in this field have emerged. In particular DNA, owing to its inherent sophisticated self-organization and molecular recognition properties, has served widely as a scaffold for various nanotechnological self-assembly applications, with metallic and semiconducting nanoparticles, proteins, macromolecular complexes, inter alia, being assembled onto designed DNA scaffolds. Such scaffolds may typically contain multiple branch-points and comprise a number of DNA molecules selfassembled into the desired configuration. Previously, several studies have used synthetic methods to produce the constituent DNA of the scaffolds, but this typically constrains the size of the complexes. For applications that require larger self-assembling DNA complexes, several tens of nanometers or more, other techniques need to be employed. In this article, we discuss a generic technique to generate large branched DNA macromolecular complexes

  18. Advanced tissue engineering scaffold design for regeneration of the complex hierarchical periodontal structure.

    Science.gov (United States)

    Costa, Pedro F; Vaquette, Cédryck; Zhang, Qiyi; Reis, Rui L; Ivanovski, Saso; Hutmacher, Dietmar W

    2014-03-01

    This study investigated the ability of an osteoconductive biphasic scaffold to simultaneously regenerate alveolar bone, periodontal ligament and cementum. A biphasic scaffold was built by attaching a fused deposition modelled bone compartment to a melt electrospun periodontal compartment. The bone compartment was coated with a calcium phosphate (CaP) layer for increasing osteoconductivity, seeded with osteoblasts and cultured in vitro for 6 weeks. The resulting constructs were then complemented with the placement of PDL cell sheets on the periodontal compartment, attached to a dentin block and subcutaneously implanted into athymic rats for 8 weeks. Scanning electron microscopy, X-ray diffraction, alkaline phosphatase and DNA content quantification, confocal laser microscopy, micro computerized tomography and histological analysis were employed to evaluate the scaffold's performance. The in vitro study showed that alkaline phosphatase activity was significantly increased in the CaP-coated samples and they also displayed enhanced mineralization. In the in vivo study, significantly more bone formation was observed in the coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted vascularization of the cell sheets, and periodontal attachment was achieved at the dentin interface. This work demonstrates that the combination of cell sheet technology together with an osteoconductive biphasic scaffold could be utilized to address the limitations of current periodontal regeneration techniques. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Crystal structure of human cyclin-dependent kinase-2 complex with MK2 inhibitor TEI-I01800: insight into the selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Fujino, Aiko; Fukushima, Kei; Kubota, Takaharu; Kosugi, Tomomi; Takimoto-Kamimura, Midori, E-mail: m.kamimura@teijin.co.jp [Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo 191-8512 (Japan)

    2013-11-01

    The Gly-rich loop of cyclin-dependent kinase 2 (CDK2) bound to TEI-I01800 as an MK2 specific inhibitor forms a β-sheet which is a common structure in CDK2–ligand complexes. Here, the reason why TEI-I01800 does not become a strong inhibitor against CDK2 based on the conformation of TEI-I01800 is presented. Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the MK2–TEI-I01800 complex has been reported; its Gly-rich loop was found to form an α-helix, not a β-sheet as has been observed for other Ser/Thr kinases. TEI-I01800 is 177-fold selective against MK2 compared with CDK2; in order to understand the inhibitory mechanism of TEI-I01800, the cyclin-dependent kinase 2 (CDK2) complex structure with TEI-I01800 was determined at 2.0 Å resolution. Interestingly, the Gly-rich loop of CDK2 formed a β-sheet that was different from that of MK2. In MK2, TEI-I01800 changed the secondary structure of the Gly-rich loop from a β-sheet to an α-helix by collision between Leu70 and a p-ethoxyphenyl group at the 7-position and bound to MK2. However, for CDK2, TEI-I01800 bound to CDK2 without this structural change and lost the interaction with the substituent at the 7-position. In summary, the results of this study suggest that the reason for the selectivity of TEI-I01800 is the favourable conformation of TEI-I01800 itself, making it suitable for binding to the α-form MK2.

  20. p130Cas scaffolds the signalosome to direct adaptor-effector cross talk during Kaposi's sarcoma-associated herpesvirus trafficking in human microvascular dermal endothelial cells.

    Science.gov (United States)

    Bandyopadhyay, Chirosree; Veettil, Mohanan Valiya; Dutta, Sujoy; Chandran, Bala

    2014-12-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. Eukaryotic cell adaptor molecules, without any intrinsic

  1. Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Wieczorek Andrew S

    2012-12-01

    Full Text Available Abstract Background The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. Results We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. Conclusions The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study

  2. Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

    Science.gov (United States)

    Provenzani, Riccardo; Tarvainen, Ilari; Brandoli, Giulia; Lempinen, Antti; Artes, Sanna; Turku, Ainoleena; Jäntti, Maria Helena; Talman, Virpi; Yli-Kauhaluoma, Jari; Tuominen, Raimo K; Boije Af Gennäs, Gustav

    2018-01-01

    Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain-targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

  3. Bioactive Scaffolds for Regeneration of Cartilage and Subchondral Bone Interface

    Science.gov (United States)

    Deng, Cuijun; Zhu, Huiying; Li, Jiayi; Feng, Chun; Yao, Qingqiang; Wang, Liming; Chang, Jiang; Wu, Chengtie

    2018-01-01

    The cartilage lesion resulting from osteoarthritis (OA) always extends into subchondral bone. It is of great importance for simultaneous regeneration of two tissues of cartilage and subchondral bone. 3D-printed Sr5(PO4)2SiO4 (SPS) bioactive ceramic scaffolds may achieve the aim of regenerating both of cartilage and subchondral bone. We hypothesized that strontium (Sr) and silicon (Si) ions released from SPS scaffolds play a crucial role in osteochondral defect reconstruction. Methods: SPS bioactive ceramic scaffolds were fabricated by a 3D-printing method. The SEM and ICPAES were used to investigate the physicochemical properties of SPS scaffolds. The proliferation and maturation of rabbit chondrocytes stimulated by SPS bioactive ceramics were measured in vitro. The stimulatory effect of SPS scaffolds for cartilage and subchondral bone regeneration was investigated in vivo. Results: SPS scaffolds significantly stimulated chondrocyte proliferation, and SPS extracts distinctly enhanced the maturation of chondrocytes and preserved chondrocytes from OA. SPS scaffolds markedly promoted the regeneration of osteochondral defects. The complex interface microstructure between cartilage and subchondral bone was obviously reconstructed. The underlying mechanism may be related to Sr and Si ions stimulating cartilage regeneration by activating HIF pathway and promoting subchondral bone reconstruction through activating Wnt pathway, as well as preserving chondrocytes from OA via inducing autophagy and inhibiting hedgehog pathway. Conclusion: Our findings suggest that SPS scaffolds can help osteochondral defect reconstruction and well reconstruct the complex interface between cartilage and subchondral bone, which represents a promising strategy for osteochondral defect regeneration. PMID:29556366

  4. Extrusion-based 3D printing of poly(propylene fumarate) scaffolds with hydroxyapatite gradients

    Science.gov (United States)

    Trachtenberg, Jordan E.; Placone, Jesse K.; Smith, Brandon T.; Fisher, John P.; Mikos, Antonios G.

    2017-01-01

    The primary focus of this work is to present the current challenges of printing scaffolds with concentration gradients of nanoparticles with an aim to improve the processing of these scaffolds. Furthermore, we address how print fidelity is related to material composition and emphasize the importance of considering this relationship when developing complex scaffolds for bone implants. The ability to create complex tissues is becoming increasingly relevant in the tissue engineering community. For bone tissue engineering applications, this work demonstrates the ability to use extrusion-based printing techniques to control the spatial deposition of hydroxyapatite (HA) nanoparticles in a 3D composite scaffold. In doing so, we combined the benefits of synthetic, degradable polymers, such as poly(propylene fumarate) (PPF), with osteoconductive HA nanoparticles that provide robust compressive mechanical properties. Furthermore, the final 3D printed scaffolds consisted of well-defined layers with interconnected pores, two critical features for a successful bone implant. To demonstrate a controlled gradient of HA, thermogravimetric analysis was carried out to quantify HA on a per-layer basis. Moreover, we non-destructively evaluated the tendency of HA particles to aggregate within PPF using micro-computed tomography (µCT). This work provides insight for proper fabrication and characterization of composite scaffolds containing particle gradients and has broad applicability for future efforts in fabricating complex scaffolds for tissue engineering applications. PMID:28125380

  5. Building bone tissue: matrices and scaffolds in physiology and biotechnology

    Directory of Open Access Journals (Sweden)

    Riminucci M.

    2003-01-01

    Full Text Available Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

  6. Simulations as Scaffolds in Science Education

    DEFF Research Database (Denmark)

    Renken, Maggie; Peffer, Melanie; Otrel-Cass, Kathrin

    This book outlines key issues for addressing the grand challenges posed to educators, developers, and researchers interested in the intersection of simulations and science education. To achieve this, the authors explore the use of computer simulations as instructional scaffolds that provide...... strategies and support when students are faced with the need to acquire new skills or knowledge. The monograph aims to provide insight into what research has reported on navigating the complex process of inquiry- and problem-based science education and whether computer simulations as instructional scaffolds...

  7. Scaffold Diversity from N-Acyliminium Ions

    DEFF Research Database (Denmark)

    Wu, Peng; Nielsen, Thomas E

    2017-01-01

    N-Acyliminium ions are powerful reactive species for the formation of carbon-carbon and carbon-heteroatom bonds. Strategies relying on intramolecular reactions of N-acyliminium intermediates, also referred to as N-acyliminium ion cyclization reactions, have been employed for the construction...... of structurally diverse scaffolds, ranging from simple bicyclic skeletons to complex polycyclic systems and natural-product-like compounds. This review aims to provide an overview of cyclization reactions of N-acyliminium ions derived from various precursors for the assembly of structurally diverse scaffolds...

  8. Spiral-structured, nanofibrous, 3D scaffolds for bone tissue engineering.

    Science.gov (United States)

    Wang, Junping; Valmikinathan, Chandra M; Liu, Wei; Laurencin, Cato T; Yu, Xiaojun

    2010-05-01

    Polymeric nanofiber matrices have already been widely used in tissue engineering. However, the fabrication of nanofibers into complex three-dimensional (3D) structures is restricted due to current manufacturing techniques. To overcome this limitation, we have incorporated nanofibers onto spiral-structured 3D scaffolds made of poly (epsilon-caprolactone) (PCL). The spiral structure with open geometries, large surface areas, and porosity will be helpful for improving nutrient transport and cell penetration into the scaffolds, which are otherwise limited in conventional tissue-engineered scaffolds for large bone defects repair. To investigate the effect of structure and fiber coating on the performance of the scaffolds, three groups of scaffolds including cylindrical PCL scaffolds, spiral PCL scaffolds (without fiber coating), and spiral-structured fibrous PCL scaffolds (with fiber coating) have been prepared. The morphology, porosity, and mechanical properties of the scaffolds have been characterized. Furthermore, human osteoblast cells are seeded on these scaffolds, and the cell attachment, proliferation, differentiation, and mineralized matrix deposition on the scaffolds are evaluated. The results indicated that the spiral scaffolds possess porosities within the range of human trabecular bone and an appropriate pore structure for cell growth, and significantly lower compressive modulus and strength than cylindrical scaffolds. When compared with the cylindrical scaffolds, the spiral-structured scaffolds demonstrated enhanced cell proliferation, differentiation, and mineralization and allowed better cellular growth and penetration. The incorporation of nanofibers onto spiral scaffolds further enhanced cell attachment, proliferation, and differentiation. These studies suggest that spiral-structured nanofibrous scaffolds may serve as promising alternatives for bone tissue engineering applications. Copyright 2009 Wiley Periodicals, Inc.

  9. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  10. Multi-scale mechanical characterization of scaffolds for heart valve tissue engineering

    NARCIS (Netherlands)

    Argento, G.; Simonet, M.; Oomens, C.W.J.; Baaijens, F.P.T.

    2012-01-01

    Electrospinning is a promising technology to produce scaffolds for cardiovascular tissue engineering. Each electrospun scaffold is characterized by a complex micro-scale structure that is responsible for its macroscopic mechanical behavior. In this study, we focus on the development and the

  11. Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

    Science.gov (United States)

    Gershlak, Joshua R; Hernandez, Sarah; Fontana, Gianluca; Perreault, Luke R; Hansen, Katrina J; Larson, Sara A; Binder, Bernard Y K; Dolivo, David M; Yang, Tianhong; Dominko, Tanja; Rolle, Marsha W; Weathers, Pamela J; Medina-Bolivar, Fabricio; Cramer, Carole L; Murphy, William L; Gaudette, Glenn R

    2017-05-01

    Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. The dual kinase complex FAK-Src as a promising therapeutic target in cancer

    Directory of Open Access Journals (Sweden)

    Victoria Bolós

    2010-06-01

    Full Text Available Victoria Bolós1,*, Joan Manuel Gasent2,*, Sara López-Tarruella3, Enrique Grande1,#1Pfizer Oncology, Madrid, Spain; 2Hospital Gral. Universitario Marina Alta, Oncology Department, Denia Alicante, 3,#Hospital Clínico San Carlos, Oncology Department, ∗These authors contributed equally to this work, #Center affiliated to the Red Temática de Investigación Cooperativa (RD06/0020/0021. Instituto de Salud Carlos III (ISCIII, Spanish Ministry of Science and InnovationAbstract: Focal adhesion kinase (FAK and steroid receptor coactivator (Src are intracellular (nonreceptor tyrosine kinases that physically and functionally interact to promote a variety of cellular responses. Plenty of reports have already suggested an additional central role for this complex in cancer through its ability to promote proliferation and anoikis resistance in tumor cells. An important role for the FAK/Src complex in tumor angiogenesis has also been established. Furthermore, FAK and Src have been associated with solid tumor metastasis through their ability to promote the epithelial mesenchymal transition. In fact, a strong correlation between increased FAK/Src expression/phosphorylation and the invasive phenotype in human tumors has been found. Additionally, an association for FAK/Src with resistances to the current anticancer therapies has already been established. Currently, novel anticancer agents that target FAK or Src are under development in a broad variety of solid tumors. In this article we will review the normal cellular functions of the FAK/Src complex as an effector of integrin and/or tyrosine kinase receptor signaling. We will also collect data about their role in cancer and we will summarize the most recent data from the FAK and Src inhibitors under clinical and preclinical development. Furthermore, the association of both these proteins with chemotherapy and hormonal therapy resistances, as a rationale for new combined therapeutic approaches with these novel

  13. Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment

    OpenAIRE

    Ceccaldi, Caroline; Bushkalova, Raya; Alfarano, Chiara; Lairez, Olivier; Calise, Denis; Bourin, Philippe; Frugier, Céline; Rouzaud-Laborde, Charlotte; Cussac, Daniel; Parini, Angelo; Sallerin, Brigitte; Girod Fullana, Sophie

    2014-01-01

    Three-dimensional (3D) scaffolds hold great potential for stem cell-based therapies. Indeed, recent results have shown that biomimetic scaffolds may enhance cell survival and promote an increase in the concentration of therapeutic cells at the injury site. The aim of this work was to engineer an original polymeric scaffold based on the respective beneficial effects of alginate and chitosan. Formulations were made from various alginate/chitosan ratios to form opposite-charge polyelectrolyte co...

  14. A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

    Science.gov (United States)

    Moroco, Jamie A; Baumgartner, Matthew P; Rust, Heather L; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S; Camacho, Carlos J; Smithgall, Thomas E

    2015-08-01

    The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. © 2014 John Wiley & Sons A/S.

  15. Scaffold translation: barriers between concept and clinic.

    Science.gov (United States)

    Hollister, Scott J; Murphy, William L

    2011-12-01

    Translation of scaffold-based bone tissue engineering (BTE) therapies to clinical use remains, bluntly, a failure. This dearth of translated tissue engineering therapies (including scaffolds) remains despite 25 years of research, research funding totaling hundreds of millions of dollars, over 12,000 papers on BTE and over 2000 papers on BTE scaffolds alone in the past 10 years (PubMed search). Enabling scaffold translation requires first an understanding of the challenges, and second, addressing the complete range of these challenges. There are the obvious technical challenges of designing, manufacturing, and functionalizing scaffolds to fill the Form, Fixation, Function, and Formation needs of bone defect repair. However, these technical solutions should be targeted to specific clinical indications (e.g., mandibular defects, spine fusion, long bone defects, etc.). Further, technical solutions should also address business challenges, including the need to obtain regulatory approval, meet specific market needs, and obtain private investment to develop products, again for specific clinical indications. Finally, these business and technical challenges present a much different model than the typical research paradigm, presenting the field with philosophical challenges in terms of publishing and funding priorities that should be addressed as well. In this article, we review in detail the technical, business, and philosophical barriers of translating scaffolds from Concept to Clinic. We argue that envisioning and engineering scaffolds as modular systems with a sliding scale of complexity offers the best path to addressing these translational challenges. © Mary Ann Liebert, Inc.

  16. Advanced computer-aided design for bone tissue-engineering scaffolds.

    Science.gov (United States)

    Ramin, E; Harris, R A

    2009-04-01

    The design of scaffolds with an intricate and controlled internal structure represents a challenge for tissue engineering. Several scaffold-manufacturing techniques allow the creation of complex architectures but with little or no control over the main features of the channel network such as the size, shape, and interconnectivity of each individual channel, resulting in intricate but random structures. The combined use of computer-aided design (CAD) systems and layer-manufacturing techniques allows a high degree of control over these parameters with few limitations in terms of achievable complexity. However, the design of complex and intricate networks of channels required in CAD is extremely time-consuming since manually modelling hundreds of different geometrical elements, all with different parameters, may require several days to design individual scaffold structures. An automated design methodology is proposed by this research to overcome these limitations. This approach involves the investigation of novel software algorithms, which are able to interact with a conventional CAD program and permit the automated design of several geometrical elements, each with a different size and shape. In this work, the variability of the parameters required to define each geometry has been set as random, but any other distribution could have been adopted. This methodology has been used to design five cubic scaffolds with interconnected pore channels that range from 200 to 800 microm in diameter, each with an increased complexity of the internal geometrical arrangement. A clinical case study, consisting of an integration of one of these geometries with a craniofacial implant, is then presented.

  17. Silk fibroin porous scaffolds for nucleus pulposus tissue engineering

    International Nuclear Information System (INIS)

    Zeng, Chao; Yang, Qiang; Zhu, Meifeng; Du, Lilong; Zhang, Jiamin; Ma, Xinlong; Xu, Baoshan; Wang, Lianyong

    2014-01-01

    Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38 ± 5.12% and pore size of 165.00 ± 8.25 μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell–scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue. - Highlights: • Paraffin microsphere-leaching method is used to fabricate silk fibroin scaffold. • The scaffold has appropriate mechanical property, porosity and pore size • The scaffold supports growth and infiltration of nucleus pulposus cells. • Nucleus pulposus cells can secrete extracellular matrix in the scaffolds. • The scaffold is a potential candidate for tissue engineered nucleus pulposus

  18. Silk fibroin porous scaffolds for nucleus pulposus tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Chao; Yang, Qiang [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Tianjin Medical University, Tianjin 300070 (China); Zhu, Meifeng [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China); Du, Lilong [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Tianjin Medical University, Tianjin 300070 (China); Zhang, Jiamin [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ma, Xinlong [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Xu, Baoshan, E-mail: xubaoshan99@126.com [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Wang, Lianyong, E-mail: wly@nankai.edu.cn [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2014-04-01

    Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38 ± 5.12% and pore size of 165.00 ± 8.25 μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell–scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue. - Highlights: • Paraffin microsphere-leaching method is used to fabricate silk fibroin scaffold. • The scaffold has appropriate mechanical property, porosity and pore size • The scaffold supports growth and infiltration of nucleus pulposus cells. • Nucleus pulposus cells can secrete extracellular matrix in the scaffolds. • The scaffold is a potential candidate for tissue engineered nucleus pulposus.

  19. PHBV/PAM scaffolds with local oriented structure through UV polymerization for tissue engineering.

    Science.gov (United States)

    Ke, Yu; Wu, Gang; Wang, Yingjun

    2014-01-01

    Locally oriented tissue engineering scaffolds can provoke cellular orientation and direct cell spread and migration, offering an exciting potential way for the regeneration of the complex tissue. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with locally oriented hydrophilic polyacrylamide (PAM) inside the macropores of the scaffolds were achieved through UV graft polymerization. The interpenetrating PAM chains enabled good interconnectivity of PHBV/PAM scaffolds that presented a lower porosity and minor diameter of pores than PHBV scaffolds. The pores with diameter below 100  μm increased to 82.15% of PHBV/PAM scaffolds compared with 31.5% of PHBV scaffolds. PHBV/PAM scaffold showed a much higher compressive elastic modulus than PHBV scaffold due to PAM stuffing. At 5 days of culturing, sheep chondrocytes spread along the similar direction in the macropores of PHBV/PAM scaffolds. The locally oriented PAM chains might guide the attachment and spreading of chondrocytes and direct the formation of microfilaments via contact guidance.

  20. Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase.adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

    International Nuclear Information System (INIS)

    Renosto, F.; Martin, R.L.; Segel, I.H.

    1989-01-01

    At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no ''substrate channeling'' of APS between the two sulfate-activating enzymes. APS kinase had no effect on the [S]0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound [ 35 S]APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a ''3'-phosphoadenosine 5'-phosphosulfate synthetase'' complex

  1. Integrin-linked kinase interactions with ELMO2 modulate cell polarity.

    Science.gov (United States)

    Ho, Ernest; Irvine, Tames; Vilk, Gregory J A; Lajoie, Gilles; Ravichandran, Kodi S; D'Souza, Sudhir J A; Dagnino, Lina

    2009-07-01

    Cell polarization is a key prerequisite for directed migration during development, tissue regeneration, and metastasis. Integrin-linked kinase (ILK) is a scaffold protein essential for cell polarization, but very little is known about the precise mechanisms whereby ILK modulates polarization in normal epithelia. Elucidating these mechanisms is essential to understand tissue morphogenesis, transformation, and repair. Here we identify a novel ILK protein complex that includes Engulfment and Cell Motility 2 (ELMO2). We also demonstrate the presence of RhoG in ILK-ELMO2 complexes, and the localization of this multiprotein species specifically to the leading lamellipodia of polarized cells. Significantly, the ability of RhoG to bind ELMO is crucial for ILK induction of cell polarization, and the joint expression of ILK and ELMO2 synergistically promotes the induction of front-rear polarity and haptotactic migration. This places RhoG-ELMO2-ILK complexes in a key position for the development of cell polarity and forward movement. Although ILK is a component of many diverse multiprotein species that may contribute to cell polarization, expression of dominant-negative ELMO2 mutants is sufficient to abolish the ability of ILK to promote cell polarization. Thus, its interaction with ELMO2 and RhoG is essential for the ability of ILK to induce front-rear cell polarity.

  2. DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels.

    Science.gov (United States)

    Kurokawa, Tatsuki; Kiyonaka, Shigeki; Nakata, Eiji; Endo, Masayuki; Koyama, Shohei; Mori, Emiko; Tran, Nam Ha; Dinh, Huyen; Suzuki, Yuki; Hidaka, Kumi; Kawata, Masaaki; Sato, Chikara; Sugiyama, Hiroshi; Morii, Takashi; Mori, Yasuo

    2018-03-01

    In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K + channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K + channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to co-ordinate assembly of signalling complexes downstream from MAVS.

    Science.gov (United States)

    Gu, Lili; Fullam, Anthony; McCormack, Niamh; Höhn, Yvette; Schröder, Martina

    2017-02-15

    The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon (IFN) induction downstream from antiviral pattern recognition receptors. It binds to TANK-binding kinase 1 and IκB-kinase-ε (IKKε), the two key kinases mediating activation of IFN regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKKε activation downstream from RIG-I and then links the activated kinase to IRF3. In the present study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TNF receptor-associated factor 3 (TRAF3) mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai virus (SeV) infection, both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the mitochondrial antiviral signalling (MAVS) adaptor, DDX3, IκB-kinase-ε (IKKε), TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting that it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented the formation of TRAF3-MAVS and TRAF3-IKKε complexes. Based on our data, we propose that early TRAF3 ubiquitination is required for the formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that co-ordinates assembly of different TRAF3, IKKε and IRF3-containing signalling complexes downstream from MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  4. 3D Printing of Scaffolds for Tissue Regeneration Applications

    Science.gov (United States)

    Do, Anh-Vu; Khorsand, Behnoush; Geary, Sean M.; Salem, Aliasger K.

    2015-01-01

    The current need for organ and tissue replacement, repair and regeneration for patients is continually growing such that supply is not meeting the high demand primarily due to a paucity of donors as well as biocompatibility issues that lead to immune rejection of the transplant. In an effort to overcome these drawbacks, scientists working in the field of tissue engineering and regenerative medicine have investigated the use of scaffolds as an alternative to transplantation. These scaffolds are designed to mimic the extracellular matrix (ECM) by providing structural support as well as promoting attachment, proliferation, and differentiation with the ultimate goal of yielding functional tissues or organs. Initial attempts at developing scaffolds were problematic and subsequently inspired a growing interest in 3D printing as a mode for generating scaffolds. Utilizing three-dimensional printing (3DP) technologies, ECM-like scaffolds can be produced with a high degree of complexity and precision, where fine details can be included at a micron level. In this review, we discuss the criteria for printing viable and functional scaffolds, scaffolding materials, and 3DP technologies used to print scaffolds for tissue engineering. A hybrid approach, employing both natural and synthetic materials, as well as multiple printing processes may be the key to yielding an ECM-like scaffold with high mechanical strength, porosity, interconnectivity, biocompatibility, biodegradability, and high processability. Creating such biofunctional scaffolds could potentially help to meet the demand by patients for tissues and organs without having to wait or rely on donors for transplantation. PMID:26097108

  5. Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1.

    Science.gov (United States)

    Gaude, H; Aznar, N; Delay, A; Bres, A; Buchet-Poyau, K; Caillat, C; Vigouroux, A; Rogon, C; Woods, A; Vanacker, J-M; Höhfeld, J; Perret, C; Meyer, P; Billaud, M; Forcet, C

    2012-03-22

    LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.

  6. Biomimetic nanoclay scaffolds for bone tissue engineering

    Science.gov (United States)

    Ambre, Avinash Harishchandra

    Tissue engineering offers a significant potential alternative to conventional methods for rectifying tissue defects by evoking natural regeneration process via interactions between cells and 3D porous scaffolds. Imparting adequate mechanical properties to biodegradable scaffolds for bone tissue engineering is an important challenge and extends from molecular to macroscale. This work focuses on the use of sodium montmorillonite (Na-MMT) to design polymer composite scaffolds having enhanced mechanical properties along with multiple interdependent properties. Materials design beginning at the molecular level was used in which Na-MMT clay was modified with three different unnatural amino acids and further characterized using Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD). Based on improved bicompatibility with human osteoblasts (bone cells) and intermediate increase in d-spacing of MMT clay (shown by XRD), 5-aminovaleric acid modified clay was further used to prepare biopolymer (chitosan-polygalacturonic acid complex) scaffolds. Osteoblast proliferation in biopolymer scaffolds containing 5-aminovaleric acid modified clay was similar to biopolymer scaffolds containing hydroxyapatite (HAP). A novel process based on biomineralization in bone was designed to prepare 5-aminovaleric acid modified clay capable of imparting multiple properties to the scaffolds. Bone-like apatite was mineralized in modified clay and a novel nanoclay-HAP hybrid (in situ HAPclay) was obtained. FTIR spectroscopy indicated a molecular level organic-inorganic association between the intercalated 5-aminovaleric acid and mineralized HAP. Osteoblasts formed clusters on biopolymer composite films prepared with different weight percent compositions of in situ HAPclay. Human MSCs formed mineralized nodules on composite films and mineralized extracellular matrix (ECM) in composite scaffolds without the use of osteogenic supplements. Polycaprolactone (PCL), a synthetic polymer, was

  7. Heterogeneity of Scaffold Biomaterials in Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Lauren Edgar

    2016-05-01

    Full Text Available Tissue engineering (TE offers a potential solution for the shortage of transplantable organs and the need for novel methods of tissue repair. Methods of TE have advanced significantly in recent years, but there are challenges to using engineered tissues and organs including but not limited to: biocompatibility, immunogenicity, biodegradation, and toxicity. Analysis of biomaterials used as scaffolds may, however, elucidate how TE can be enhanced. Ideally, biomaterials should closely mimic the characteristics of desired organ, their function and their in vivo environments. A review of biomaterials used in TE highlighted natural polymers, synthetic polymers, and decellularized organs as sources of scaffolding. Studies of discarded organs supported that decellularization offers a remedy to reducing waste of donor organs, but does not yet provide an effective solution to organ demand because it has shown varied success in vivo depending on organ complexity and physiological requirements. Review of polymer-based scaffolds revealed that a composite scaffold formed by copolymerization is more effective than single polymer scaffolds because it allows copolymers to offset disadvantages a single polymer may possess. Selection of biomaterials for use in TE is essential for transplant success. There is not, however, a singular biomaterial that is universally optimal.

  8. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

  9. Fragment-based lead discovery of small molecule inhibitors for the EPHA4 receptor tyrosine kinase

    NARCIS (Netherlands)

    van Linden, O.P.J.; Farenc, C; Zoutman, W.H.; Hameetman, L; Wijtmans, M.; Leurs, R.; Tensen, C.P.; Siegal, G.; de Esch, I.J.P.

    2011-01-01

    The in silico identification, optimization and crystallographic characterization of a 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine scaffold as an inhibitor for the EPHA4 receptor tyrosine kinase is described. A database containing commercially available compounds was subjected to an in

  10. Biomimetic mineral-organic composite scaffolds with controlled internal architecture.

    Science.gov (United States)

    Manjubala, I; Woesz, Alexander; Pilz, Christine; Rumpler, Monika; Fratzl-Zelman, Nadja; Roschger, Paul; Stampfl, Juergen; Fratzl, Peter

    2005-12-01

    Bone and cartilage generation by three-dimensional scaffolds is one of the promising techniques in tissue engineering. One approach is to generate histologically and functionally normal tissue by delivering healthy cells in biocompatible scaffolds. These scaffolds provide the necessary support for cells to proliferate and maintain their differentiated function, and their architecture defines the ultimate shape. Rapid prototyping (RP) is a technology by which a complex 3-dimensional (3D) structure can be produced indirectly from computer aided design (CAD). The present study aims at developing a 3D organic-inorganic composite scaffold with defined internal architecture by a RP method utilizing a 3D printer to produce wax molds. The composite scaffolds consisting of chitosan and hydroxyapatite were prepared using soluble wax molds. The behaviour and response of MC3T3-E1 pre-osteoblast cells on the scaffolds was studied. During a culture period of two and three weeks, cell proliferation and in-growth were observed by phase contrast light microscopy, histological staining and electron microscopy. The Giemsa and Gömöri staining of the cells cultured on scaffolds showed that the cells proliferated not only on the surface, but also filled the micro pores of the scaffolds and produced extracellular matrix within the pores. The electron micrographs showed that the cells covering the surface of the struts were flattened and grew from the periphery into the middle region of the pores.

  11. Hierarchical scaffolds enhance osteogenic differentiation of human Wharton’s jelly derived stem cells

    International Nuclear Information System (INIS)

    Canha-Gouveia, Analuce; Rita Costa-Pinto, Ana; Martins, Albino M; Sousa, Rui A; Reis, Rui L; Neves, Nuno M; Silva, Nuno A; Salgado, António J; Sousa, Nuno; Faria, Susana

    2015-01-01

    Hierarchical structures, constituted by polymeric nano and microfibers, have been considered promising scaffolds for tissue engineering strategies, mainly because they mimic, in some way, the complexity and nanoscale detail observed in real organs. The chondrogenic potential of these scaffolds has been previously demonstrated, but their osteogenic potential is not yet corroborated. In order to assess if a hierarchical structure, with nanoscale details incorporated, is an improved scaffold for bone tissue regeneration, we evaluate cell adhesion, proliferation, and osteogenic differentiation of human Wharton’s jelly derived stem cells (hWJSCs), seeded into hierarchical fibrous scaffolds. Biological data corroborates that hierarchical fibrous scaffolds show an enhanced cell entrapment when compared to rapid prototyped scaffolds without nanofibers. Furthermore, upregulation of bone specific genes and calcium phosphate deposition confirms the successful osteogenic differentiation of hWJSCs on these scaffolds. These results support our hypothesis that a scaffold with hierarchical structure, in conjugation with hWJSCs, represents a possible feasible strategy for bone tissue engineering applications. (paper)

  12. An integrative study to identify novel scaffolds for sphingosine kinase 1 inhibitors

    Czech Academy of Sciences Publication Activity Database

    Vettorazzi, M.; Angelina, E.; Lima, S.; Goněc, T.; Otevřel, J.; Marvanová, P.; Padrtová, T.; Mokrý, P.; Bobáľ, P.; Acosta, L. M.; Palma, A.; Cobo, J.; Bobálová, Janette; Csollei, J.; Malik, I.; Alvarez, S.; Spiegel, S.; Jampílek, J.; Enriz, R. D.

    2017-01-01

    Roč. 139, OCT (2017), s. 461-481 ISSN 0223-5234 Institutional support: RVO:68081715 Keywords : sphingosine kinase 1 inhibitors * bioassays * molecular modelling * virtual screening Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 4.519, year: 2016

  13. The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation.

    Science.gov (United States)

    Sun, X J; Pons, S; Asano, T; Myers, M G; Glasheen, E; White, M F

    1996-05-03

    Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

  14. Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

    Directory of Open Access Journals (Sweden)

    Inger Lindin

    2014-03-01

    Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

  15. The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling.

    Science.gov (United States)

    Nyati, Shyam; Schinske-Sebolt, Katrina; Pitchiaya, Sethuramasundaram; Chekhovskiy, Katerina; Chator, Areeb; Chaudhry, Nauman; Dosch, Joseph; Van Dort, Marcian E; Varambally, Sooryanarayana; Kumar-Sinha, Chandan; Nyati, Mukesh Kumar; Ray, Dipankar; Walter, Nils G; Yu, Hongtao; Ross, Brian Dale; Rehemtulla, Alnawaz

    2015-01-06

    Transforming growth factor-β (TGF-β) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-β, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-β signaling. BUB1 interacted with TGFBRI in the presence of TGF-β and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-β-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-β signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-β signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-β signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-β pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion. Copyright © 2015, American Association for the Advancement of Science.

  16. A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

    KAUST Repository

    Smirnova, Ekaterina; Kwan, Jamie J.; Siu, Ryan; Gao, Xin; Zoidl, Georg; Demeler, Borries; Saridakis, Vivian; Donaldson, Logan W.

    2016-01-01

    Background: CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions.

  17. A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

    KAUST Repository

    Smirnova, Ekaterina

    2016-08-22

    Background: CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions.

  18. Current trends in the design of scaffolds for computer-aided tissue engineering.

    Science.gov (United States)

    Giannitelli, S M; Accoto, D; Trombetta, M; Rainer, A

    2014-02-01

    Advances introduced by additive manufacturing have significantly improved the ability to tailor scaffold architecture, enhancing the control over microstructural features. This has led to a growing interest in the development of innovative scaffold designs, as testified by the increasing amount of research activities devoted to the understanding of the correlation between topological features of scaffolds and their resulting properties, in order to find architectures capable of optimal trade-off between often conflicting requirements (such as biological and mechanical ones). The main aim of this paper is to provide a review and propose a classification of existing methodologies for scaffold design and optimization in order to address key issues and help in deciphering the complex link between design criteria and resulting scaffold properties. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  19. Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

    Science.gov (United States)

    Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

    2006-11-24

    The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

  20. Noninvasive Evaluation of Injectable Chitosan/Nano-Hydroxyapatite/Collagen Scaffold via Ultrasound

    Directory of Open Access Journals (Sweden)

    Yan Chen

    2012-01-01

    Full Text Available To meet the challenges of designing an in situ forming scaffold and regenerating bone with complex three-dimensional (3D structures, an in situ forming hydrogel scaffold based on nano-hydroxyapatite (nHA, collagen (Col, and chitosan (CS was synthesized. Currently, only a limited number of techniques are available to mediate and visualize the injection process of the injectable biomaterials directly and noninvasively. In this study, the potential of ultrasound for the quantitative in vivo evaluation of tissue development in CS/nHAC scaffold was evaluated. The CS/nHAC scaffold was injected into rat subcutaneous tissue and evaluated for 28 days. Quantitative measurements of the gray-scale value, volume, and blood flow of the scaffold were evaluated using diagnostic technique. This study demonstrates that ultrasound can be used to noninvasively and nondestructively monitor and evaluate the in vivo characteristics of injectable bone scaffold. In comparison to the CS, the CS/nHAC scaffold showed a greater stiffness, less degradation rate, and better blood supply in the in vivo evaluation. In conclusion, the diagnostic ultrasound method is a good tool to evaluate the in vivo formation of injectable bone scaffolds and facilitates the broad use to monitor tissue development and remodeling in bone tissue engineering.

  1. Selective targeting of the mTORC1/2 protein kinase complexes leads to antileukemic effects in vitro and in vivo

    International Nuclear Information System (INIS)

    Schuster, K; Zheng, J; Arbini, A A; Zhang, C C; Scaglioni, P P

    2011-01-01

    The BCR/ABL tyrosine kinase promotes leukemogenesis through activation of several targets that include the phosphoinositide 3-kinase (PI3K). Tyrosine kinase inhibitors (TKIs), which target BCR/ABL, induce striking clinical responses. However, therapy with TKIs is associated with limitations such as drug intolerance, inability to universally eradicate the disease and emergence of BCR/ABL drug-resistant mutants. To overcome these limitations, we tested whether inhibition of the PI3K/target of rapamycin (mTOR) signaling pathway has antileukemic effect in primary hematopoietic stem cells and BA/F3 cells expressing the BCR/ABL oncoprotein. We determined that dual inhibition of PI3K/mTOR causes growth arrest and apoptosis leading to profound antileukemic effects both in vitro and in vivo. We also established that pharmacologic inhibition of the mTORC1/mTORC2 complexes is sufficient to cause these antileukemic effects. Our results support the development of inhibitors of the mTORC1/2 complexes for the therapy of leukemias that either express BCR/ABL or display deregulation of the PI3K/mTOR signaling pathway

  2. [Development of computer aided forming techniques in manufacturing scaffolds for bone tissue engineering].

    Science.gov (United States)

    Wei, Xuelei; Dong, Fuhui

    2011-12-01

    To review recent advance in the research and application of computer aided forming techniques for constructing bone tissue engineering scaffolds. The literature concerning computer aided forming techniques for constructing bone tissue engineering scaffolds in recent years was reviewed extensively and summarized. Several studies over last decade have focused on computer aided forming techniques for bone scaffold construction using various scaffold materials, which is based on computer aided design (CAD) and bone scaffold rapid prototyping (RP). CAD include medical CAD, STL, and reverse design. Reverse design can fully simulate normal bone tissue and could be very useful for the CAD. RP techniques include fused deposition modeling, three dimensional printing, selected laser sintering, three dimensional bioplotting, and low-temperature deposition manufacturing. These techniques provide a new way to construct bone tissue engineering scaffolds with complex internal structures. With rapid development of molding and forming techniques, computer aided forming techniques are expected to provide ideal bone tissue engineering scaffolds.

  3. Characterization of Electrospun Nanofibrous Scaffolds for Nanobiomedical Applications

    Science.gov (United States)

    Emul, E.; Saglam, S.; Ates, H.; Korkusuz, F.; Saglam, N.

    2016-08-01

    The electrospinning method is employed in the production of porous fiber scaffolds, and the usage of electrospun scaffolds especially as drug carrier and bone reconstructive material such as implants is promising for future applications in tissue engineering. The number of publications has grown very rapidly in this field through the fabrication of complex scaffolds, novel approaches in nanotechnology, and improvements of imaging methods. Hence, characterization of these materials has also grown significantly important for getting satisfied and accurate results. This advantageous and versatile method is ideal for mimicking bone extracellular matrix, and many biodegradable and biocompatible polymers are preferred in the field of bone reconstruction. In this study, gelatin, gelatin/nanohydroxyapatite (nHAp) and gelatin/PLLA/nHAp scaffolds were fabricated by the electrospinning process. These composite fibers showed clear and continuous morphology according to observation through a scanning electron microscope and their component analyses were also determined by Fourier transform infrared spectrometer analyses. These characterization experiments revealed the great effects of the electrospinning method for biomedical applications and have an especially important role in bone reconstruction and production of implant coating material.

  4. Strategic Design and Fabrication of Engineered Scaffolds for Articular Cartilage Repair

    Science.gov (United States)

    Izadifar, Zohreh; Chen, Xiongbiao; Kulyk, William

    2012-01-01

    Damage to articular cartilage can eventually lead to osteoarthritis (OA), a debilitating, degenerative joint disease that affects millions of people around the world. The limited natural healing ability of cartilage and the limitations of currently available therapies make treatment of cartilage defects a challenging clinical issue. Hopes have been raised for the repair of articular cartilage with the help of supportive structures, called scaffolds, created through tissue engineering (TE). Over the past two decades, different designs and fabrication techniques have been investigated for developing TE scaffolds suitable for the construction of transplantable artificial cartilage tissue substitutes. Advances in fabrication technologies now enable the strategic design of scaffolds with complex, biomimetic structures and properties. In particular, scaffolds with hybrid and/or biomimetic zonal designs have recently been developed for cartilage tissue engineering applications. This paper reviews critical aspects of the design of engineered scaffolds for articular cartilage repair as well as the available advanced fabrication techniques. In addition, recent studies on the design of hybrid and zonal scaffolds for use in cartilage tissue repair are highlighted. PMID:24955748

  5. Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade

    Directory of Open Access Journals (Sweden)

    Alessi Dario R

    2003-09-01

    Full Text Available Abstract Background The AMP-activated protein kinase (AMPK cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRADα/β and MO25α/β. Results We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRADα and MO25α, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRADα/β and MO25α/β activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRADα or STRADβ and MO25α or MO25β are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1, but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. Conclusions These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

  6. Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

    Science.gov (United States)

    Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

    2017-08-01

    Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

  7. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    Energy Technology Data Exchange (ETDEWEB)

    Reidick, Christina [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany); El Magraoui, Fouzi; Meyer, Helmut E. [Biomedical Research, Human Brain Proteomics II, Leibniz-Institut für Analytische Wissenschaften-ISAS, Dortmund 44139 (Germany); Stenmark, Harald [Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo 0310 (Norway); Platta, Harald W., E-mail: harald.platta@rub.de [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)

    2014-12-23

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

  8. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    International Nuclear Information System (INIS)

    Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

    2014-01-01

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept

  9. Preferential Selectivity of Inhibitors with Human Tau Protein Kinase Gsk3 Elucidates Their Potential Roles for Off-Target Alzheimer’s Therapy

    Directory of Open Access Journals (Sweden)

    Jagadeesh Kumar Dasappa

    2013-01-01

    Full Text Available Alzheimer’s disease (AD is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides (A and neurofibrillary tangles (NFTs. The abnormal phosphorylation of tau leads to the formation of NFTs produced by the action of tau kinases, resulting in the loss of neurons and synapse, leading to dementia. Hence, tau kinases have become potential drug target candidates for small molecule inhibitors. With an aim to explore the identification of a common inhibitor, this investigation was undertaken towards analyzing all 10 tau kinases which are implicated in phosphorylation of AD. A set of 7 inhibitors with varied scaffolds were collected from the Protein Data Bank (PDB. The analysis, involving multiple sequence alignment, 3D structural alignment, catalytic active site overlap, and docking studies, has enabled elucidation of the pharmacophoric patterns for the class of 7 inhibitors. Our results divulge that tau protein kinases share a specific set of conserved structural elements for the binding of inhibitors and ATP, respectively. The scaffold of 3-aminopyrrolidine (inhibitor 6 exhibits high preferential affinity with GSK3. Surprisingly, the PDB does not contain the structural details of GSK3 with this specific inhibitor. Thus, our investigations provide vital clues towards design of novel off-target drugs for Alzheimer’s.

  10. Neuron membrane trafficking and protein kinases involved in autism and ADHD.

    Science.gov (United States)

    Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

    2015-01-30

    A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  11. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  12. Highly porous scaffolds of PEDOT:PSS for bone tissue engineering.

    Science.gov (United States)

    Guex, Anne Géraldine; Puetzer, Jennifer L; Armgarth, Astrid; Littmann, Elena; Stavrinidou, Eleni; Giannelis, Emmanuel P; Malliaras, George G; Stevens, Molly M

    2017-10-15

    Conjugated polymers have been increasingly considered for the design of conductive materials in the field of regenerative medicine. However, optimal scaffold properties addressing the complexity of the desired tissue still need to be developed. The focus of this study lies in the development and evaluation of a conductive scaffold for bone tissue engineering. In this study PEDOT:PSS scaffolds were designed and evaluated in vitro using MC3T3-E1 osteogenic precursor cells, and the cells were assessed for distinct differentiation stages and the expression of an osteogenic phenotype. Ice-templated PEDOT:PSS scaffolds presented high pore interconnectivity with a median pore diameter of 53.6±5.9µm and a total pore surface area of 7.72±1.7m 2 ·g -1 . The electrical conductivity, based on I-V curves, was measured to be 140µS·cm -1 with a reduced, but stable conductivity of 6.1µS·cm -1 after 28days in cell culture media. MC3T3-E1 gene expression levels of ALPL, COL1A1 and RUNX2 were significantly enhanced after 4weeks, in line with increased extracellular matrix mineralisation, and osteocalcin deposition. These results demonstrate that a porous material, based purely on PEDOT:PSS, is suitable as a scaffold for bone tissue engineering and thus represents a promising candidate for regenerative medicine. Tissue engineering approaches have been increasingly considered for the repair of non-union fractions, craniofacial reconstruction or large bone defect replacements. The design of complex biomaterials and successful engineering of 3-dimensional tissue constructs is of paramount importance to meet this clinical need. Conductive scaffolds, based on conjugated polymers, present interesting candidates to address the piezoelectric properties of bone tissue and to induce enhanced osteogenesis upon implantation. However, conductive scaffolds have not been investigated in vitro in great measure. To this end, we have developed a highly porous, electrically conductive scaffold

  13. Rapid-prototyped PLGA/β-TCP/hydroxyapatite nanocomposite scaffolds in a rabbit femoral defect model

    International Nuclear Information System (INIS)

    Kim, Jinku; McBride, Sean; Alvarez-Urena, Pedro; Song, Young-Hye; Hollinger, Jeffrey O; Tellis, Brandi; Dean, David D; Sylvia, Victor L; Elgendy, Hoda; Ong, Joo

    2012-01-01

    Bone tissue engineering scaffolds composed of poly(d,l-lactide:glycolide) (DL-PLGA) and β-tricalcium phosphate (β-TCP) nanocomposites were prepared and characterized. Scaffolds with two specific architectures were produced via fused deposition modeling (FDM), a type of extrusion freeform fabrication. Microfilaments deposited at angles of 0° and 90° were designated as the ‘simple’ scaffold architecture, while those deposited at angles alternating between 0°, 90°, 45° and −45° were designated as the ‘complex’ scaffold architecture. In addition, the simple and complex scaffolds were coated with hydroxyapatite (HA). The surface morphology of the scaffolds was assessed before and after HA coating and uniform distribution of HA coating on the surface was observed by scanning electron microscopy. The scaffolds were implanted into rabbit femoral unicortical bone defects according to four treatment groups based on pore structure and HA coating. After 6 and 12 weeks, scaffolds and host bone were recovered and processed for histology. Data suggest that all configurations of the scaffolds integrated with the host bone and were biocompatible and thus may offer an exciting new scaffold platform for delivery of biologicals for bone regeneration. (paper)

  14. In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Whu, Shu Wen [Department of Orthopaedic Surgery, Chang Gung Memorial Hospital at Keelung, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Hung, Kun-Che; Hsieh, Kuo-Huang [Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan (China); Chen, Chih-Hwa [Department of Orthopaedic Surgery, Chang Gung Memorial Hospital at Keelung, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Tsai, Ching-Lin, E-mail: tsaicl@ntuh.gov.tw [Department of Orthopaedics, National Taiwan University Hospital, Taipei, Taiwan (China); Hsu, Shan-hui, E-mail: shhsu@ntu.edu.tw [Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan (China)

    2013-07-01

    Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1{sub WSC}) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1{sub WSC} promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1{sub WSC} constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1{sub WSC} constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1{sub WSC} scaffolds may enhance the cartilage regeneration in vitro and in vivo. - Highlights: • We developed a chitosan–gelatin scaffold crosslinked with carbodiimide. • Neocartilage formation was more evident in crosslinked vs. non-crosslinked scaffolds. • Histological features of tide line and lacunae were observed in vivo at 6.5 months. • Compressive

  15. A Stabilized Demethoxyviridin Derivative Inhibits PI3 kinase

    Science.gov (United States)

    Yuan, Hushan; Pupo, Monica T.; Blois, Joe; Smith, Adam; Weissleder, Ralph; Clardy, Jon; Josephson, Lee

    2009-01-01

    The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26 min vs Wm’s half-life of 3470 min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1 min. To overcome Dmv’s instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218 min in PBS and 64 min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC50 of 44 nM, compared to Wm’s IC50 of 12 nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems. PMID:19523825

  16. Recent Advances in the Development and Application of Radiolabeled Kinase Inhibitors for PET Imaging

    Directory of Open Access Journals (Sweden)

    Vadim Bernard-Gauthier

    2015-12-01

    Full Text Available Over the last 20 years, intensive investigation and multiple clinical successes targeting protein kinases, mostly for cancer treatment, have identified small molecule kinase inhibitors as a prominent therapeutic class. In the course of those investigations, radiolabeled kinase inhibitors for positron emission tomography (PET imaging have been synthesized and evaluated as diagnostic imaging probes for cancer characterization. Given that inhibitor coverage of the kinome is continuously expanding, in vivo PET imaging will likely find increasing applications for therapy monitoring and receptor density studies both in- and outside of oncological conditions. Early investigated radiolabeled inhibitors, which are mostly based on clinically approved tyrosine kinase inhibitor (TKI isotopologues, have now entered clinical trials. Novel radioligands for cancer and PET neuroimaging originating from novel but relevant target kinases are currently being explored in preclinical studies. This article reviews the literature involving radiotracer design, radiochemistry approaches, biological tracer evaluation and nuclear imaging results of radiolabeled kinase inhibitors for PET reported between 2010 and mid-2015. Aspects regarding the usefulness of pursuing selective vs. promiscuous inhibitor scaffolds and the inherent challenges associated with intracellular enzyme imaging will be discussed.

  17. hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

    International Nuclear Information System (INIS)

    Berglund, Fredrik M.; Clarke, Paul R.

    2009-01-01

    Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

  18. WiseScaffolder: an algorithm for the semi-automatic scaffolding of Next Generation Sequencing data.

    Science.gov (United States)

    Farrant, Gregory K; Hoebeke, Mark; Partensky, Frédéric; Andres, Gwendoline; Corre, Erwan; Garczarek, Laurence

    2015-09-03

    The sequencing depth provided by high-throughput sequencing technologies has allowed a rise in the number of de novo sequenced genomes that could potentially be closed without further sequencing. However, genome scaffolding and closure require costly human supervision that often results in genomes being published as drafts. A number of automatic scaffolders were recently released, which improved the global quality of genomes published in the last few years. Yet, none of them reach the efficiency of manual scaffolding. Here, we present an innovative semi-automatic scaffolder that additionally helps with chimerae resolution and generates valuable contig maps and outputs for manual improvement of the automatic scaffolding. This software was tested on the newly sequenced marine cyanobacterium Synechococcus sp. WH8103 as well as two reference datasets used in previous studies, Rhodobacter sphaeroides and Homo sapiens chromosome 14 (http://gage.cbcb.umd.edu/). The quality of resulting scaffolds was compared to that of three other stand-alone scaffolders: SSPACE, SOPRA and SCARPA. For all three model organisms, WiseScaffolder produced better results than other scaffolders in terms of contiguity statistics (number of genome fragments, N50, LG50, etc.) and, in the case of WH8103, the reliability of the scaffolds was confirmed by whole genome alignment against a closely related reference genome. We also propose an efficient computer-assisted strategy for manual improvement of the scaffolding, using outputs generated by WiseScaffolder, as well as for genome finishing that in our hands led to the circularization of the WH8103 genome. Altogether, WiseScaffolder proved more efficient than three other scaffolders for both prokaryotic and eukaryotic genomes and is thus likely applicable to most genome projects. The scaffolding pipeline described here should be of particular interest to biologists wishing to take advantage of the high added value of complete genomes.

  19. The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair.

    Science.gov (United States)

    Breslin, Claire; Mani, Rajam S; Fanta, Mesfin; Hoch, Nicolas; Weinfeld, Michael; Caldecott, Keith W

    2017-09-29

    The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  1. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  2. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  3. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

  4. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  5. Development of Pharmacophore Model for Indeno[1,2-b]indoles as Human Protein Kinase CK2 Inhibitors and Database Mining

    Directory of Open Access Journals (Sweden)

    Samer Haidar

    2017-01-01

    Full Text Available Protein kinase CK2, initially designated as casein kinase 2, is an ubiquitously expressed serine/threonine kinase. This enzyme, implicated in many cellular processes, is highly expressed and active in many tumor cells. A large number of compounds has been developed as inhibitors comprising different backbones. Beside others, structures with an indeno[1,2-b]indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors of human protein kinase CK2, we report here on the generation of common feature pharmacophore model to further explain the binding requirements for human CK2 inhibitors. Nine common chemical features of indeno[1,2-b]indole-type CK2 inhibitors were determined using MOE software (Chemical Computing Group, Montreal, Canada. This pharmacophore model was used for database mining with the aim to identify novel scaffolds for developing new potent and selective CK2 inhibitors. Using this strategy several structures were selected by searching inside the ZINC compound database. One of the selected compounds was bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[b]xanthene-7,10,12-trione, a natural compound which is produced by several kinds of fungi. This compound was tested on human recombinant CK2 and turned out to be an active inhibitor with an IC50 value of 1.24 µM.

  6. Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S. (Lodz - Poland); (UV)

    2012-09-11

    The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-a-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 {angstrom} resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the aD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

  7. Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

    Science.gov (United States)

    Chapman, Timothy M.; Bouloc, Nathalie; Buxton, Roger S.; Chugh, Jasveen; Lougheed, Kathryn E.A.; Osborne, Simon A.; Saxty, Barbara; Smerdon, Stephen J.; Taylor, Debra L.; Whalley, David

    2012-01-01

    A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained. PMID:22469702

  8. Theoretical Insights Reveal Novel Motions in Csk's SH3 Domain That Control Kinase Activation.

    Directory of Open Access Journals (Sweden)

    Sulyman Barkho

    Full Text Available The Src family of tyrosine kinases (SFKs regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk. Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk's activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk.

  9. A brief review of extrusion-based tissue scaffold bio-printing.

    Science.gov (United States)

    Ning, Liqun; Chen, Xiongbiao

    2017-08-01

    Extrusion-based bio-printing has great potential as a technique for manipulating biomaterials and living cells to create three-dimensional (3D) scaffolds for damaged tissue repair and function restoration. Over the last two decades, advances in both engineering techniques and life sciences have evolved extrusion-based bio-printing from a simple technique to one able to create diverse tissue scaffolds from a wide range of biomaterials and cell types. However, the complexities associated with synthesis of materials for bio-printing and manipulation of multiple materials and cells in bio-printing pose many challenges for scaffold fabrication. This paper presents an overview of extrusion-based bio-printing for scaffold fabrication, focusing on the prior-printing considerations (such as scaffold design and materials/cell synthesis), working principles, comparison to other techniques, and to-date achievements. This paper also briefly reviews the recent development of strategies with regard to hydrogel synthesis, multi-materials/cells manipulation, and process-induced cell damage in extrusion-based bio-printing. The key issue and challenges for extrusion-based bio-printing are also identified and discussed along with recommendations for future, aimed at developing novel biomaterials and bio-printing systems, creating patterned vascular networks within scaffolds, and preserving the cell viability and functions in scaffold bio-printing. The address of these challenges will significantly enhance the capability of extrusion-based bio-printing. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase.

    Science.gov (United States)

    Wu, Tzung-Ju; Wang, Xiaowen; Zhang, Yanjie; Meng, Linghua; Kerrigan, John E; Burley, Stephen K; Zheng, X F Steven

    2015-04-21

    Protein kinases are therapeutic targets for human cancer. However, "gatekeeper" mutations in tyrosine kinases cause acquired clinical resistance, limiting long-term treatment benefits. mTOR is a key cancer driver and drug target. Numerous small-molecule mTOR kinase inhibitors have been developed, with some already in human clinical trials. Given our clinical experience with targeted therapeutics, acquired drug resistance in mTOR is thought likely, but not yet documented. Herein, we describe identification of a hot spot (L2185) for drug-resistant mutations, which is distinct from the gatekeeper site, and a chemical scaffold refractory to drug-resistant mutations. We also provide new insights into mTOR kinase structure and function. The hot spot mutations are potentially useful as surrogate biomarkers for acquired drug resistance in ongoing clinical trials and future treatments and for the design of the next generation of mTOR-targeted drugs. Our study provides a foundation for further research into mTOR kinase function and targeting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

    Science.gov (United States)

    Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

    2005-10-28

    SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

  12. Human amniotic epithelial cells combined with silk fibroin scaffold in the repair of spinal cord injury

    Directory of Open Access Journals (Sweden)

    Ting-gang Wang

    2016-01-01

    Full Text Available Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithelial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the transplant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial cells combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.

  13. Indirect three-dimensional printing of synthetic polymer scaffold based on thermal molding process

    International Nuclear Information System (INIS)

    Park, Jeong Hun; Jung, Jin Woo; Cho, Dong-Woo; Kang, Hyun-Wook

    2014-01-01

    One of the major issues in tissue engineering has been the development of three-dimensional (3D) scaffolds, which serve as a structural template for cell growth and extracellular matrix formation. In scaffold-based tissue engineering, 3D printing (3DP) technology has been successfully applied for the fabrication of complex 3D scaffolds by using both direct and indirect techniques. In principle, direct 3DP techniques rely on the straightforward utilization of the final scaffold materials during the actual scaffold fabrication process. In contrast, indirect 3DP techniques use a negative mold based on a scaffold design, to which the desired biomaterial is cast and then sacrificed to obtain the final scaffold. Such indirect 3DP techniques generally impose a solvent-based process for scaffold fabrication, resulting in a considerable increase in the fabrication time and poor mechanical properties. In addition, the internal architecture of the resulting scaffold is affected by the properties of the biomaterial solution. In this study, we propose an advanced indirect 3DP technique using projection-based micro-stereolithography and an injection molding system (IMS) in order to address these challenges. The scaffold was fabricated by a thermal molding process using IMS to overcome the limitation of the solvent-based molding process in indirect 3DP techniques. The results indicate that the thermal molding process using an IMS has achieved a substantial reduction in scaffold fabrication time and has also provided the scaffold with higher mechanical modulus and strength. In addition, cell adhesion and proliferation studies have indicated no significant difference in cell activity between the scaffolds prepared by solvent-based and thermal molding processes. (paper)

  14. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    KAUST Repository

    Walkiewicz, Katarzyna Wiktoria

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  16. The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling

    NARCIS (Netherlands)

    Stegmann, Martin; Monaghan, Jacqueline; Smakowska-Luzan, Elwira; Rovenich, Hanna; Lehner, Anita; Holton, Nicholas; Belkhadir, Youssef; Zipfel, Cyril

    2017-01-01

    In plants, perception of invading pathogens involves cell-surface immune receptor kinases. Here, we report that the Arabidopsis SITE-1 PROTEASE (S1P) cleaves endogenous RAPID ALKALINIZATION FACTOR (RALF) propeptides to inhibit plant immunity. This inhibition is mediated by the malectin-like receptor

  17. Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14.

    Science.gov (United States)

    Jonkers, Wilfried; Fischer, Monika S; Do, Hung P; Starr, Trevor L; Glass, N Louise

    2016-05-01

    In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5. Copyright © 2016 by the Genetics Society of America.

  18. Cartilage Tissue Engineering with Silk Fibroin Scaffolds Fabricated by Indirect Additive Manufacturing Technology.

    Science.gov (United States)

    Chen, Chih-Hao; Liu, Jolene Mei-Jun; Chua, Chee-Kai; Chou, Siaw-Meng; Shyu, Victor Bong-Hang; Chen, Jyh-Ping

    2014-03-13

    Advanced tissue engineering (TE) technology based on additive manufacturing (AM) can fabricate scaffolds with a three-dimensional (3D) environment suitable for cartilage regeneration. Specifically, AM technology may allow the incorporation of complex architectural features. The present study involves the fabrication of 3D TE scaffolds by an indirect AM approach using silk fibroin (SF). From scanning electron microscopic observations, the presence of micro-pores and interconnected channels within the scaffold could be verified, resulting in a TE scaffold with both micro- and macro-structural features. The intrinsic properties, such as the chemical structure and thermal characteristics of SF, were preserved after the indirect AM manufacturing process. In vitro cell culture within the SF scaffold using porcine articular chondrocytes showed a steady increase in cell numbers up to Day 14. The specific production (per cell basis) of the cartilage-specific extracellular matrix component (collagen Type II) was enhanced with culture time up to 12 weeks, indicating the re-differentiation of chondrocytes within the scaffold. Subcutaneous implantation of the scaffold-chondrocyte constructs in nude mice also confirmed the formation of ectopic cartilage by histological examination and immunostaining.

  19. Cartilage Tissue Engineering with Silk Fibroin Scaffolds Fabricated by Indirect Additive Manufacturing Technology

    Directory of Open Access Journals (Sweden)

    Chih-Hao Chen

    2014-03-01

    Full Text Available Advanced tissue engineering (TE technology based on additive manufacturing (AM can fabricate scaffolds with a three-dimensional (3D environment suitable for cartilage regeneration. Specifically, AM technology may allow the incorporation of complex architectural features. The present study involves the fabrication of 3D TE scaffolds by an indirect AM approach using silk fibroin (SF. From scanning electron microscopic observations, the presence of micro-pores and interconnected channels within the scaffold could be verified, resulting in a TE scaffold with both micro- and macro-structural features. The intrinsic properties, such as the chemical structure and thermal characteristics of SF, were preserved after the indirect AM manufacturing process. In vitro cell culture within the SF scaffold using porcine articular chondrocytes showed a steady increase in cell numbers up to Day 14. The specific production (per cell basis of the cartilage-specific extracellular matrix component (collagen Type II was enhanced with culture time up to 12 weeks, indicating the re-differentiation of chondrocytes within the scaffold. Subcutaneous implantation of the scaffold-chondrocyte constructs in nude mice also confirmed the formation of ectopic cartilage by histological examination and immunostaining.

  20. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  1. Dissection of Protein Kinase Pathways in Live Cells Using Photoluminescent Probes: Surveillance or Interrogation?

    Directory of Open Access Journals (Sweden)

    Darja Lavogina

    2018-04-01

    Full Text Available Protein kinases catalyze phosphorylation, a small yet crucial modification that affects participation of the substrate proteins in the intracellular signaling pathways. The activity of 538 protein kinases encoded in human genome relies upon spatiotemporally controlled mechanisms, ensuring correct progression of virtually all physiological processes on the cellular level—from cell division to cell death. The aberrant functioning of protein kinases is linked to a wide spectrum of major health issues including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory diseases, etc. Hence, significant effort of scientific community has been dedicated to the dissection of protein kinase pathways in their natural milieu. The combination of recent advances in the field of light microscopy, the wide variety of genetically encoded or synthetic photoluminescent scaffolds, and the techniques for intracellular delivery of cargoes has enabled design of a plethora of probes that can report activation of target protein kinases in human live cells. The question remains: how much do we bias intracellular signaling of protein kinases by monitoring it? This review seeks answers to this question by analyzing different classes of probes according to their general structure, mechanism of recognition of biological target, and optical properties necessary for the reporting of intracellular events.

  2. Scaffold architecture and fibrin gels promote meniscal cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Pawelec, K. M., E-mail: pawelec.km@gmail.com, E-mail: jw626@cam.ac.uk; Best, S. M.; Cameron, R. E. [Cambridge Centre for Medical Materials, Materials Science and Metallurgy Department, University of Cambridge, Cambridge CB3 0FS (United Kingdom); Wardale, R. J., E-mail: pawelec.km@gmail.com, E-mail: jw626@cam.ac.uk [Division of Trauma and Orthopaedic Surgery, Department of Surgery, University of Cambridge, Cambridge CB2 2QQ (United Kingdom)

    2015-01-01

    Stability of the knee relies on the meniscus, a complex connective tissue with poor healing ability. Current meniscal tissue engineering is inadequate, as the signals for increasing meniscal cell proliferation have not been established. In this study, collagen scaffold structure, isotropic or aligned, and fibrin gel addition were tested. Metabolic activity was promoted by fibrin addition. Cellular proliferation, however, was significantly increased by both aligned architectures and fibrin addition. None of the constructs impaired collagen type I production or triggered adverse inflammatory responses. It was demonstrated that both fibrin gel addition and optimized scaffold architecture effectively promote meniscal cell proliferation.

  3. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    Energy Technology Data Exchange (ETDEWEB)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  4. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    International Nuclear Information System (INIS)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-01-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified

  5. The Drosophila PNG kinase complex regulates the translation of cyclin B.

    Science.gov (United States)

    Vardy, Leah; Orr-Weaver, Terry L

    2007-01-01

    The Drosophila PAN GU (PNG) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of PNG activity, but PNG is required for translation from egg activation. We find that although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for PNG-mediated control of cyclin B translation and of the early embryonic cell cycles. We present evidence that PNG functions instead as an antagonist of PUMILIO-dependent translational repression. Our data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that PNG-mediated derepression of translation is an important alternative mechanism in Drosophila.

  6. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties

    Energy Technology Data Exchange (ETDEWEB)

    Sloman, David L.; Noucti, Njamkou; Altman, Michael D.; Chen, Dapeng; Mislak, Andrea C.; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C.; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G. (Merck)

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer’s disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

  7. Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

    Science.gov (United States)

    Almeida, Henrique V; Sathy, Binulal N; Dudurych, Ivan; Buckley, Conor T; O'Brien, Fergal J; Kelly, Daniel J

    2017-01-01

    Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

  8. Efficient Computational Design of a Scaffold for Cartilage Cell Regeneration

    DEFF Research Database (Denmark)

    Tajsoleiman, Tannaz; Jafar Abdekhodaie, Mohammad; Gernaey, Krist V.

    2018-01-01

    Due to the sensitivity of mammalian cell cultures, understanding the influence of operating conditions during a tissue generation procedure is crucial. In this regard, a detailed study of scaffold based cell culture under a perfusion flow is presented with the aid of mathematical modelling...... and computational fluid dynamics (CFD). With respect to the complexity of the case study, this work focuses solely on the effect of nutrient and metabolite concentrations, and the possible influence of fluid-induced shear stress on a targeted cell (cartilage) culture. The simulation set up gives the possibility...... of predicting the cell culture behavior under various operating conditions and scaffold designs. Thereby, the exploitation of the predictive simulation into a newly developed stochastic routine provides the opportunity of exploring improved scaffold geometry designs. This approach was applied on a common type...

  9. Ischemic preconditioning negatively regulates plenty of SH3s-mixed lineage kinase 3-Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt.

    Science.gov (United States)

    Zhang, Q-G; Han, D; Xu, J; Lv, Q; Wang, R; Yin, X-H; Xu, T-L; Zhang, G-Y

    2006-12-01

    Activation of Akt/protein kinase B has been recently reported to play an important role in ischemic tolerance. We here demonstrate that the decreased protein expression and phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) underlie the increased Akt-Ser-473 phosphorylation in the hippocampal CA1 subfield in ischemic preconditioning (IPC). Co-immunoprecipitation analysis reveals that Akt physically interacts with Rac1, a small Rho family GTPase required for mixed lineage kinase 3 (MLK3) autophosphorylation, and both this interaction and Rac1-Ser-71 phosphorylation induced by Akt are promoted in preconditioned rats. In addition, we show that Akt activation results in the disassembly of the plenty of SH3s (POSH)-MLK3-Rac1 signaling complex and down-regulation of the activation of MLK3/c-Jun N-terminal kinase (JNK) pathway. Akt activation results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c, and activation of caspase-3. The expression of Fas ligand is also decreased in the CA1 region. Akt activation protects against apoptotic neuronal death as shown in TUNEL staining following IPC. Intracerebral infusion of LY294002 before IPC reverses the increase in Akt phosphorylation and the decrease in JNK signaling activation, as well as the neuroprotective action of IPC. Our results suggest that activation of pro-apoptotic MLK3/JNK3 cascade can be suppressed through activating anti-apoptotic phosphoinositide 3-kinase/Akt pathway induced by a sublethal ischemic insult, which provides a functional link between Akt and the JNK family of stress-activated kinases in ischemic tolerance.

  10. Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing.

    Science.gov (United States)

    Reader, John C; Matthews, Thomas P; Klair, Suki; Cheung, Kwai-Ming J; Scanlon, Jane; Proisy, Nicolas; Addison, Glynn; Ellard, John; Piton, Nelly; Taylor, Suzanne; Cherry, Michael; Fisher, Martin; Boxall, Kathy; Burns, Samantha; Walton, Michael I; Westwood, Isaac M; Hayes, Angela; Eve, Paul; Valenti, Melanie; de Haven Brandon, Alexis; Box, Gary; van Montfort, Rob L M; Williams, David H; Aherne, G Wynne; Raynaud, Florence I; Eccles, Suzanne A; Garrett, Michelle D; Collins, Ian

    2011-12-22

    Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.

  11. Scaffolds of PDLLA/bioglass 58S produced via selective laser sintering

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Rafaela do Vale; Salmoria, Gean Vitor; Moura, Marcela Oliveira Caldeira de; Aragones, Aguedo; Fredel, Marcio Celso, E-mail: rafaelavpereira@gmail.com [Universidade Federal de Santa Catarina (UFSC), Florianopolis, SC (Brazil)

    2014-08-15

    Scaffolds of PDLLA were produced to be implemented in maxillofacial surgeries inducing bone repair and regeneration. To prepare these scaffolds, bioglass (BG58S) was synthesized by sol-gel method, in order to be applied as osteoconductive dispersed particles in PDLLA matrix. Once presenting greater facility on parts fabrication, this polymeric matrix enables complex geometries production besides presenting compatible degradation rate for scaffold absorption and bone regeneration. Scaffolds production was performed by selective laser sintering in order to obtain tailored-made parts. FTIR and XRD analyses were carried out to observe the composition and evaluate the presence of crystallized phases in bioglass, obtaining Wollastonite. SEM was used to observe the BG particle distribution in PDLLA matrix and flexural test was performed to evaluate the composite mechanical properties. Results showed that was possible to obtain pieces using SLS method and with addition of 10%wt BG to polymeric matrix, flexural modulus and strength increased regarding to pure polymer. (author)

  12. A Scaffold Analysis Tool Using Mate-Pair Information in Genome Sequencing

    Directory of Open Access Journals (Sweden)

    Pan-Gyu Kim

    2008-01-01

    Full Text Available We have developed a Windows-based program, ConPath, as a scaffold analyzer. ConPath constructs scaffolds by ordering and orienting separate sequence contigs by exploiting the mate-pair information between contig-pairs. Our algorithm builds directed graphs from link information and traverses them to find the longest acyclic graphs. Using end read pairs of fixed-sized mate-pair libraries, ConPath determines relative orientations of all contigs, estimates the gap size of each adjacent contig pair, and reports wrong assembly information by validating orientations and gap sizes. We have utilized ConPath in more than 10 microbial genome projects, including Mannheimia succiniciproducens and Vibro vulnificus, where we verified contig assembly and identified several erroneous contigs using the four types of error defined in ConPath. Also, ConPath supports some convenient features and viewers that permit investigation of each contig in detail; these include contig viewer, scaffold viewer, edge information list, mate-pair list, and the printing of complex scaffold structures.

  13. Scaffolded biology.

    Science.gov (United States)

    Minelli, Alessandro

    2016-09-01

    Descriptions and interpretations of the natural world are dominated by dichotomies such as organism vs. environment, nature vs. nurture, genetic vs. epigenetic, but in the last couple of decades strong dissatisfaction with those partitions has been repeatedly voiced and a number of alternative perspectives have been suggested, from perspectives such as Dawkins' extended phenotype, Turner's extended organism, Oyama's Developmental Systems Theory and Odling-Smee's niche construction theory. Last in time is the description of biological phenomena in terms of hybrids between an organism (scaffolded system) and a living or non-living scaffold, forming unit systems to study processes such as reproduction and development. As scaffold, eventually, we can define any resource used by the biological system, especially in development and reproduction, without incorporating it as happens in the case of resources fueling metabolism. Addressing biological systems as functionally scaffolded systems may help pointing to functional relationships that can impart temporal marking to the developmental process and thus explain its irreversibility; revisiting the boundary between development and metabolism and also regeneration phenomena, by suggesting a conceptual framework within which to investigate phenomena of regular hypermorphic regeneration such as characteristic of deer antlers; fixing a periodization of development in terms of the times at which a scaffolding relationship begins or is terminated; and promoting plant galls to legitimate study objects of developmental biology.

  14. Hybrid 3D-2D printing for bone scaffolds fabrication

    Science.gov (United States)

    Seleznev, V. A.; Prinz, V. Ya

    2017-02-01

    It is a well-known fact that bone scaffold topography on micro- and nanometer scale influences the cellular behavior. Nano-scale surface modification of scaffolds allows the modulation of biological activity for enhanced cell differentiation. To date, there has been only a limited success in printing scaffolds with micro- and nano-scale features exposed on the surface. To improve on the currently available imperfect technologies, in our paper we introduce new hybrid technologies based on a combination of 2D (nano imprint) and 3D printing methods. The first method is based on using light projection 3D printing and simultaneous 2D nanostructuring of each of the layers during the formation of the 3D structure. The second method is based on the sequential integration of preliminarily created 2D nanostructured films into a 3D printed structure. The capabilities of the developed hybrid technologies are demonstrated with the example of forming 3D bone scaffolds. The proposed technologies can be used to fabricate complex 3D micro- and nanostructured products for various fields.

  15. Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

    Directory of Open Access Journals (Sweden)

    Jason M van Rooyen

    Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

  16. Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase

    International Nuclear Information System (INIS)

    Trewhella, J.; Blumenthal, D.K.; Rokop, S.E.; Seeger, P.A.

    1990-01-01

    Small-angle X-ray and neutron scattering have been used to study the solution structures of calmodulin complexed with synthetic peptides corresponding to residues 342-366 and 301-326, designated PhK5 and PhK13, respectively, in the regulatory domain of the catalytic subunit of skeletal muscle phosphorylase kinase. The scattering data show that binding of PhK5 to calmodulin induces a dramatic contraction of calmodulin, similar to that previously observed when calmodulin is complexed with the calmodulin-binding domain peptide from rabbit skeletal muscle myosin light chain kinase. In contrast, calmodulin remains extended upon binding PhK13. In the presence of both peptides, calmodulin also remains extended. Apparently, the presence of PhK13 inhibits calmodulin from undergoing the PhK5-induced contraction. These data indicate that there is a fundamentally different type of calmodulin-target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase

  17. Repopulating Decellularized Kidney Scaffolds: An Avenue for Ex Vivo Organ Generation

    Directory of Open Access Journals (Sweden)

    Robert A. McKee

    2016-03-01

    Full Text Available Recent research has shown that fully developed organs can be decellularized, resulting in a complex scaffold and extracellular matrix (ECM network capable of being populated with other cells. This work has resulted in a growing field in bioengineering focused on the isolation, characterization, and modification of organ derived acellular scaffolds and their potential to sustain and interact with new cell populations, a process termed reseeding. In this review, we cover contemporary advancements in the bioengineering of kidney scaffolds including novel work showing that reseeded donor scaffolds can be transplanted and can function in recipients using animal models. Several major areas of the field are taken into consideration, including the decellularization process, characterization of acellular and reseeded scaffolds, culture conditions, and cell sources. Finally, we discuss future avenues based on the advent of 3D bioprinting and recent developments in kidney organoid cultures as well as animal models of renal genesis. The ongoing mergers and collaborations between these fields hold the potential to produce functional kidneys that can be generated ex vivo and utilized for kidney transplantations in patients suffering with renal disease.

  18. Thiophene Scaffold as Prospective Central Nervous System Agent: A Review.

    Science.gov (United States)

    Deep, Aakash; Narasimhan, Balasubramanian; Aggarwal, Swati; Kaushik, Dhirender; Sharma, Arun K

    2016-01-01

    Heterocyclic compounds are extensively dispersed in nature and are vital for life. Various investigational approaches towards Structural Activity Relationship that focus upon the exploration of optimized candidates have become vastly important. Literature studies tell that for a series of compounds that are imperative in industrial and medicinal chemistry, thiophene acts as parent. Among various classes of heterocyclic compounds that have potential central nervous system activity, thiophene is the most important one. In the largely escalating chemical world of heterocyclic compounds showing potential pharmacological character, thiophene nucleus has been recognized as the budding entity. Seventeen Papers were included in this review article to define the central nervous system potential of thiophene. This review article enlightens the rationalized use and scope of thiophene scaffold as novel central nervous system activity such as anticonvulsant, acetylcholinesterase inhibitor, cyclin-dependent kinase 5 (cdk5/p25) inhibitors, CNS depressant, capability to block norepinephrine, serotonin and dopamine reuptake by their respective transporters etc. The Finding of this review confirm the importance of thiophene scaffold as potential central nervous system agents. From this outcome, ideas for future molecular modifications leading to the novel derivatives with better constructive pharmacological potential may be derived.

  19. 3D printing nano conductive multi-walled carbon nanotube scaffolds for nerve regeneration

    Science.gov (United States)

    Lee, Se-Jun; Zhu, Wei; Nowicki, Margaret; Lee, Grace; Nyoung Heo, Dong; Kim, Junghoon; Zuo, Yi Y.; Zhang, Lijie Grace

    2018-02-01

    Objective. Nanomaterials, such as carbon nanotubes (CNTs), have been introduced to modify the surface properties of scaffolds, thus enhancing the interaction between the neural cells and biomaterials. In addition to superior electrical conductivity, CNTs can provide nanoscale structures similar to those present in the natural neural environment. The primary objective of this study is to investigate the proliferative capability and differential potential of neural stem cells (NSCs) seeded on a CNT incorporated scaffold. Approach. Amine functionalized multi-walled carbon nanotubes (MWCNTs) were incorporated with a PEGDA polymer to provide enhanced electrical properties as well as nanofeatures on the surface of the scaffold. A stereolithography 3D printer was employed to fabricate a well-dispersed MWCNT-hydrogel composite neural scaffold with a tunable porous structure. 3D printing allows easy fabrication of complex 3D scaffolds with extremely intricate microarchitectures and controlled porosity. Main results. Our results showed that MWCNT-incorporated scaffolds promoted neural stem cell proliferation and early neuronal differentiation when compared to those scaffolds without the MWCNTs. Furthermore, biphasic pulse stimulation with 500 µA current promoted neuronal maturity quantified through protein expression analysis by quantitative polymerase chain reaction. Significance. Results of this study demonstrated that an electroconductive MWCNT scaffold, coupled with electrical stimulation, may have a synergistic effect on promoting neurite outgrowth for therapeutic application in nerve regeneration.

  20. Flexible control of cellular encapsulation, permeability, and release in a droplet-templated bifunctional copolymer scaffold.

    Science.gov (United States)

    Chen, Qiushui; Chen, Dong; Wu, Jing; Lin, Jin-Ming

    2016-11-01

    Designing cell-compatible, bio-degradable, and stimuli-responsive hydrogels is very important for biomedical applications in cellular delivery and micro-scale tissue engineering. Here, we report achieving flexible control of cellular microencapsulation, permeability, and release by rationally designing a diblock copolymer, alginate-conjugated poly(N-isopropylacrylamide) (Alg-co-PNiPAM). We use the microfluidic technique to fabricate the bifunctional copolymers into thousands of mono-disperse droplet-templated hydrogel microparticles for controlled encapsulation and triggered release of mammalian cells. In particular, the grafting PNiPAM groups in the synthetic cell-laden microgels produce lots of nano-aggregates into hydrogel networks at elevated temperature, thereafter enhancing the permeability of microparticle scaffolds. Importantly, the hydrogel scaffolds are readily fabricated via on-chip quick gelation by triggered release of Ca 2+ from the Ca-EDTA complex; it is also quite exciting that very mild release of microencapsulated cells is achieved via controlled degradation of hydrogel scaffolds through a simple strategy of competitive affinity of Ca 2+ from the Ca-Alginate complex. This finding suggests that we are able to control cellular encapsulation and release through ion-induced gelation and degradation of the hydrogel scaffolds. Subsequently, we demonstrate a high viability of microencapsulated cells in the microgel scaffolds.

  1. Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

    Science.gov (United States)

    Schiering, Nikolaus; Knapp, Stefan; Marconi, Marina; Flocco, Maria M; Cui, Jean; Perego, Rita; Rusconi, Luisa; Cristiani, Cinzia

    2003-10-28

    The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

  2. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions.

    Science.gov (United States)

    Walkiewicz, Katarzyna W; Girault, Jean-Antoine; Arold, Stefan T

    2015-10-01

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein 'nanomachines' to become activated in a site-specific manner. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Fabrication of a Highly Aligned Neural Scaffold via a Table Top Stereolithography 3D Printing and Electrospinning.

    Science.gov (United States)

    Lee, Se-Jun; Nowicki, Margaret; Harris, Brent; Zhang, Lijie Grace

    2017-06-01

    Three-dimensional (3D) bioprinting is a rapidly emerging technique in the field of tissue engineering to fabricate extremely intricate and complex biomimetic scaffolds in the range of micrometers. Such customized 3D printed constructs can be used for the regeneration of complex tissues such as cartilage, vessels, and nerves. However, the 3D printing techniques often offer limited control over the resolution and compromised mechanical properties due to short selection of printable inks. To address these limitations, we combined stereolithography and electrospinning techniques to fabricate a novel 3D biomimetic neural scaffold with a tunable porous structure and embedded aligned fibers. By employing two different types of biofabrication methods, we successfully utilized both synthetic and natural materials with varying chemical composition as bioink to enhance biocompatibilities and mechanical properties of the scaffold. The resulting microfibers composed of polycaprolactone (PCL) polymer and PCL mixed with gelatin were embedded in 3D printed hydrogel scaffold. Our results showed that 3D printed scaffolds with electrospun fibers significantly improve neural stem cell adhesion when compared to those without the fibers. Furthermore, 3D scaffolds embedded with aligned fibers showed an enhancement in cell proliferation relative to bare control scaffolds. More importantly, confocal microscopy images illustrated that the scaffold with PCL/gelatin fibers greatly increased the average neurite length and directed neurite extension of primary cortical neurons along the fiber. The results of this study demonstrate the potential to create unique 3D neural tissue constructs by combining 3D bioprinting and electrospinning techniques.

  4. Parallel fabrication of macroporous scaffolds.

    Science.gov (United States)

    Dobos, Andrew; Grandhi, Taraka Sai Pavan; Godeshala, Sudhakar; Meldrum, Deirdre R; Rege, Kaushal

    2018-07-01

    Scaffolds generated from naturally occurring and synthetic polymers have been investigated in several applications because of their biocompatibility and tunable chemo-mechanical properties. Existing methods for generation of 3D polymeric scaffolds typically cannot be parallelized, suffer from low throughputs, and do not allow for quick and easy removal of the fragile structures that are formed. Current molds used in hydrogel and scaffold fabrication using solvent casting and porogen leaching are often single-use and do not facilitate 3D scaffold formation in parallel. Here, we describe a simple device and related approaches for the parallel fabrication of macroporous scaffolds. This approach was employed for the generation of macroporous and non-macroporous materials in parallel, in higher throughput and allowed for easy retrieval of these 3D scaffolds once formed. In addition, macroporous scaffolds with interconnected as well as non-interconnected pores were generated, and the versatility of this approach was employed for the generation of 3D scaffolds from diverse materials including an aminoglycoside-derived cationic hydrogel ("Amikagel"), poly(lactic-co-glycolic acid) or PLGA, and collagen. Macroporous scaffolds generated using the device were investigated for plasmid DNA binding and cell loading, indicating the use of this approach for developing materials for different applications in biotechnology. Our results demonstrate that the device-based approach is a simple technology for generating scaffolds in parallel, which can enhance the toolbox of current fabrication techniques. © 2018 Wiley Periodicals, Inc.

  5. Magnetic resonance imaging-three-dimensional printing technology fabricates customized scaffolds for brain tissue engineering

    Institute of Scientific and Technical Information of China (English)

    Feng Fu; Chong Chen; Sai Zhang; Ming-liang Zhao; Xiao-hong Li; Zhe Qin; Chao Xu; Xu-yi Chen; Rui-xin Li; Li-na Wang; Ding-wei Peng; Hong-tao Sun; Yue Tu

    2017-01-01

    Conventional fabrication methods lack the ability to control both macro- and micro-structures of generated scaffolds. Three-dimensional printing is a solid free-form fabrication method that provides novel ways to create customized scaffolds with high precision and accuracy. In this study, an electrically controlled cortical impactor was used to induce randomized brain tissue defects. The overall shape of scaffolds was designed using rat-specific anatomical data obtained from magnetic resonance imaging, and the internal structure was created by computer- aided design. As the result of limitations arising from insufficient resolution of the manufacturing process, we magnified the size of the cavity model prototype five-fold to successfully fabricate customized collagen-chitosan scaffolds using three-dimensional printing. Results demonstrated that scaffolds have three-dimensional porous structures, high porosity, highly specific surface areas, pore connectivity and good internal characteristics. Neural stem cells co-cultured with scaffolds showed good viability, indicating good biocompatibility and biodegradability. This technique may be a promising new strategy for regenerating complex damaged brain tissues, and helps pave the way toward personalized medicine.

  6. Teachers' Use of Linguistic Scaffolding to Support the Academic Language Development of First-Grade Emergent Bilingual Students

    Science.gov (United States)

    Lucero, Audrey

    2014-01-01

    Research suggests that teachers need to scaffold emergent bilingual students as they develop the complex language associated with school success. This may especially be true in dual language settings, where children are learning two languages simultaneously. In this study, therefore, I investigate the linguistic scaffolding practices of…

  7. Crystal structures of T. b. rhodesiense adenosine kinase complexed with inhibitor and activator: implications for catalysis and hyperactivation.

    Directory of Open Access Journals (Sweden)

    Sabine Kuettel

    2011-05-01

    Full Text Available BACKGROUND: The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP utilizing adenosine triphosphate (ATP as the preferred phosphoryl donor. METHODS AND FINDINGS: Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK: the structure of TbrAK in complex with the bisubstrate inhibitor P(1,P(5-di(adenosine-5'-pentaphosphate (AP5A at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]morpholine (compound 1 at 2.8 Å resolution. CONCLUSIONS: The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.

  8. Discovery of potent 1H-imidazo[4,5-b]pyridine-based c-Met kinase inhibitors via mechanism-directed structural optimization.

    Science.gov (United States)

    An, Xiao-De; Liu, Hongyan; Xu, Zhong-Liang; Jin, Yi; Peng, Xia; Yao, Ying-Ming; Geng, Meiyu; Long, Ya-Qiu

    2015-02-01

    Starting from our previously identified novel c-Met kinase inhibitors bearing 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one scaffold, a global structural exploration was conducted to furnish an optimal binding motif for further development, directed by the enzyme inhibitory mechanism. First round SAR study picked two imidazonaphthyridinone frameworks with 1,8- and 3,5-disubstitution pattern as class I and class II c-Met kinase inhibitors, respectively. Further structural optimization on type II inhibitors by truncation of the imidazonaphthyridinone core and incorporation of an N-phenyl cyclopropane-1,1-dicarboxamide pharmacophore led to the discovery of novel imidazopyridine-based c-Met kinase inhibitors, displaying nanomolar enzyme inhibitory activity and improved Met kinase selectivity. More significantly, the new chemotype c-Met kinase inhibitors effectively inhibited Met phosphorylation and its downstream signaling as well as the proliferation of Met-dependent EBC-1 human lung cancer cells at submicromolar concentrations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Precision extruding deposition (PED) fabrication of polycaprolactone (PCL) scaffolds for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Shor, Lauren; Gueceri, Selcuk; Chang, Robert; Sun Wei [Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA (United States); Gordon, Jennifer; Kang Qian; Hartsock, Langdon; An Yuehuei [Department of Orthopedic Surgery, Medical University of South Carolina, Charleston, SC (United States)], E-mail: st963bya@drexel.edu, E-mail: guceri@drexel.edu, E-mail: rcc34@drexel.edu, E-mail: sunwei@drexel.edu, E-mail: kangqk@musc.edu, E-mail: hartsock@musc.edu, E-mail: any@musc.edu

    2009-03-01

    Bone tissue engineering is an emerging field providing viable substitutes for bone regeneration. Recent advances have allowed scientists and engineers to develop scaffolds for guided bone growth. However, success requires scaffolds to have specific macroscopic geometries and internal architectures conducive to biological and biophysical functions. Freeform fabrication provides an effective process tool to manufacture three-dimensional porous scaffolds with complex shapes and designed properties. A novel precision extruding deposition (PED) technique was developed to fabricate polycaprolactone (PCL) scaffolds. It was possible to manufacture scaffolds with a controlled pore size of 350 {mu}m with designed structural orientations using this method. The scaffold morphology, internal micro-architecture and mechanical properties were evaluated using scanning electron microscopy (SEM), micro-computed tomography (micro-CT) and mechanical testing, respectively. An in vitro cell-scaffold interaction study was carried out using primary fetal bovine osteoblasts. Specifically, the cell proliferation and differentiation was evaluated by Alamar Blue assay for cell metabolic activity, alkaline phosphatase activity and osteoblast production of calcium. An in vivo study was performed on nude mice to determine the capability of osteoblast-seeded PCL to induce osteogenesis. Each scaffold was implanted subcutaneously in nude mice and, following sacrifice, was explanted at one of a series of time intervals. The explants were then evaluated histologically for possible areas of osseointegration. Microscopy and radiological examination showed multiple areas of osseous ingrowth suggesting that the osteoblast-seeded PCL scaffolds evoke osteogenesis in vivo. These studies demonstrated the viability of the PED process to fabricate PCL scaffolds having the necessary mechanical properties, structural integrity, and controlled pore size and interconnectivity desired for bone tissue engineering.

  10. Precision extruding deposition (PED) fabrication of polycaprolactone (PCL) scaffolds for bone tissue engineering

    International Nuclear Information System (INIS)

    Shor, Lauren; Gueceri, Selcuk; Chang, Robert; Sun Wei; Gordon, Jennifer; Kang Qian; Hartsock, Langdon; An Yuehuei

    2009-01-01

    Bone tissue engineering is an emerging field providing viable substitutes for bone regeneration. Recent advances have allowed scientists and engineers to develop scaffolds for guided bone growth. However, success requires scaffolds to have specific macroscopic geometries and internal architectures conducive to biological and biophysical functions. Freeform fabrication provides an effective process tool to manufacture three-dimensional porous scaffolds with complex shapes and designed properties. A novel precision extruding deposition (PED) technique was developed to fabricate polycaprolactone (PCL) scaffolds. It was possible to manufacture scaffolds with a controlled pore size of 350 μm with designed structural orientations using this method. The scaffold morphology, internal micro-architecture and mechanical properties were evaluated using scanning electron microscopy (SEM), micro-computed tomography (micro-CT) and mechanical testing, respectively. An in vitro cell-scaffold interaction study was carried out using primary fetal bovine osteoblasts. Specifically, the cell proliferation and differentiation was evaluated by Alamar Blue assay for cell metabolic activity, alkaline phosphatase activity and osteoblast production of calcium. An in vivo study was performed on nude mice to determine the capability of osteoblast-seeded PCL to induce osteogenesis. Each scaffold was implanted subcutaneously in nude mice and, following sacrifice, was explanted at one of a series of time intervals. The explants were then evaluated histologically for possible areas of osseointegration. Microscopy and radiological examination showed multiple areas of osseous ingrowth suggesting that the osteoblast-seeded PCL scaffolds evoke osteogenesis in vivo. These studies demonstrated the viability of the PED process to fabricate PCL scaffolds having the necessary mechanical properties, structural integrity, and controlled pore size and interconnectivity desired for bone tissue engineering

  11. Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

    Science.gov (United States)

    Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

    2006-09-08

    The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

  12. Crystal structure of human protein kinase CK2

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I

    2001-01-01

    The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalyt...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit...

  13. Analog series-based scaffolds: computational design and exploration of a new type of molecular scaffolds for medicinal chemistry

    Science.gov (United States)

    Dimova, Dilyana; Stumpfe, Dagmar; Hu, Ye; Bajorath, Jürgen

    2016-01-01

    Aim: Computational design of and systematic search for a new type of molecular scaffolds termed analog series-based scaffolds. Materials & methods: From currently available bioactive compounds, analog series were systematically extracted, key compounds identified and new scaffolds isolated from them. Results: Using our computational approach, more than 12,000 scaffolds were extracted from bioactive compounds. Conclusion: A new scaffold definition is introduced and a computational methodology developed to systematically identify such scaffolds, yielding a large freely available scaffold knowledge base. PMID:28116132

  14. Kinase activity determination of specific AMPK complexes/heterotrimers in the skeletal muscle

    DEFF Research Database (Denmark)

    Birk, Jesper Bratz; Wojtaszewski, Jørgen

    2018-01-01

    Measuring the kinase activity of the 5'-AMP-activated protein kinase (AMPK) is an essential part of understanding the regulation of this metabolic master switch. The AMPK heterotrimer can exist in 12 different constellations with potentially diverse activation patterns. It is therefore important ...

  15. Spatial control of bone formation using a porous polymer scaffold co-delivering anabolic rhBMP-2 and anti-resorptive agents

    Directory of Open Access Journals (Sweden)

    NYC Yu

    2014-01-01

    Full Text Available Current clinical delivery of recombinant human bone morphogenetic proteins (rhBMPs utilises freeze-dried collagen. Despite effective new bone generation, rhBMP via collagen can be limited by significant complications due to inflammation and uncontrolled bone formation. This study aimed to produce an alternative rhBMP local delivery system to permit more controllable and superior rhBMP-induced bone formation. Cylindrical porous poly(lactic-co-glycolic acid (PLGA scaffolds were manufactured by thermally-induced phase separation. Scaffolds were encapsulated with anabolic rhBMP-2 (20 µg ± anti-resorptive agents: zoledronic acid (5 µg ZA, ZA pre-adsorbed onto hydroxyapatite microparticles, (5 µg ZA/2 % HA or IkappaB kinase (IKK inhibitor (10 µg PS-1145. Scaffolds were inserted in a 6-mm critical-sized femoral defect in Wistar rats, and compared against rhBMP-2 via collagen. The regenerate region was examined at 6 weeks by 3D microCT and descriptive histology. MicroCT and histology revealed rhBMP-induced bone was more restricted in the PLGA scaffolds than collagen scaffolds (-92.3 % TV, p < 0.01. The regenerate formed by PLGA + rhBMP-2/ZA/HA showed comparable bone volume to rhBMP-2 via collagen, and bone mineral density was +9.1 % higher (p < 0.01. Local adjunct ZA/HA or PS-1145 significantly enhanced PLGA + rhBMP-induced bone formation by +78.2 % and +52.0 %, respectively (p ≤ 0.01. Mechanistically, MG-63 human osteoblast-like cells showed cellular invasion and proliferation within PLGA scaffolds. In conclusion, PLGA scaffolds enabled superior spatial control of rhBMP-induced bone formation over clinically-used collagen. The PLGA scaffold has the potential to avoid uncontrollable bone formation-related safety issues and to customise bone shape by scaffold design. Moreover, local treatment with anti-resorptive agents incorporated within the scaffold further augmented rhBMP-induced bone formation.

  16. Use of Interim Scaffolding and Neotissue Development to Produce a Scaffold-Free Living Hyaline Cartilage Graft.

    Science.gov (United States)

    Lau, Ting Ting; Leong, Wenyan; Peck, Yvonne; Su, Kai; Wang, Dong-An

    2015-01-01

    The fabrication of three-dimensional (3D) constructs relies heavily on the use of biomaterial-based scaffolds. These are required as mechanical supports as well as to translate two-dimensional cultures to 3D cultures for clinical applications. Regardless of the choice of scaffold, timely degradation of scaffolds is difficult to achieve and undegraded scaffold material can lead to interference in further tissue development or morphogenesis. In cartilage tissue engineering, hydrogel is the highly preferred scaffold material as it shares many similar characteristics with native cartilaginous matrix. Hence, we employed gelatin microspheres as porogens to create a microcavitary alginate hydrogel as an interim scaffold to facilitate initial chondrocyte 3D culture and to establish a final scaffold-free living hyaline cartilaginous graft (LhCG) for cartilage tissue engineering.

  17. Adhesion and growth of human bone marrow mesenchymal stem cells on precise-geometry 3D organic–inorganic composite scaffolds for bone repair

    International Nuclear Information System (INIS)

    Chatzinikolaidou, Maria; Rekstyte, Sima; Danilevicius, Paulius; Pontikoglou, Charalampos; Papadaki, Helen; Farsari, Maria; Vamvakaki, Maria

    2015-01-01

    Engineering biomaterial scaffolds that promote attachment and growth of mesenchymal stem cells in three dimensions is a crucial parameter for successful bone tissue engineering. Towards this direction, a lot of research effort has focused recently into the development of three-dimensional porous scaffolds, aiming to elicit positive cellular behavior. However, the fabrication of three-dimensional tissue scaffolds with a precise geometry and complex micro- and nano-features, supporting cell in-growth remains a challenge. In this study we report on a positive cellular response of human bone marrow-derived (BM) mesenchymal stem cells (MSCs) onto hybrid material scaffolds consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide, and 2-(dimethylamino)ethyl methacrylate (DMAEMA). First, we use Direct fs Laser Writing, a 3D scaffolding technology to fabricate the complex structures. Subsequently, we investigate the morphology, viability and proliferation of BM-MSCs onto the hybrid scaffolds and examine the cellular response from different donors. Finally, we explore the effect of the materials' chemical composition on cell proliferation, employing three different material surfaces: (i) a hybrid consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide and 50 mol% DMAEMA, (ii) a hybrid material comprising methacryloxypropyl trimethoxysilane and zirconium propoxide, and (iii) a purely organic polyDMAEMA. Our results show a strong adhesion of BM-MSCs onto the hybrid material containing 50% DMAEMA from the first 2 h after seeding, and up to several days, and a proliferation increase after 14 and 21 days, similar to the polystyrene control, independent of cell donor. These findings support the potential use of our proposed cell–material combination in bone tissue engineering. - Graphical abstract: Scanning electron microscopy image depicting cell adhesion of bone marrow mesenchymal stem cells into a pore of a hybrid Direct Laser Writing

  18. Adhesion and growth of human bone marrow mesenchymal stem cells on precise-geometry 3D organic–inorganic composite scaffolds for bone repair

    Energy Technology Data Exchange (ETDEWEB)

    Chatzinikolaidou, Maria, E-mail: mchatzin@materials.uoc.gr [Department of Materials Science and Technology, University of Crete (Greece); Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH) (Greece); Rekstyte, Sima; Danilevicius, Paulius [Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH) (Greece); Pontikoglou, Charalampos; Papadaki, Helen [Hematology Laboratory, School of Medicine, University of Crete (Greece); Farsari, Maria [Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH) (Greece); Vamvakaki, Maria [Department of Materials Science and Technology, University of Crete (Greece); Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH) (Greece)

    2015-03-01

    Engineering biomaterial scaffolds that promote attachment and growth of mesenchymal stem cells in three dimensions is a crucial parameter for successful bone tissue engineering. Towards this direction, a lot of research effort has focused recently into the development of three-dimensional porous scaffolds, aiming to elicit positive cellular behavior. However, the fabrication of three-dimensional tissue scaffolds with a precise geometry and complex micro- and nano-features, supporting cell in-growth remains a challenge. In this study we report on a positive cellular response of human bone marrow-derived (BM) mesenchymal stem cells (MSCs) onto hybrid material scaffolds consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide, and 2-(dimethylamino)ethyl methacrylate (DMAEMA). First, we use Direct fs Laser Writing, a 3D scaffolding technology to fabricate the complex structures. Subsequently, we investigate the morphology, viability and proliferation of BM-MSCs onto the hybrid scaffolds and examine the cellular response from different donors. Finally, we explore the effect of the materials' chemical composition on cell proliferation, employing three different material surfaces: (i) a hybrid consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide and 50 mol% DMAEMA, (ii) a hybrid material comprising methacryloxypropyl trimethoxysilane and zirconium propoxide, and (iii) a purely organic polyDMAEMA. Our results show a strong adhesion of BM-MSCs onto the hybrid material containing 50% DMAEMA from the first 2 h after seeding, and up to several days, and a proliferation increase after 14 and 21 days, similar to the polystyrene control, independent of cell donor. These findings support the potential use of our proposed cell–material combination in bone tissue engineering. - Graphical abstract: Scanning electron microscopy image depicting cell adhesion of bone marrow mesenchymal stem cells into a pore of a hybrid Direct Laser Writing

  19. LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

    Science.gov (United States)

    Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

    2016-01-13

    Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

  20. A novel three-dimensional scaffold for regenerative endodontics: materials and biological characterizations.

    Science.gov (United States)

    Bottino, Marco C; Yassen, Ghaeth H; Platt, Jeffrey A; Labban, Nawaf; Windsor, L Jack; Spolnik, Kenneth J; Bressiani, Ana H A

    2015-11-01

    An electrospun nanocomposite fibrous material holds promise as a scaffold, as well as a drug-delivery device to aid in root maturogenesis and the regeneration of the pulp-dentine complex. A novel three-dimensional (3D) nanocomposite scaffold composed of polydioxanone (PDS II®) and halloysite nanotubes (HNTs) was designed and fabricated by electrospinning. Morphology, structure, mechanical properties and cell compatibility studies were carried out to evaluate the effects of HNTs incorporation (0.5-10 wt% relative to PDS w/w). Overall, a 3D porous network was seen in the different fabricated electrospun scaffolds, regardless of the HNT content. The incorporation of HNTs at 10 wt% led to a significant (p endodontics. Copyright © 2013 John Wiley & Sons, Ltd.

  1. Dispensing-based bioprinting of mechanically-functional hybrid scaffolds with vessel-like channels for tissue engineering applications - A brief review.

    Science.gov (United States)

    Naghieh, Saman; Sarker, Md; Izadifar, Mohammad; Chen, Xiongbiao

    2018-02-01

    Over the past decades, significant progress has been achieved in the field of tissue engineering (TE) to restore/repair damaged tissues or organs and, in this regard, scaffolds made from biomaterials have played a critical role. Notably, recent advances in biomaterials and three-dimensional (3D) printing have enabled the manipulation of two or more biomaterials of distinct, yet complementary, mechanical and/or biological properties to form so-called hybrid scaffolds mimicking native tissues. Among various biomaterials, hydrogels synthesized to incorporate living cells and/or biological molecules have dominated due to their hydrated tissue-like environment. Moreover, dispensing-based bioprinting has evolved to the point that it can now be used to create hybrid scaffolds with complex structures. However, the complexities associated with multi-material bioprinting and synthesis of hydrogels used for hybrid scaffolds pose many challenges for their fabrication. This paper presents a brief review of dispensing-based bioprinting of hybrid scaffolds for TE applications. The focus is on the design and fabrication of hybrid scaffolds, including imaging techniques, potential biomaterials, physical architecture, mechanical properties, cell viability, and the importance of vessel-like channels. The key issues and challenges for dispensing-based bioprinting of hybrid scaffolds are also identified and discussed along with recommendations for future research directions. Addressing these issues will significantly enhance the design and fabrication of hybrid scaffolds to and pave the way for translating them into clinical applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Templated self-assembly of quantum dots from aqueous solution using protein scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Blum, Amy Szuchmacher [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Soto, Carissa M [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Wilson, Charmaine D [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Whitley, Jessica L [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Moore, Martin H [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Sapsford, Kim E [George Mason University, 10910 University Boulevard, Manassas, VA 20110 (United States); Lin, Tianwei [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Chatterji, Anju [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Johnson, John E [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Ratna, Banahalli R [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States)

    2006-10-28

    Short, histidine-containing peptides can be conjugated to lysine-containing protein scaffolds to controllably attach quantum dots (QDs) to the scaffold, allowing for generic attachment of quantum dots to any protein without the use of specially engineered domains. This technique was used to bind quantum dots from aqueous solution to both chicken IgG and cowpea mosaic virus (CPMV), a 30 nm viral particle. These quantum dot-protein assemblies were studied in detail. The IgG-QD complexes were shown to retain binding specificity to their antigen after modification. The CPMV-QD complexes have a local concentration of quantum dots greater than 3000 nmol ml{sup -1}, and show a 15% increase in fluorescence quantum yield over free quantum dots in solution.

  3. * Fabrication and Characterization of Biphasic Silk Fibroin Scaffolds for Tendon/Ligament-to-Bone Tissue Engineering.

    Science.gov (United States)

    Font Tellado, Sònia; Bonani, Walter; Balmayor, Elizabeth R; Foehr, Peter; Motta, Antonella; Migliaresi, Claudio; van Griensven, Martijn

    2017-08-01

    Tissue engineering is an attractive strategy for tendon/ligament-to-bone interface repair. The structure and extracellular matrix composition of the interface are complex and allow for a gradual mechanical stress transfer between tendons/ligaments and bone. Thus, scaffolds mimicking the structural features of the native interface may be able to better support functional tissue regeneration. In this study, we fabricated biphasic silk fibroin scaffolds designed to mimic the gradient in collagen molecule alignment present at the interface. The scaffolds had two different pore alignments: anisotropic at the tendon/ligament side and isotropic at the bone side. Total porosity ranged from 50% to 80% and the majority of pores (80-90%) were ligament, enthesis, and cartilage markers significantly changed depending on pore alignment in each region of the scaffolds. In conclusion, the biphasic scaffolds fabricated in this study show promising features for tendon/ligament-to-bone tissue engineering.

  4. Functionally graded electrospun scaffolds with tunable mechanical properties for vascular tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Vinoy [Center for Nanoscale Materials and Biointegration (CNMB), Department of Physics, University of Alabama at Birmingham (UAB), AL 35294 (United States); Zhang Xing [Department of Biomedical Engineering, School of Engineering, University of Alabama at Birmingham (UAB), AL 35294 (United States); Catledge, Shane A [Center for Nanoscale Materials and Biointegration (CNMB), Department of Physics, University of Alabama at Birmingham (UAB), AL 35294 (United States); Vohra, Yogesh K [Center for Nanoscale Materials and Biointegration (CNMB), Department of Physics, University of Alabama at Birmingham (UAB), AL 35294 (United States)

    2007-12-15

    Electrospun tubular scaffolds (4 mm inner diameter) based on bio-artificial blends of polyglyconate (Maxon (registered) ) and proteins such as gelatin and elastin having a spatially designed multilayer structure were prepared for use as vascular tissue scaffolds. Scanning electron microscopy analysis of scaffolds showed a random nanofibrous morphology with fiber diameter in the range of 200-400 nm for protein-blended Maxon, which mimics the nanoscale dimensions of collagen (50-500 nm). The scaffolds have a well interconnected pore structure and porosity up to 82%, with protein blending and multi-layering in contrast to electrospun Maxon (registered) scaffolds (67%). Fourier-transform infrared spectroscopy, x-ray diffraction and differential scanning calorimetry results confirmed the blended composition and crystallinity of fibers. Uniaxial tensile testing revealed a strength of 14.46 {+-} 0.42 MPa and a modulus of 15.44 {+-} 2.53 MPa with a failure strain of 322.5 {+-} 10% for a pure Maxon (registered) scaffold. The blending of polyglyconate with biopolymers decreased the tensile properties in general, with an exception of the tensile modulus (48.38 {+-} 2 MPa) of gelatin/Maxon mesh, which was higher than that of the pure Maxon (registered) scaffold. Trilayered tubular scaffolds of gelatin/elastin, gelatin/elastin/Maxon and gelatin/Maxon (GE-GEM-GM) that mimic the complex trilayer matrix structure of natural artery have been prepared by sequential electrospinning. Tensile testing under dry conditions revealed a tensile strength of 2.71 {+-} 0.2 MPa and a modulus of 20.4 {+-} 3 MPa with a failure strain of 140 {+-} 10%. However, GE-GEM-GM scaffolds tested under wet conditions after soaking in a phosphate buffered saline medium at 37 {sup 0}C for 24 h exhibited mechanical properties (2.5 MPa tensile strength and 9 MPa tensile modulus) comparable to those of native femoral artery.

  5. Functionally graded electrospun scaffolds with tunable mechanical properties for vascular tissue regeneration

    International Nuclear Information System (INIS)

    Thomas, Vinoy; Zhang Xing; Catledge, Shane A; Vohra, Yogesh K

    2007-01-01

    Electrospun tubular scaffolds (4 mm inner diameter) based on bio-artificial blends of polyglyconate (Maxon (registered) ) and proteins such as gelatin and elastin having a spatially designed multilayer structure were prepared for use as vascular tissue scaffolds. Scanning electron microscopy analysis of scaffolds showed a random nanofibrous morphology with fiber diameter in the range of 200-400 nm for protein-blended Maxon, which mimics the nanoscale dimensions of collagen (50-500 nm). The scaffolds have a well interconnected pore structure and porosity up to 82%, with protein blending and multi-layering in contrast to electrospun Maxon (registered) scaffolds (67%). Fourier-transform infrared spectroscopy, x-ray diffraction and differential scanning calorimetry results confirmed the blended composition and crystallinity of fibers. Uniaxial tensile testing revealed a strength of 14.46 ± 0.42 MPa and a modulus of 15.44 ± 2.53 MPa with a failure strain of 322.5 ± 10% for a pure Maxon (registered) scaffold. The blending of polyglyconate with biopolymers decreased the tensile properties in general, with an exception of the tensile modulus (48.38 ± 2 MPa) of gelatin/Maxon mesh, which was higher than that of the pure Maxon (registered) scaffold. Trilayered tubular scaffolds of gelatin/elastin, gelatin/elastin/Maxon and gelatin/Maxon (GE-GEM-GM) that mimic the complex trilayer matrix structure of natural artery have been prepared by sequential electrospinning. Tensile testing under dry conditions revealed a tensile strength of 2.71 ± 0.2 MPa and a modulus of 20.4 ± 3 MPa with a failure strain of 140 ± 10%. However, GE-GEM-GM scaffolds tested under wet conditions after soaking in a phosphate buffered saline medium at 37 0 C for 24 h exhibited mechanical properties (2.5 MPa tensile strength and 9 MPa tensile modulus) comparable to those of native femoral artery

  6. Antimicrobial Cu-bearing stainless steel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Qiang, E-mail: mfqwang@163.com [School of Stomatology, China Medical University, Shenyang 110002 (China); Ren, Ling [Institute of Metal Research, Chinese Academy of Sciences (China); Li, Xiaopeng [School of Mechanical and Chemical Engineering, The University of Western Australia (Australia); Zhang, Shuyuan [Institute of Metal Research, Chinese Academy of Sciences (China); Sercombe, Timothy B., E-mail: tim.sercombe@uwa.edu.au [School of Mechanical and Chemical Engineering, The University of Western Australia (Australia); Yang, Ke, E-mail: kyang@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences (China)

    2016-11-01

    Copper-bearing stainless steel scaffolds with two different structures (Body Centered Cubic and Gyroid labyrinth) at two solid fractions (25% and 40%) were fabricated from both 316L powder and a mixture of 316L and elemental Cu powder using selective laser melting, and relative 316L scaffolds were served as control group. After processing, the antimicrobial testing demonstrated that the 316L-Cu scaffolds presented excellent antimicrobial activity against Escherichia coli and Staphylococcus aureus, and the cell viability assay indicated that there was no cytotoxic effect of 316L-Cu scaffolds on rat marrow mesenchymal stem cells. As such, these have the potential to reduce implant-associated infections. The Cu was also found to homogeneously distribute within the microstructure by scanning electronic microcopy. The addition of Cu would not significantly affect its strength and stiffness compared to 316L scaffold, and the stiffness of all the scaffolds (3-20GPa) is similar to that of bone and much less than that of bulk stainless steel. Consequently, fabrication of such low stiffness porous structures, especially coupled with the addition of antimicrobial Cu, may provide a new direction for medical stainless steels. - Highlights: • 316L-Cu scaffolds were fabricated by using selective laser melting (SLM). • 316L-Cu scaffolds showed satisfied antimicrobial activities. • 316L-Cu scaffolds have no cytotoxic effect on normal cells. • Other properties of 316L-Cu scaffolds were similar to 316L scaffolds. • 316L-Cu scaffolds have the potential to be used in orthopedic applications.

  7. Antimicrobial Cu-bearing stainless steel scaffolds

    International Nuclear Information System (INIS)

    Wang, Qiang; Ren, Ling; Li, Xiaopeng; Zhang, Shuyuan; Sercombe, Timothy B.; Yang, Ke

    2016-01-01

    Copper-bearing stainless steel scaffolds with two different structures (Body Centered Cubic and Gyroid labyrinth) at two solid fractions (25% and 40%) were fabricated from both 316L powder and a mixture of 316L and elemental Cu powder using selective laser melting, and relative 316L scaffolds were served as control group. After processing, the antimicrobial testing demonstrated that the 316L-Cu scaffolds presented excellent antimicrobial activity against Escherichia coli and Staphylococcus aureus, and the cell viability assay indicated that there was no cytotoxic effect of 316L-Cu scaffolds on rat marrow mesenchymal stem cells. As such, these have the potential to reduce implant-associated infections. The Cu was also found to homogeneously distribute within the microstructure by scanning electronic microcopy. The addition of Cu would not significantly affect its strength and stiffness compared to 316L scaffold, and the stiffness of all the scaffolds (3-20GPa) is similar to that of bone and much less than that of bulk stainless steel. Consequently, fabrication of such low stiffness porous structures, especially coupled with the addition of antimicrobial Cu, may provide a new direction for medical stainless steels. - Highlights: • 316L-Cu scaffolds were fabricated by using selective laser melting (SLM). • 316L-Cu scaffolds showed satisfied antimicrobial activities. • 316L-Cu scaffolds have no cytotoxic effect on normal cells. • Other properties of 316L-Cu scaffolds were similar to 316L scaffolds. • 316L-Cu scaffolds have the potential to be used in orthopedic applications.

  8. Mono- and Dinuclear Manganese Carbonyls Supported by 1,8-Disubstituted (L = Py, SMe, SH) Anthracene Ligand Scaffolds.

    Science.gov (United States)

    Manes, Taylor A; Rose, Michael J

    2016-06-06

    Presented herein is a synthetic scheme to generate symmetric and asymmetric ligands based on a 1,8-disubstituted anthracene scaffold. The metal-binding scaffolds were prepared by aryl chloride activation of 1,8-dichloroanthracene using Suzuki-type couplings facilitated by [Pd(dba)2] as a Pd source; the choice of cocatalyst (XPhos or SPhos) yielded symmetrically or asymmetrically substituted scaffolds (respectively): namely, Anth-SMe2 (3), Anth-N2 (4), and Anth-NSMe (6). The ligands exhibit a nonplanar geometry in the solid state (X-ray), owing to steric hindrance between the anthracene scaffold and the coupled aryl units. To determine the flexibility and binding characteristics of the anthracene-based ligands, the symmetric scaffolds were complexed with [Mn(CO)5Br] to afford the mononuclear species [(Anth-SMe2)Mn(CO)3Br] (8) and [(Anth-N2)Mn(CO)3Br] (9), in which the donor moieties chelate the Mn center in a cis fashion. The asymmetric ligand Anth-NSMe (6) binds preferentially through the py moieties, affording the bis-ligated complex [(Anth-NSMe)2Mn(CO)3Br] (10), wherein the thioether-S donors remain unbound. Alternatively, deprotection of the thioether in 6 affords the free thiol ligand Anth-NSH (7), which more readily binds the Mn center. Complexation of 7 ultimately affords the mixed-valence Mn(I)/Mn(II) dimer of formula [(Anth-NS)3Mn2(CO)3] (11), which exhibits a fac-{Mn(CO)3} unit supported by a triad of bridging thiolates, which are in turn ligated to a supporting Mn(II) center (EPR: |D| = 0.053 cm(-1), E/|D| = 0.3, Aiso = -150 MHz). All of the metal complexes have been characterized by single-crystal X-ray diffraction, IR spectroscopy and NMR/EPR measurements-all of which demonstrate that the meta-linked, anthracene-based ligand scaffold is a viable approach for the coordination of metal carbonyls.

  9. Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Ken; Levell, Julian; Yoon, Taeyong; Kohls, Darcy; Yowe, David; Rigel, Dean F.; Imase, Hidetomo; Yuan, Jun; Yasoshima, Kayo; DiPetrillo, Keith; Monovich, Lauren; Xu, Lingfei; Zhu, Meicheng; Kato, Mitsunori; Jain, Monish; Idamakanti, Neeraja; Taslimi, Paul; Kawanami, Toshio; Argikar, Upendra A.; Kunjathoor, Vidya; Xie, Xiaoling; Yagi, Yukiko I.; Iwaki, Yuki; Robinson, Zachary; Park, Hyi-Man (Novartis)

    2017-08-03

    The observed structure–activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

  10. Computational design of new molecular scaffolds for medicinal chemistry, part II: generalization of analog series-based scaffolds

    Science.gov (United States)

    Dimova, Dilyana; Stumpfe, Dagmar; Bajorath, Jürgen

    2018-01-01

    Aim: Extending and generalizing the computational concept of analog series-based (ASB) scaffolds. Materials & methods: Methodological modifications were introduced to further increase the coverage of analog series (ASs) and compounds by ASB scaffolds. From bioactive compounds, ASs were systematically extracted and second-generation ASB scaffolds isolated. Results: More than 20,000 second-generation ASB scaffolds with single or multiple substitution sites were extracted from active compounds, achieving more than 90% coverage of ASs. Conclusion: Generalization of the ASB scaffold approach has yielded a large knowledge base of scaffold-capturing compound series and target information. PMID:29379641

  11. Hybrid Scaffolds for Tissue Regeneration: Chemotaxis and Physical Confinement as Sources of Biomimesis

    Directory of Open Access Journals (Sweden)

    Simone Sprio

    2012-01-01

    Full Text Available Biomineralization is a complex ensemble of concomitant phenomena, driving the development of vertebrate and invertebrate organisms, particularly the formation of human bone tissue. In such a process collagen molecules assemble and organize in a complex 3-D structure and simultaneously mineralize with nearly amorphous apatite nanoparticles, whose heterogeneous nucleation, growth, and specific orientation are mediated by various chemical, physical, morphological, and structural control mechanisms, activated by the organic matrix at different size levels. The present work investigates on in-lab biomineralization processes, performed to synthesize hybrid hydroxyapatite/collagen scaffolds for bone and osteochondral regeneration. The synthesis processes are carried out by soft-chemistry procedures, with the purpose to activate all the different control mechanisms at the basis of new bone formation in vivo, so as to achieve scaffolds with high biomimesis, that is, physical, chemical, morphological, and ultrastructural properties very close to the newly formed human bone. Deep analysis of cell behaviour in contact with such hybrid scaffolds confirms their strong affinity with human bone, which in turn determines high regenerative properties in vivo.

  12. Polo-like kinase phosphorylation determines Caenorhabditis elegans centrosome size and density by biasing SPD-5 toward an assembly-competent conformation

    Directory of Open Access Journals (Sweden)

    Oliver Wueseke

    2016-10-01

    Full Text Available Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM. In Caenorhabditis elegans embryos, the mitotic PCM expands by Polo-like kinase 1 (PLK-1 phosphorylation-accelerated assembly of SPD-5 molecules into supramolecular scaffolds. However, how PLK-1 phosphorylation regulates SPD-5 assembly is not known. We found that a mutant version of SPD-5 that is insensitive to PLK-1 phosphorylation (SPD-54A could localize to PCM but was unable to rescue the reduction in PCM size and density when wild-type SPD-5 levels were decreased. In vitro, purified SPD-54A self-assembled into functional supramolecular scaffolds over long time scales, suggesting that phosphorylation only controls the rate of SPD-5 scaffold assembly. Furthermore, the SPD-5 scaffold, once assembled, remained intact and supported microtubule nucleation in the absence of PLK-1 activity in vivo. We conclude that PLK-1 is required for rapid assembly of the PCM scaffold but not for scaffold maintenance or function. Based on this idea, we developed a theoretical model that adequately predicted PCM growth rates in different mutant conditions in vivo. We propose that PLK-1 phosphorylation-dependent conversion of SPD-5 into an assembly-competent form underlies PCM formation in vivo and that the rate of this conversion determines final PCM size and density.

  13. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Directory of Open Access Journals (Sweden)

    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  14. Cell–scaffold interaction within engineered tissue

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiping; Liu, Yuanyuan, E-mail: Yuanyuan_liu@shu.edu.cn; Jiang, Zhenglong; Chen, Weihua; Yu, Yongzhe; Hu, Qingxi

    2014-05-01

    The structure of a tissue engineering scaffold plays an important role in modulating tissue growth. A novel gelatin–chitosan (Gel–Cs) scaffold with a unique structure produced by three-dimensional printing (3DP) technology combining with vacuum freeze-drying has been developed for tissue-engineering applications. The scaffold composed of overall construction, micro-pore, surface morphology, and effective mechanical property. Such a structure meets the essential design criteria of an ideal engineered scaffold. The favorable cell–matrix interaction supports the active biocompatibility of the structure. The structure is capable of supporting cell attachment and proliferation. Cells seeded into this structure tend to maintain phenotypic shape and secreted large amounts of extracellular matrix (ECM) and the cell growth decreased the mechanical properties of scaffold. This novel biodegradable scaffold has potential applications for tissue engineering based upon its unique structure, which acts to support cell growth. - Highlights: • The scaffold is not only for providing a surface for cell residence but also for determining cell phenotype and retaining structural integrity. • The mechanical property of scaffold can be affected by activities of cell. • The scaffold provides a microenvironment for cell attachment, growth, and migration.

  15. Self-Imitation and Environmental Scaffolding for Robot Teaching

    Directory of Open Access Journals (Sweden)

    Joe Saunders

    2007-03-01

    Full Text Available Imitative learning and learning by observation are social mechanisms that allow a robot to acquire knowledge from a human or another robot. However to be able to obtain skills in this way the robot faces many complex issues, one of which is that of finding solutions to the correspondence problem. Evolutionary predecessors to observational imitation may have been self-imitation where an agent avoids the complexities of the correspondence problem by learning and replicating actions it has experienced through the manipulation of its body. We investigate how a robotic control and teaching system using self-imitation can be constructed with reference to psychological models of motor control and ideas from social scaffolding seen in animals. Within these scaffolded environments sets of competencies can be built by constructing hierarchical state/action memory maps of the robot's interaction within that environment. The scaffolding process provides a mechanism to enable learning to be scaled up. The resulting system allows a human trainer to teach a robot new skills and modify skills that the robot may possess. Additionally the system allows the robot to notify the trainer when it is being taught skills it already has in its repertoire and to direct and focus its attention and sensor resources to relevant parts of the skill being executed. We argue that these mechanisms may be a first step towards the transformation from self-imitation to observational imitation. The system is validated on a physical pioneer robot that is taught using self-imitation to track, follow and point to a patterned object.

  16. Self-imitation and Environmental Scaffolding for Robot Teaching

    Directory of Open Access Journals (Sweden)

    Chrystopher L. Nehaniv

    2008-11-01

    Full Text Available Imitative learning and learning by observation are social mechanisms that allow a robot to acquire knowledge from a human or another robot. However to be able to obtain skills in this way the robot faces many complex issues, one of which is that of finding solutions to the correspondence problem. Evolutionary predecessors to observational imitation may have been self-imitation where an agent avoids the complexities of the correspondence problem by learning and replicating actions it has experienced through the manipulation of its body. We investigate how a robotic control and teaching system using self-imitation can be constructed with reference to psychological models of motor control and ideas from social scaffolding seen in animals. Within these scaffolded environments sets of competencies can be built by constructing hierarchical state/action memory maps of the robot's interaction within that environment. The scaffolding process provides a mechanism to enable learning to be scaled up. The resulting system allows a human trainer to teach a robot new skills and modify skills that the robot may possess. Additionally the system allows the robot to notify the trainer when it is being taught skills it already has in its repertoire and to direct and focus its attention and sensor resources to relevant parts of the skill being executed. We argue that these mechanisms may be a first step towards the transformation from self-imitation to observational imitation. The system is validated on a physical pioneer robot that is taught using self-imitation to track, follow and point to a patterned object.

  17. In Vitro Activation of the IκB Kinase Complex by Human T-cell Leukemia Virus Type-1 Tax*

    Science.gov (United States)

    Mukherjee, Sohini; Negi, Veera S.; Keitany, Gladys; Tanaka, Yuetsu; Orth, Kim

    2008-01-01

    Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IκB kinase (IKK), resulting in constitutive activation of NFκB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFκB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation. PMID:18223255

  18. Using Scaffolds in Problem-Based Hypermedia

    Science.gov (United States)

    Su, Yuyan; Klein, James D.

    2010-01-01

    This study investigated the use of scaffolds in problem-based hypermedia. Three hundred and twelve undergraduate students enrolled in a computer literacy course worked in project teams to use a hypermedia PBL program focused on designing a personal computer. The PBL program included content scaffolds, metacognitive scaffolds, or no scaffolds.…

  19. Bioactive polymeric scaffolds for tissue engineering

    Directory of Open Access Journals (Sweden)

    Scott Stratton

    2016-12-01

    Full Text Available A variety of engineered scaffolds have been created for tissue engineering using polymers, ceramics and their composites. Biomimicry has been adopted for majority of the three-dimensional (3D scaffold design both in terms of physicochemical properties, as well as bioactivity for superior tissue regeneration. Scaffolds fabricated via salt leaching, particle sintering, hydrogels and lithography have been successful in promoting cell growth in vitro and tissue regeneration in vivo. Scaffold systems derived from decellularization of whole organs or tissues has been popular due to their assured biocompatibility and bioactivity. Traditional scaffold fabrication techniques often failed to create intricate structures with greater resolution, not reproducible and involved multiple steps. The 3D printing technology overcome several limitations of the traditional techniques and made it easier to adopt several thermoplastics and hydrogels to create micro-nanostructured scaffolds and devices for tissue engineering and drug delivery. This review highlights scaffold fabrication methodologies with a focus on optimizing scaffold performance through the matrix pores, bioactivity and degradation rate to enable tissue regeneration. Review highlights few examples of bioactive scaffold mediated nerve, muscle, tendon/ligament and bone regeneration. Regardless of the efforts required for optimization, a shift in 3D scaffold uses from the laboratory into everyday life is expected in the near future as some of the methods discussed in this review become more streamlined.

  20. Decellularized Human Dental Pulp as a Scaffold for Regenerative Endodontics.

    Science.gov (United States)

    Song, J S; Takimoto, K; Jeon, M; Vadakekalam, J; Ruparel, N B; Diogenes, A

    2017-06-01

    Teeth undergo postnatal organogenesis relatively late in life and only complete full maturation a few years after the crown first erupts in the oral cavity. At this stage, development can be arrested if the tooth organ is damaged by either trauma or caries. Regenerative endodontic procedures (REPs) are a treatment alternative to conventional root canal treatment for immature teeth. These procedures rely on the transfer of apically positioned stem cells, including stem cells of the apical papilla (SCAP), into the root canal system. Although clinical success has been reported for these procedures, the predictability of expected outcomes and the organization of the newly formed tissues are affected by the lack of an available suitable scaffold that mimics the complexity of the dental pulp extracellular matrix (ECM). In this study, we evaluated 3 methods of decellularization of human dental pulp to be used as a potential autograft scaffold. Tooth slices of human healthy extracted third molars were decellularized by 3 different methods. One of the methods generated the maximum observed decellularization with minimal impact on the ECM composition and organization. Furthermore, recellularization of the scaffold supported the proliferation of SCAP throughout the scaffold with differentiation into odontoblast-like cells near the dentinal walls. Thus, this study reports that human dental pulp from healthy extracted teeth can be successfully decellularized, and the resulting scaffold supports the proliferation and differentiation of SCAP. The future application of this form of an autograft in REPs can fulfill a yet unmet need for a suitable scaffold, potentially improving clinical outcomes and ultimately promoting the survival and function of teeth with otherwise poor prognosis.

  1. Substrate-specific reorganization of the conformational ensemble of CSK implicates novel modes of kinase function.

    Directory of Open Access Journals (Sweden)

    Michael A Jamros

    Full Text Available Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.

  2. Fabrication of individual alginate-TCP scaffolds for bone tissue engineering by means of powder printing.

    Science.gov (United States)

    Castilho, Miguel; Rodrigues, Jorge; Pires, Inês; Gouveia, Barbara; Pereira, Manuel; Moseke, Claus; Groll, Jürgen; Ewald, Andrea; Vorndran, Elke

    2015-01-06

    The development of polymer-calcium phosphate composite scaffolds with tailored architectures and properties has great potential for bone regeneration. Herein, we aimed to improve the functional performance of brittle ceramic scaffolds by developing a promising biopolymer-ceramic network. For this purpose, two strategies, namely, direct printing of a powder composition consisting of a 60:40 mixture of α/β-tricalcium phosphate (TCP) powder and alginate powder or vacuum infiltration of printed TCP scaffolds with an alginate solution, were tracked. Results of structural characterization revealed that the scaffolds printed with 2.5 wt% alginate-modified TCP powders presented a uniformly distributed and interfusing alginate TCP network. Mechanical results indicated a significant increase in strength, energy to failure and reliability of powder-modified scaffolds with an alginate content in the educts of 2.5 wt% when compared to pure TCP, as well as to TCP scaffolds containing 5 wt% or 7.5 wt% in the educts, in both dry and wet states. Culture of human osteoblast cells on these scaffolds also demonstrated a great improvement of cell proliferation and cell viability. While in the case of powder-mixed alginate TCP scaffolds, isolated alginate gels were formed between the calcium phosphate crystals, the vacuum-infiltration strategy resulted in the covering of the surface and internal pores of the TCP scaffold with a thin alginate film. Furthermore, the prediction of the scaffolds' critical fracture conditions under more complex stress states by the applied Mohr fracture criterion confirmed the potential of the powder-modified scaffolds with 2.5 wt% alginate in the educts as structural biomaterial for bone tissue engineering.

  3. 3D printing for the design and fabrication of polymer-based gradient scaffolds.

    Science.gov (United States)

    Bracaglia, Laura G; Smith, Brandon T; Watson, Emma; Arumugasaamy, Navein; Mikos, Antonios G; Fisher, John P

    2017-07-01

    To accurately mimic the native tissue environment, tissue engineered scaffolds often need to have a highly controlled and varied display of three-dimensional (3D) architecture and geometrical cues. Additive manufacturing in tissue engineering has made possible the development of complex scaffolds that mimic the native tissue architectures. As such, architectural details that were previously unattainable or irreproducible can now be incorporated in an ordered and organized approach, further advancing the structural and chemical cues delivered to cells interacting with the scaffold. This control over the environment has given engineers the ability to unlock cellular machinery that is highly dependent upon the intricate heterogeneous environment of native tissue. Recent research into the incorporation of physical and chemical gradients within scaffolds indicates that integrating these features improves the function of a tissue engineered construct. This review covers recent advances on techniques to incorporate gradients into polymer scaffolds through additive manufacturing and evaluate the success of these techniques. As covered here, to best replicate different tissue types, one must be cognizant of the vastly different types of manufacturing techniques available to create these gradient scaffolds. We review the various types of additive manufacturing techniques that can be leveraged to fabricate scaffolds with heterogeneous properties and discuss methods to successfully characterize them. Additive manufacturing techniques have given tissue engineers the ability to precisely recapitulate the native architecture present within tissue. In addition, these techniques can be leveraged to create scaffolds with both physical and chemical gradients. This work offers insight into several techniques that can be used to generate graded scaffolds, depending on the desired gradient. Furthermore, it outlines methods to determine if the designed gradient was achieved. This review

  4. STRUCTURAL ASPECTS OF STRONG INHIBITION AND ROLE OF SCAFFOLD FOR SERINE PROTEASE INHIBITORS

    Directory of Open Access Journals (Sweden)

    Jhimli Dasgupta

    2011-12-01

    Full Text Available Canonical serine protease inhibitors inhibit their cognate enzymes by binding tightly at the enzyme active site in a substrate-like manner, being cleaved extremely slowly compared to a true substrate. They interact with cognate enzymes through P3-P2 region of the inhibitory loop while the scaffold hardly makes any contact. Neighbouring scaffolding residues like arginine or asparagine shape-up the inhibitory loop and religate the cleaved scissile bond. The specificity of the inhibitor can be altered by mutating the hyper solvent accessible P1 residue without changing loop-scaffold interactions. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECIL-WCIS , ETIL-WCIS , and STIL-WCIS , where the inhibitory loops of ECI, ETI, and STI were placed on the scaffold of their homologue WCI. Results showed that although ECIL-WCIS and STIL-WCIS behave like inhibitors, ETIL-WCIS behaves like a substrate. Crystal structure of ETIL-WCIS and its comparison with ETI indicated that three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETIL-WCIS leading to a loss of canonical conformation, explaining its substrate-like behaviour. Furthermore, complex structures of the inhibitors with their cognate enzymes indicate that rigidification of the inhibitory loop at the enzyme active site is necessary for efficient inhibition.

  5. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  6. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  7. Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-07-17

    Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

  8. DNA-scaffolded nanoparticle structures

    Energy Technology Data Exchange (ETDEWEB)

    Hoegberg, Bjoern; Olin, Haakan [Department of Engineering Physics and Mathematics, Mid Sweden University, SE-851 70 Sundsvall, Sweden (Sweden)

    2007-03-15

    DNA self-assembly is a powerful route to the production of very small, complex structures. When used in combination with nanoparticles it is likely to become a key technology in the production of nanoelectronics in the future. Previously, demonstrated nanoparticle assemblies have mainly been periodic and highly symmetric arrays, unsuited as building blocks for any complex circuits. With the invention of DNA-scaffolded origami reported earlier this year (Rothemund P W K 2006 Nature 440 (7082) 297-302), a new route to complex nanostructures using DNA has been opened. Here, we give a short review of the field and present the current status of our experiments were DNA origami is used in conjunction with nanoparticles. Gold nanoparticles are functionalized with thiolated single stranded DNA. Strands that are complementary to the gold particle strands can be positioned on the self-assembled DNA-structure in arbitrary patterns. This property should allow an accurate positioning of the particles by letting them hybridize on the lattice. We report on our recent experiments on this system and discuss open problems and future applications.

  9. DNA-scaffolded nanoparticle structures

    International Nuclear Information System (INIS)

    Hoegberg, Bjoern; Olin, Haakan

    2007-01-01

    DNA self-assembly is a powerful route to the production of very small, complex structures. When used in combination with nanoparticles it is likely to become a key technology in the production of nanoelectronics in the future. Previously, demonstrated nanoparticle assemblies have mainly been periodic and highly symmetric arrays, unsuited as building blocks for any complex circuits. With the invention of DNA-scaffolded origami reported earlier this year (Rothemund P W K 2006 Nature 440 (7082) 297-302), a new route to complex nanostructures using DNA has been opened. Here, we give a short review of the field and present the current status of our experiments were DNA origami is used in conjunction with nanoparticles. Gold nanoparticles are functionalized with thiolated single stranded DNA. Strands that are complementary to the gold particle strands can be positioned on the self-assembled DNA-structure in arbitrary patterns. This property should allow an accurate positioning of the particles by letting them hybridize on the lattice. We report on our recent experiments on this system and discuss open problems and future applications

  10. Semiotic scaffolding

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2015-01-01

    Life processes at all levels (from the genetic to the behavioral) are coordinated by semiotic interactions between cells, tissues, membranes, organs, or individuals and tuned through evolution to stabilize important functions. A stabilizing dynamics based on a system of semiotic scaffoldings impl...... semiotic scaffolding is not, of course, exclusive for phylogenetic and ontogenetic development, it is also an important dynamical element in cultural evolution.......Life processes at all levels (from the genetic to the behavioral) are coordinated by semiotic interactions between cells, tissues, membranes, organs, or individuals and tuned through evolution to stabilize important functions. A stabilizing dynamics based on a system of semiotic scaffoldings...... (the representamen) and the effect. Semiotic interaction patterns therefore provide fast and versatile mechanisms for adaptations, mechanisms that depend on communication and “learning” rather than on genetic preformation. Seen as a stabilizing agency supporting the emergence of higher-order structure...

  11. Functionalization of 3D scaffolds with protein-releasing biomaterials for intracellular delivery.

    Science.gov (United States)

    Seras-Franzoso, Joaquin; Steurer, Christoph; Roldán, Mònica; Vendrell, Meritxell; Vidaurre-Agut, Carla; Tarruella, Anna; Saldaña, Laura; Vilaboa, Nuria; Parera, Marc; Elizondo, Elisa; Ratera, Imma; Ventosa, Nora; Veciana, Jaume; Campillo-Fernández, Alberto J; García-Fruitós, Elena; Vázquez, Esther; Villaverde, Antonio

    2013-10-10

    Appropriate combinations of mechanical and biological stimuli are required to promote proper colonization of substrate materials in regenerative medicine. In this context, 3D scaffolds formed by compatible and biodegradable materials are under continuous development in an attempt to mimic the extracellular environment of mammalian cells. We have here explored how novel 3D porous scaffolds constructed by polylactic acid, polycaprolactone or chitosan can be decorated with bacterial inclusion bodies, submicron protein particles formed by releasable functional proteins. A simple dipping-based decoration method tested here specifically favors the penetration of the functional particles deeper than 300μm from the materials' surface. The functionalized surfaces support the intracellular delivery of biologically active proteins to up to more than 80% of the colonizing cells, a process that is slightly influenced by the chemical nature of the scaffold. The combination of 3D soft scaffolds and protein-based sustained release systems (Bioscaffolds) offers promise in the fabrication of bio-inspired hybrid matrices for multifactorial control of cell proliferation in tissue engineering under complex architectonic setting-ups. © 2013.

  12. Highly Concentrated Alginate-Gellan Gum Composites for 3D Plotting of Complex Tissue Engineering Scaffolds

    Directory of Open Access Journals (Sweden)

    Ashwini Rahul Akkineni

    2016-04-01

    Full Text Available In tissue engineering, additive manufacturing (AM technologies have brought considerable progress as they allow the fabrication of three-dimensional (3D structures with defined architecture. 3D plotting is a versatile, extrusion-based AM technology suitable for processing a wide range of biomaterials including hydrogels. In this study, composites of highly concentrated alginate and gellan gum were prepared in order to combine the excellent printing properties of alginate with the favorable gelling characteristics of gellan gum. Mixtures of 16.7 wt % alginate and 2 or 3 wt % gellan gum were found applicable for 3D plotting. Characterization of the resulting composite scaffolds revealed an increased stiffness in the wet state (15%–20% higher Young’s modulus and significantly lower volume swelling in cell culture medium compared to pure alginate scaffolds (~10% vs. ~23%. Cytocompatibility experiments with human mesenchymal stem cells (hMSC revealed that cell attachment was improved—the seeding efficiency was ~2.5–3.5 times higher on the composites than on pure alginate. Additionally, the composites were shown to support hMSC proliferation and early osteogenic differentiation. In conclusion, print fidelity of highly concentrated alginate-gellan gum composites was comparable to those of pure alginate; after plotting and crosslinking, the scaffolds possessed improved qualities regarding shape fidelity, mechanical strength, and initial cell attachment making them attractive for tissue engineering applications.

  13. Biliverdin reductase: more than a namesake - the reductase, its Peptide fragments, and biliverdin regulate activity of the three classes of protein kinase C.

    Science.gov (United States)

    Gibbs, Peter E M; Tudor, Cicerone; Maines, Mahin D

    2012-01-01

    The expanse of human biliverdin reductase (hBVR) functions in the cells is arguably unmatched by any single protein. hBVR is a Ser/Thr/Tyr-kinase, a scaffold protein, a transcription factor, and an intracellular transporter of gene regulators. hBVR is an upstream activator of the insulin/IGF-1 signaling pathway and of protein kinase C (PKC) kinases in the two major arms of the pathway. In addition, it is the sole means for generating the antioxidant bilirubin-IXα. hBVR is essential for activation of ERK1/2 kinases by upstream MAPKK-MEK and by PKCδ, as well as the nuclear import and export of ERK1/2. Small fragments of hBVR are potent activators and inhibitors of the ERK kinases and PKCs: as such, they suggest the potential application of BVR-based technology in therapeutic settings. Presently, we have reviewed the function of hBVR in cell signaling with an emphasis on regulation of PKCδ activity.

  14. SHOP: scaffold hopping by GRID-based similarity searches

    DEFF Research Database (Denmark)

    Bergmann, Rikke; Linusson, Anna; Zamora, Ismael

    2007-01-01

    A new GRID-based method for scaffold hopping (SHOP) is presented. In a fully automatic manner, scaffolds were identified in a database based on three types of 3D-descriptors. SHOP's ability to recover scaffolds was assessed and validated by searching a database spiked with fragments of known...... scaffolds were in the 31 top-ranked scaffolds. SHOP also identified new scaffolds with substantially different chemotypes from the queries. Docking analysis indicated that the new scaffolds would have similar binding modes to those of the respective query scaffolds observed in X-ray structures...

  15. Structural determinants of hydration, mechanics and fluid flow in freeze-dried collagen scaffolds.

    Science.gov (United States)

    Offeddu, G S; Ashworth, J C; Cameron, R E; Oyen, M L

    2016-09-01

    Freeze-dried scaffolds provide regeneration templates for a wide range of tissues, due to their flexibility in physical and biological properties. Control of structure is crucial for tuning such properties, and therefore scaffold functionality. However, the common approach of modeling these scaffolds as open-cell foams does not fully account for their structural complexity. Here, the validity of the open-cell model is examined across a range of physical characteristics, rigorously linking morphology to hydration and mechanical properties. Collagen scaffolds with systematic changes in relative density were characterized using Scanning Electron Microscopy, X-ray Micro-Computed Tomography and spherical indentation analyzed in a time-dependent poroelastic framework. Morphologically, all scaffolds were mid-way between the open- and closed-cell models, approaching the closed-cell model as relative density increased. Although pore size remained constant, transport pathway diameter decreased. Larger collagen fractions also produced greater volume swelling on hydration, although the change in pore diameter was constant, and relatively small at ∼6%. Mechanically, the dry and hydrated scaffold moduli varied quadratically with relative density, as expected of open-cell materials. However, the increasing pore wall closure was found to determine the time-dependent nature of the hydrated scaffold response, with a decrease in permeability producing increasingly elastic rather than viscoelastic behavior. These results demonstrate that characterizing the deviation from the open-cell model is vital to gain a full understanding of scaffold biophysical properties, and provide a template for structural studies of other freeze-dried biomaterials. Freeze-dried collagen sponges are three-dimensional microporous scaffolds that have been used for a number of exploratory tissue engineering applications. The characterization of the structure-properties relationships of these scaffolds is

  16. Alginate based scaffolds for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Valente, J.F.A.; Valente, T.A.M. [CICS-UBI - Centro de Investigacao em Ciencias da Saude, Faculdade de Ciencias da Saude, Universidade da Beira Interior, Covilha (Portugal); Alves, P.; Ferreira, P. [CIEPQPF, Departamento de Engenharia Quimica, Universidade de Coimbra, Polo II, Pinhal de Marrocos, 3030-290 Coimbra (Portugal); Silva, A. [Centro de Ciencia e Tecnologia Aeroespaciais, Universidade da Beira Interior, Covilha (Portugal); Correia, I.J., E-mail: icorreia@ubi.pt [CICS-UBI - Centro de Investigacao em Ciencias da Saude, Faculdade de Ciencias da Saude, Universidade da Beira Interior, Covilha (Portugal)

    2012-12-01

    The design and production of scaffolds for bone tissue regeneration is yet unable to completely reproduce the native bone properties. In the present study new alginate microparticle and microfiber aggregated scaffolds were produced to be applied in this area of regenerative medicine. The scaffolds' mechanical properties were characterized by thermo mechanical assays. Their morphological characteristics were evaluated by isothermal nitrogen adsorption and scanning electron microscopy. The density of both types of scaffolds was determined by helium pycnometry and mercury intrusion porosimetry. Furthermore, scaffolds' cytotoxic profiles were evaluated in vitro by seeding human osteoblast cells in their presence. The results obtained showed that scaffolds have good mechanical and morphological properties compatible with their application as bone substitutes. Moreover, scaffold's biocompatibility was confirmed by the observation of cell adhesion and proliferation after 5 days of being seeded in their presence and by non-radioactive assays. - Highlights: Black-Right-Pointing-Pointer Design and production of scaffolds for bone tissue regeneration. Black-Right-Pointing-Pointer Microparticle and microfiber alginate scaffolds were produced through a particle aggregation technique; Black-Right-Pointing-Pointer Scaffolds' mechanically and biologically properties were characterized through in vitro studies;.

  17. CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

    Science.gov (United States)

    Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

    2000-09-12

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

  18. CIKS, a connection to IκB kinase and stress-activated protein kinase

    Science.gov (United States)

    Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

    2000-01-01

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

  19. Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Kolodkin-Gal, I; Elsholz, AKW; Muth, C; Girguis, PR; Kolter, R; Losick, R

    2013-04-29

    Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.

  20. Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

    Science.gov (United States)

    Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

    2013-01-01

    Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

  1. Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines Inhibit Mammalian and Protozoan DYRK & CLK Kinases

    Directory of Open Access Journals (Sweden)

    Nadège Loaëc

    2017-10-01

    Full Text Available A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton, physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases and CLKs (cdc2-like kinases and potential pharmacological leads for the treatment of several diseases, including Alzheimer’s disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.

  2. Drug-likeness analysis of traditional Chinese medicines: 2. Characterization of scaffold architectures for drug-like compounds, non-drug-like compounds, and natural compounds from traditional Chinese medicines.

    Science.gov (United States)

    Tian, Sheng; Li, Youyong; Wang, Junmei; Xu, Xiaojie; Xu, Lei; Wang, Xiaohong; Chen, Lei; Hou, Tingjun

    2013-01-21

    In order to better understand the structural features of natural compounds from traditional Chinese medicines, the scaffold architectures of drug-like compounds in MACCS-II Drug Data Report (MDDR), non-drug-like compounds in Available Chemical Directory (ACD), and natural compounds in Traditional Chinese Medicine Compound Database (TCMCD) were explored and compared. First, the different scaffolds were extracted from ACD, MDDR and TCMCD by using three scaffold representations, including Murcko frameworks, Scaffold Tree, and ring systems with different complexity and side chains. Then, by examining the accumulative frequency of the scaffolds in each dataset, we observed that the Level 1 scaffolds of the Scaffold Tree offer advantages over the other scaffold architectures to represent the scaffold diversity of the compound libraries. By comparing the similarity of the scaffold architectures presented in MDDR, ACD and TCMCD, structural overlaps were observed not only between MDDR and TCMCD but also between MDDR and ACD. Finally, Tree Maps were used to cluster the Level 1 scaffolds of the Scaffold Tree and visualize the scaffold space of the three datasets. The analysis of the scaffold architectures of MDDR, ACD and TCMCD shows that, on average, drug-like molecules in MDDR have the highest diversity while natural compounds in TCMCD have the highest complexity. According to the Tree Maps, it can be observed that the Level 1 scaffolds present in MDDR have higher diversity than those presented in TCMCD and ACD. However, some representative scaffolds in MDDR with high frequency show structural similarities to those in TCMCD and ACD, suggesting that some scaffolds in TCMCD and ACD may be potentially drug-like fragments for fragment-based and de novo drug design.

  3. Fabrication of individual alginate-TCP scaffolds for bone tissue engineering by means of powder printing

    International Nuclear Information System (INIS)

    Castilho, Miguel; Rodrigues, Jorge; Pires, Inês; Gouveia, Barbara; Pereira, Manuel; Moseke, Claus; Groll, Jürgen; Ewald, Andrea; Vorndran, Elke

    2015-01-01

    The development of polymer-calcium phosphate composite scaffolds with tailored architectures and properties has great potential for bone regeneration. Herein, we aimed to improve the functional performance of brittle ceramic scaffolds by developing a promising biopolymer–ceramic network. For this purpose, two strategies, namely, direct printing of a powder composition consisting of a 60:40 mixture of α/β-tricalcium phosphate (TCP) powder and alginate powder or vacuum infiltration of printed TCP scaffolds with an alginate solution, were tracked. Results of structural characterization revealed that the scaffolds printed with 2.5 wt% alginate-modified TCP powders presented a uniformly distributed and interfusing alginate TCP network. Mechanical results indicated a significant increase in strength, energy to failure and reliability of powder-modified scaffolds with an alginate content in the educts of 2.5 wt% when compared to pure TCP, as well as to TCP scaffolds containing 5 wt% or 7.5 wt% in the educts, in both dry and wet states. Culture of human osteoblast cells on these scaffolds also demonstrated a great improvement of cell proliferation and cell viability. While in the case of powder-mixed alginate TCP scaffolds, isolated alginate gels were formed between the calcium phosphate crystals, the vacuum-infiltration strategy resulted in the covering of the surface and internal pores of the TCP scaffold with a thin alginate film. Furthermore, the prediction of the scaffolds’ critical fracture conditions under more complex stress states by the applied Mohr fracture criterion confirmed the potential of the powder-modified scaffolds with 2.5 wt% alginate in the educts as structural biomaterial for bone tissue engineering. (paper)

  4. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    Science.gov (United States)

    Liu, Fang

    -coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

  5. Modifying bone scaffold architecture in vivo with permanent magnets to facilitate fixation of magnetic scaffolds.

    Science.gov (United States)

    Panseri, S; Russo, A; Sartori, M; Giavaresi, G; Sandri, M; Fini, M; Maltarello, M C; Shelyakova, T; Ortolani, A; Visani, A; Dediu, V; Tampieri, A; Marcacci, M

    2013-10-01

    The fundamental elements of tissue regeneration are cells, biochemical signals and the three-dimensional microenvironment. In the described approach, biomineralized-collagen biomaterial functions as a scaffold and provides biochemical stimuli for tissue regeneration. In addition superparamagnetic nanoparticles were used to magnetize the biomaterials with direct nucleation on collagen fibres or impregnation techniques. Minimally invasive surgery was performed on 12 rabbits to implant cylindrical NdFeB magnets in close proximity to magnetic scaffolds within the lateral condyles of the distal femoral epiphyses. Under this static magnetic field we demonstrated, for the first time in vivo, that the ability to modify the scaffold architecture could influence tissue regeneration obtaining a well-ordered tissue. Moreover, the association between NdFeB magnet and magnetic scaffolds represents a potential technique to ensure scaffold fixation avoiding micromotion at the tissue/biomaterial interface. © 2013.

  6. Protein kinase A governs oxidative phosphorylation kinetics and oxidant emitting potential at complex I

    Directory of Open Access Journals (Sweden)

    Daniel Stephen Lark

    2015-11-01

    Full Text Available The mitochondrial electron transport system (ETS is responsible for setting and maintaining both the energy and redox charges throughout the cell. Reversible phosphorylation of mitochondrial proteins, particularly via the soluble adenylyl cyclase (sAC/cyclic AMP (cAMP/Protein kinase A (PKA axis, has recently been revealed as a potential mechanism regulating the ETS. However, the governance of cAMP/PKA signaling and its implications on ETS function are incompletely understood. In contrast to prior reports using exogenous bicarbonate, we provide evidence that endogenous CO2 produced by increased tricarboxylic acid (TCA cycle flux is insufficient to increase mitochondrial cAMP levels, and that exogenous addition of membrane permeant 8Br-cAMP does not enhance mitochondrial respiratory capacity. We also report important non-specific effects of commonly used inhibitors of sAC which preclude their use in studies of mitochondrial function. In isolated liver mitochondria, inhibition of PKA reduces complex I-, but not complex II-supported respiratory capacity. In permeabilized myofibers, inhibition of PKA lowers both the Km and Vmax for complex I-supported respiration as well as succinate-supported H2O2 emitting potential. In summary, the data provided here improve our understanding of how mitochondrial cAMP production is regulated, illustrate a need for better tools to examine the impact of sAC activity on mitochondrial biology, and suggest that cAMP/PKA signaling contributes to the governance of electron flow through complex I of the ETS.

  7. The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor.

    Science.gov (United States)

    Mehta, Anil

    2007-11-01

    This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as 'membrane transmitter to the cell' is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR.

  8. Biliverdin Reductase: More than a Namesake – The Reductase, Its Peptide Fragments, and Biliverdin Regulate Activity of the Three Classes of Protein Kinase C

    Science.gov (United States)

    Gibbs, Peter E. M.; Tudor, Cicerone; Maines, Mahin. D.

    2012-01-01

    The expanse of human biliverdin reductase (hBVR) functions in the cells is arguably unmatched by any single protein. hBVR is a Ser/Thr/Tyr-kinase, a scaffold protein, a transcription factor, and an intracellular transporter of gene regulators. hBVR is an upstream activator of the insulin/IGF-1 signaling pathway and of protein kinase C (PKC) kinases in the two major arms of the pathway. In addition, it is the sole means for generating the antioxidant bilirubin-IXα. hBVR is essential for activation of ERK1/2 kinases by upstream MAPKK-MEK and by PKCδ, as well as the nuclear import and export of ERK1/2. Small fragments of hBVR are potent activators and inhibitors of the ERK kinases and PKCs: as such, they suggest the potential application of BVR-based technology in therapeutic settings. Presently, we have reviewed the function of hBVR in cell signaling with an emphasis on regulation of PKCδ activity. PMID:22419908

  9. Benzimidazoles: an ideal privileged drug scaffold for the design of multitargeted anti-inflammatory ligands.

    Science.gov (United States)

    Kaur, Gaganpreet; Kaur, Maninder; Silakari, Om

    2014-01-01

    The recent research area endeavors to discover ultimate multi-target ligands, an increasingly feasible and attractive alternative to existing mono-targeted drugs for treatment of complex, multi-factorial inflammation process which underlays plethora of debilitated health conditions. In order to improvise this option, exploration of relevant chemical core scaffold will be an utmost need. Privileged benzimidazole scaffold being historically versatile structural motif could offer a viable starting point in the search for novel multi-target ligands against multi-factorial inflammation process since, when appropriately substituted, it can selectively modulate diverse receptors, pathways and enzymes associated with the pathogenesis of inflammation. Despite this remarkable capability, the multi-target capacity of the benzimidazole scaffold remains largely unexploited. With this in focus, the present review article attempts to provide synopsis of published research to exemplify the valuable use of benzimidazole nucleus and focus on their suitability as starting scaffold to develop multi-targeted anti-inflammatory ligands.

  10. Computational Exploration of Molecular Scaffolds in Medicinal Chemistry.

    Science.gov (United States)

    Hu, Ye; Stumpfe, Dagmar; Bajorath, Jürgen

    2016-05-12

    The scaffold concept is widely applied in medicinal chemistry. Scaffolds are mostly used to represent core structures of bioactive compounds. Although the scaffold concept has limitations and is often viewed differently from a chemical and computational perspective, it has provided a basis for systematic investigations of molecular cores and building blocks, going far beyond the consideration of individual compound series. Over the past 2 decades, alternative scaffold definitions and organization schemes have been introduced and scaffolds have been studied in a variety of ways and increasingly on a large scale. Major applications of the scaffold concept include the generation of molecular hierarchies, structural classification, association of scaffolds with biological activities, and activity prediction. This contribution discusses computational approaches for scaffold generation and analysis, with emphasis on recent developments impacting medicinal chemistry. A variety of scaffold-based studies are discussed, and a perspective on scaffold methods is provided.

  11. Silk as a biocohesive sacrificial binder in the fabrication of hydroxyapatite load bearing scaffolds.

    Science.gov (United States)

    McNamara, Stephanie L; Rnjak-Kovacina, Jelena; Schmidt, Daniel F; Lo, Tim J; Kaplan, David L

    2014-08-01

    Limitations of current clinical methods for bone repair continue to fuel the demand for a high strength, bioactive bone replacement material. Recent attempts to produce porous scaffolds for bone regeneration have been limited by the intrinsic weakness associated with high porosity materials. In this study, ceramic scaffold fabrication techniques for potential use in load-bearing bone repairs have been developed using naturally derived silk from Bombyx mori. Silk was first employed for ceramic grain consolidation during green body formation, and later as a sacrificial polymer to impart porosity during sintering. These techniques allowed preparation of hydroxyapatite (HA) scaffolds that exhibited a wide range of mechanical and porosity profiles, with some displaying unusually high compressive strength up to 152.4 ± 9.1 MPa. Results showed that the scaffolds exhibited a wide range of compressive strengths and moduli (8.7 ± 2.7 MPa to 152.4 ± 9.1 MPa and 0.3 ± 0.1 GPa to 8.6 ± 0.3 GPa) with total porosities of up to 62.9 ± 2.7% depending on the parameters used for fabrication. Moreover, HA-silk scaffolds could be molded into large, complex shapes, and further machined post-sinter to generate specific three-dimensional geometries. Scaffolds supported bone marrow-derived mesenchymal stem cell attachment and proliferation, with no signs of cytotoxicity. Therefore, silk-fabricated HA scaffolds show promise for load bearing bone repair and regeneration needs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. A Tryptoline Ring-Distortion Strategy Leads to Complex and Diverse Biologically Active Molecules from the Indole Alkaloid Yohimbine.

    Science.gov (United States)

    Paciaroni, Nicholas G; Ratnayake, Ranjala; Matthews, James H; Norwood, Verrill M; Arnold, Austin C; Dang, Long H; Luesch, Hendrik; Huigens, Robert W

    2017-03-28

    High-throughput screening (HTS) is the primary driver to current drug-discovery efforts. New therapeutic agents that enter the market are a direct reflection of the structurally simple compounds that make up screening libraries. Unlike medically relevant natural products (e.g., morphine), small molecules currently being screened have a low fraction of sp 3 character and few, if any, stereogenic centers. Although simple compounds have been useful in drugging certain biological targets (e.g., protein kinases), more sophisticated targets (e.g., transcription factors) have largely evaded the discovery of new clinical agents from screening collections. Herein, a tryptoline ring-distortion strategy is described that enables the rapid synthesis of 70 complex and diverse compounds from yohimbine (1); an indole alkaloid. The compounds that were synthesized had architecturally complex and unique scaffolds, unlike 1 and other scaffolds. These compounds were subjected to phenotypic screens and reporter gene assays, leading to the identification of new compounds that possessed various biological activities, including antiproliferative activities against cancer cells with functional hypoxia-inducible factors, nitric oxide inhibition, and inhibition and activation of the antioxidant response element. This tryptoline ring-distortion strategy can begin to address diversity problems in screening libraries, while occupying biologically relevant chemical space in areas critical to human health. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Fabrication of a nanofibrous scaffold with improved bioactivity for culture of human dermal fibroblasts for skin regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Chandrasekaran, Arun Richard; Venugopal, J; Sundarrajan, S; Ramakrishna, S, E-mail: nnijrv@nus.edu.s [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore (Singapore)

    2011-02-15

    Engineering dermal substitutes with electrospun nanofibres have lately been of prime importance for skin tissue regeneration. Simple electrospinning technology served to produce nanofibrous scaffolds morphologically and structurally similar to the extracellular matrix of native tissues. The nanofibrous scaffolds of poly(l-lactic acid)-co-poly({epsilon}-caprolactone) (PLACL) and PLACL/gelatin complexes were fabricated by the electrospinning process. These nanofibres were characterized for fibre morphology, membrane porosity, wettability and chemical properties by FTIR analysis to culture human foreskin fibroblasts for skin tissue engineering. The nanofibre diameter was obtained between 282 and 761 nm for PLACL and PLACL/gelatin scaffolds; expressions of amino and carboxyl groups and porosity up to 87% were obtained for these fibres, while they also exhibited improved hydrophilic properties after plasma treatment. The results showed that fibroblasts proliferation, morphology, CMFDA dye expression and secretion of collagen were significantly increased in plasma-treated PLACL/gelatin scaffolds compared to PLACL nanofibrous scaffolds. The obtained results prove that the plasma-treated PLACL/gelatin nanofibrous scaffold is a potential biocomposite material for skin tissue regeneration.

  14. Fabrication of a nanofibrous scaffold with improved bioactivity for culture of human dermal fibroblasts for skin regeneration

    International Nuclear Information System (INIS)

    Chandrasekaran, Arun Richard; Venugopal, J; Sundarrajan, S; Ramakrishna, S

    2011-01-01

    Engineering dermal substitutes with electrospun nanofibres have lately been of prime importance for skin tissue regeneration. Simple electrospinning technology served to produce nanofibrous scaffolds morphologically and structurally similar to the extracellular matrix of native tissues. The nanofibrous scaffolds of poly(l-lactic acid)-co-poly(ε-caprolactone) (PLACL) and PLACL/gelatin complexes were fabricated by the electrospinning process. These nanofibres were characterized for fibre morphology, membrane porosity, wettability and chemical properties by FTIR analysis to culture human foreskin fibroblasts for skin tissue engineering. The nanofibre diameter was obtained between 282 and 761 nm for PLACL and PLACL/gelatin scaffolds; expressions of amino and carboxyl groups and porosity up to 87% were obtained for these fibres, while they also exhibited improved hydrophilic properties after plasma treatment. The results showed that fibroblasts proliferation, morphology, CMFDA dye expression and secretion of collagen were significantly increased in plasma-treated PLACL/gelatin scaffolds compared to PLACL nanofibrous scaffolds. The obtained results prove that the plasma-treated PLACL/gelatin nanofibrous scaffold is a potential biocomposite material for skin tissue regeneration.

  15. Developing bioactive composite scaffolds for bone tissue engineering

    Science.gov (United States)

    Chen, Yun

    Poly(L-lactic acid) (PLLA) films were fabricated using the method of dissolving and evaporation. PLLA scaffold was prepared by solid-liquid phase separation of polymer solutions and subsequent sublimation of solvent. Bonelike apatite coating was formed on PLLA films, PLLA scaffolds and poly(glycolic acid) (PGA) scaffolds in 24 hours through an accelerated biomimetic process. The ion concentrations in the simulated body fluid (SBF) were nearly 5 times of those in human blood plasma. The apatite formed was characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The apatite formed in 5SBF was similar in morphology and composition to that formed in the classical biomimetic process employing SBF or 1.5SBF, and similar to that of natural bone. This indicated that the biomimetic apatite coating process could be accelerated by using concentrated simulated body fluid at 37°C. Besides saving time, the accelerated biomimetic process is particularly significant to biodegradable polymers. Some polymers which degrade too fast to be coated with apatite by a classical biomimetic process, for example PGA, could be coated with bone-like apatite in an accelerated biomimetic process. Collagen and apatite were co-precipitated as a composite coating on poly(L-lactic acid) (PLLA) in an accelerated biomimetic process. The incubation solution contained collagen (1g/L) and simulated body fluid (SBF) with 5 times inorganic ionic concentrations as human blood plasma. The coating formed on PLLA films and scaffolds after 24 hours incubation was characterized using EDX, XRD, FTIR, and SEM. It was shown that the coating contained carbonated bone-like apatite and collagen, the primary constituents of natural bone. SEM showed a complex composite coating of submicron bone-like apatite particulates combined with collagen fibrils. This work provided an efficient process to obtain

  16. In Vitro Degradation of PHBV Scaffolds and nHA/PHBV Composite Scaffolds Containing Hydroxyapatite Nanoparticles for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Naznin Sultana

    2012-01-01

    Full Text Available This paper investigated the long-term in vitro degradation properties of scaffolds based on biodegradable polymers and osteoconductive bioceramic/polymer composite materials for the application of bone tissue engineering. The three-dimensional porous scaffolds were fabricated using emulsion-freezing/freeze-drying technique using poly(hydroxybutyrate-co-hydroxyvalerate (PHBV which is a natural biodegradable and biocompatible polymer. Nanosized hydroxyapatite (nHA particles were successfully incorporated into the PHBV scaffolds to render the scaffolds osteoconductive. The PHBV and nHA/PHBV scaffolds were systematically evaluated using various techniques in terms of mechanical strength, porosity, porous morphology, and in vitro degradation. PHBV and nHA/PHBV scaffolds degraded over time in phosphate-buffered saline at 37°C. PHBV polymer scaffolds exhibited slow molecular weight loss and weight loss in the in vitro physiological environment. Accelerated weight loss was observed in nHA incorporated PHBV composite scaffolds. An increasing trend of crystallinity was observed during the initial period of degradation time. The compressive properties decreased more than 40% after 5-month in vitro degradation. Together with interconnected pores, high porosity, suitable mechanical properties, and slow degradation profile obtained from long-term degradation studies, the PHBV scaffolds and osteoconductive nHA/PHBV composite scaffolds showed promises for bone tissue engineering application.

  17. Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase

    Directory of Open Access Journals (Sweden)

    Tomasi Vittorio

    2010-12-01

    Full Text Available Abstract Background It has been reported that cellular prion protein (PrPc co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI anchor (secPrP and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. Results By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11 expressing caveolin-1 at high levels. Conclusions We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

  18. Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase.

    Science.gov (United States)

    Tomasi, Vittorio

    2010-12-16

    It has been reported that cellular prion protein (PrPc) co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI) anchor (secPrP) and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s) of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11) expressing caveolin-1 at high levels. We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

  19. Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Benjamin Mielich-Süss

    2017-11-01

    Full Text Available Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM, a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen.

  20. Mechanical modulation of nascent stem cell lineage commitment in tissue engineering scaffolds.

    Science.gov (United States)

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L

    2013-07-01

    Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to stem cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to "map the mechanome", defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. The design of 3D scaffold for tissue engineering using automated scaffold design algorithm.

    Science.gov (United States)

    Mahmoud, Shahenda; Eldeib, Ayman; Samy, Sherif

    2015-06-01

    Several progresses have been introduced in the field of bone regenerative medicine. A new term tissue engineering (TE) was created. In TE, a highly porous artificial extracellular matrix or scaffold is required to accommodate cells and guide their growth in three dimensions. The design of scaffolds with desirable internal and external structure represents a challenge for TE. In this paper, we introduce a new method known as automated scaffold design (ASD) for designing a 3D scaffold with a minimum mismatches for its geometrical parameters. The method makes use of k-means clustering algorithm to separate the different tissues and hence decodes the defected bone portions. The segmented portions of different slices are registered to construct the 3D volume for the data. It also uses an isosurface rendering technique for 3D visualization of the scaffold and bones. It provides the ability to visualize the transplanted as well as the normal bone portions. The proposed system proves good performance in both the segmentation results and visualizations aspects.

  2. A complex regulatory network controls aerobic ethanol oxidation in Pseudomonas aeruginosa: indication of four levels of sensor kinases and response regulators.

    Science.gov (United States)

    Mern, Demissew S; Ha, Seung-Wook; Khodaverdi, Viola; Gliese, Nicole; Görisch, Helmut

    2010-05-01

    In addition to the known response regulator ErbR (former AgmR) and the two-component regulatory system EraSR (former ExaDE), three additional regulatory proteins have been identified as being involved in controlling transcription of the aerobic ethanol oxidation system in Pseudomonas aeruginosa. Two putative sensor kinases, ErcS and ErcS', and a response regulator, ErdR, were found, all of which show significant similarity to the two-component flhSR system that controls methanol and formaldehyde metabolism in Paracoccus denitrificans. All three identified response regulators, EraR (formerly ExaE), ErbR (formerly AgmR) and ErdR, are members of the luxR family. The three sensor kinases EraS (formerly ExaD), ErcS and ErcS' do not contain a membrane domain. Apparently, they are localized in the cytoplasm and recognize cytoplasmic signals. Inactivation of gene ercS caused an extended lag phase on ethanol. Inactivation of both genes, ercS and ercS', resulted in no growth at all on ethanol, as did inactivation of erdR. Of the three sensor kinases and three response regulators identified thus far, only the EraSR (formerly ExaDE) system forms a corresponding kinase/regulator pair. Using reporter gene constructs of all identified regulatory genes in different mutants allowed the hierarchy of a hypothetical complex regulatory network to be established. Probably, two additional sensor kinases and two additional response regulators, which are hidden among the numerous regulatory genes annotated in the genome of P. aeruginosa, remain to be identified.

  3. Composite porous scaffold of PEG/PLA support improved bone matrix deposition in vitro compared to PLA-only scaffolds.

    Science.gov (United States)

    Bhaskar, Birru; Owen, Robert; Bahmaee, Hossein; Wally, Zena; Sreenivasa Rao, Parcha; Reilly, Gwendolen C

    2018-05-01

    Controllable pore size and architecture are essential properties for tissue-engineering scaffolds to support cell ingrowth colonization. To investigate the effect of polyethylene glycol (PEG) addition on porosity and bone-cell behavior, porous polylactic acid (PLA)-PEG scaffolds were developed with varied weight ratios of PLA-PEG (100/0, 90/10, 75/25) using solvent casting and porogen leaching. Sugar 200-300 µm in size was used as a porogen. To assess scaffold suitability for bone tissue engineering, MLO-A5 murine osteoblast cells were cultured and cell metabolic activity, alkaline phosphatase (ALP) activity and bone-matrix production determined using (alizarin red S staining for calcium and direct red 80 staining for collagen). It was found that metabolic activity was significantly higher over time on scaffolds containing PEG, ALP activity and mineralized matrix production were also significantly higher on scaffolds containing 25% PEG. Porous architecture and cell distribution and penetration into the scaffold were analyzed using SEM and confocal microscopy, revealing that inclusion of PEG increased pore interconnectivity and therefore cell ingrowth in comparison to pure PLA scaffolds. The results of this study confirmed that PLA-PEG porous scaffolds support mineralizing osteoblasts better than pure PLA scaffolds, indicating they have a high potential for use in bone tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1334-1340, 2018. © 2018 Wiley Periodicals, Inc.

  4. Finite-element design and optimization of a three-dimensional tetrahedral porous titanium scaffold for the reconstruction of mandibular defects.

    Science.gov (United States)

    Luo, Danmei; Rong, Qiguo; Chen, Quan

    2017-09-01

    Reconstruction of segmental defects in the mandible remains a challenge for maxillofacial surgery. The use of porous scaffolds is a potential method for repairing these defects. Now, additive manufacturing techniques provide a solution for the fabrication of porous scaffolds with specific geometrical shapes and complex structures. The goal of this study was to design and optimize a three-dimensional tetrahedral titanium scaffold for the reconstruction of mandibular defects. With a fixed strut diameter of 0.45mm and a mean cell size of 2.2mm, a tetrahedral structural porous scaffold was designed for a simulated anatomical defect derived from computed tomography (CT) data of a human mandible. An optimization method based on the concept of uniform stress was performed on the initial scaffold to realize a minimal-weight design. Geometric and mechanical comparisons between the initial and optimized scaffold show that the optimized scaffold exhibits a larger porosity, 81.90%, as well as a more homogeneous stress distribution. These results demonstrate that tetrahedral structural titanium scaffolds are feasible structures for repairing mandibular defects, and that the proposed optimization scheme has the ability to produce superior scaffolds for mandibular reconstruction with better stability, higher porosity, and less weight. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

  5. Neuronal Networks on Nanocellulose Scaffolds.

    Science.gov (United States)

    Jonsson, Malin; Brackmann, Christian; Puchades, Maja; Brattås, Karoline; Ewing, Andrew; Gatenholm, Paul; Enejder, Annika

    2015-11-01

    Proliferation, integration, and neurite extension of PC12 cells, a widely used culture model for cholinergic neurons, were studied in nanocellulose scaffolds biosynthesized by Gluconacetobacter xylinus to allow a three-dimensional (3D) extension of neurites better mimicking neuronal networks in tissue. The interaction with control scaffolds was compared with cationized nanocellulose (trimethyl ammonium betahydroxy propyl [TMAHP] cellulose) to investigate the impact of surface charges on the cell interaction mechanisms. Furthermore, coatings with extracellular matrix proteins (collagen, fibronectin, and laminin) were investigated to determine the importance of integrin-mediated cell attachment. Cell proliferation was evaluated by a cellular proliferation assay, while cell integration and neurite propagation were studied by simultaneous label-free Coherent anti-Stokes Raman Scattering and second harmonic generation microscopy, providing 3D images of PC12 cells and arrangement of nanocellulose fibrils, respectively. Cell attachment and proliferation were enhanced by TMAHP modification, but not by protein coating. Protein coating instead promoted active interaction between the cells and the scaffold, hence lateral cell migration and integration. Irrespective of surface modification, deepest cell integration measured was one to two cell layers, whereas neurites have a capacity to integrate deeper than the cell bodies in the scaffold due to their fine dimensions and amoeba-like migration pattern. Neurites with lengths of >50 μm were observed, successfully connecting individual cells and cell clusters. In conclusion, TMAHP-modified nanocellulose scaffolds promote initial cellular scaffold adhesion, which combined with additional cell-scaffold treatments enables further formation of 3D neuronal networks.

  6. The Drosophila IKK-related kinase (Ik2 and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis

    Directory of Open Access Journals (Sweden)

    Shaanan Boaz

    2008-09-01

    Full Text Available Abstract Background IkappaB kinases (IKKs regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2. Results We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization. Conclusion We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

  7. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  8. Multilayer scaffolds in orthopaedic tissue engineering.

    Science.gov (United States)

    Atesok, Kivanc; Doral, M Nedim; Karlsson, Jon; Egol, Kenneth A; Jazrawi, Laith M; Coelho, Paulo G; Martinez, Amaury; Matsumoto, Tomoyuki; Owens, Brett D; Ochi, Mitsuo; Hurwitz, Shepard R; Atala, Anthony; Fu, Freddie H; Lu, Helen H; Rodeo, Scott A

    2016-07-01

    The purpose of this study was to summarize the recent developments in the field of tissue engineering as they relate to multilayer scaffold designs in musculoskeletal regeneration. Clinical and basic research studies that highlight the current knowledge and potential future applications of the multilayer scaffolds in orthopaedic tissue engineering were evaluated and the best evidence collected. Studies were divided into three main categories based on tissue types and interfaces for which multilayer scaffolds were used to regenerate: bone, osteochondral junction and tendon-to-bone interfaces. In vitro and in vivo studies indicate that the use of stratified scaffolds composed of multiple layers with distinct compositions for regeneration of distinct tissue types within the same scaffold and anatomic location is feasible. This emerging tissue engineering approach has potential applications in regeneration of bone defects, osteochondral lesions and tendon-to-bone interfaces with successful basic research findings that encourage clinical applications. Present data supporting the advantages of the use of multilayer scaffolds as an emerging strategy in musculoskeletal tissue engineering are promising, however, still limited. Positive impacts of the use of next generation scaffolds in orthopaedic tissue engineering can be expected in terms of decreasing the invasiveness of current grafting techniques used for reconstruction of bone and osteochondral defects, and tendon-to-bone interfaces in near future.

  9. Titanate nanotube coatings on biodegradable photopolymer scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Beke, S., E-mail: szabolcs.beke@iit.it [Department of Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova (Italy); Kőrösi, L. [Department of Biotechnology, Nanophage Therapy Center, Enviroinvest Corporation, Kertváros u. 2, H-7632, Pécs (Hungary); Scarpellini, A. [Department of Nanochemistry, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova (Italy); Anjum, F.; Brandi, F. [Department of Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova (Italy)

    2013-05-01

    Rigid, biodegradable photopolymer scaffolds were coated with titanate nanotubes (TNTs) by using a spin-coating method. TNTs were synthesized by a hydrothermal process at 150 °C under 4.7 bar ambient pressure. The biodegradable photopolymer scaffolds were produced by mask-assisted excimer laser photocuring at 308 nm. For scaffold coating, a stable ethanolic TNT sol was prepared by a simple colloid chemical route without the use of any binding compounds or additives. Scanning electron microscopy along with elemental analysis revealed that the scaffolds were homogenously coated by TNTs. The developed TNT coating can further improve the surface geometry of fabricated scaffolds, and therefore it can further increase the cell adhesion. Highlights: ► Biodegradable scaffolds were produced by mask-assisted UV laser photocuring. ► Titanate nanotube deposition was carried out without binding compounds or additives. ► The titanate nanotube coating can further improve the surface geometry of scaffolds. ► These reproducible platforms will be of high importance for biological applications.

  10. Construction and characterization of an electrospun tubular scaffold for small-diameter tissue-engineered vascular grafts: a scaffold membrane approach.

    Science.gov (United States)

    Hu, Jin-Jia; Chao, Wei-Chih; Lee, Pei-Yuan; Huang, Chih-Hao

    2012-09-01

    Based on a postulate that the microstructure of a scaffold can influence that of the resulting tissue and hence its mechanical behavior, we fabricated a small-diameter tubular scaffold (∼3 mm inner diameter) that has a microstructure similar to the arterial media using a scaffold membrane approach. Scaffold membranes that contain randomly oriented, moderately aligned, or highly aligned fibers were fabricated by collecting electrospun poly([epsilon]-caprolactone) fibers on a grounded rotating drum at three different drum rotation speeds (250, 1000, and 1500 rpm). Membranes of each type were wrapped around a small-diameter mandrel to form the tubular scaffolds. Particularly, the tubular scaffolds with three different off-axis fiber angles (30, 45, and 60 degree) were formed using membranes that contain aligned fibers. These scaffolds were subjected to biaxial mechanical testing to examine the effects of fiber directions as well as the distribution of fiber orientations on their mechanical properties. The circumferential elastic modulus of the tubular scaffold was closely related to the fiber directions; the larger the off-axis fiber angle the greater the circumferential elastic modulus. The distribution of fiber orientations, on the other hand, manifested itself in the mechanical behavior via the Poisson effect. Similar to cell sheet-based vascular tissue engineering, tubular cell-seeded constructs were prepared by wrapping cell-seeded scaffold membranes, alleviating the difficulty associated with cell seeding in electrospun scaffolds. Histology of the construct illustrated that cells were aligned to the fiber directions in the construct, demonstrating the potential to control the microstructure of tissue-engineered vascular grafts using the electrospun scaffold membrane. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

    Science.gov (United States)

    Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K; Smith, Douglas Y; Söderberg, Christopher A G; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

    2016-05-06

    The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native α2β2 insulin receptor subunit complex

    International Nuclear Information System (INIS)

    Sweet, L.J.; Wilden, P.A.; Pessin, J.E.

    1986-01-01

    The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing or nondenaturing conditions. Pretreatment of 32 P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the α 2 β 2 insulin receptor complex (M/sub r/ 400,000) into the monomeric 95,000 β subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the α 2 β 2 heterotetrameric complex with essentially no αβ heterodimeric or free monomeric β subunit species present. This suggests that the insulin receptor can reoxidize into the M/sub r/ 400,000 complex after the removal of DTT by gel filtration chromatography. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT. Under the conditions the insulin receptors migrated as the M/sub r/ 400,000 α 2 β 2 complex. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced α 2 β 2 insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native α 2 β 2 insulin receptor subunit complex

  13. SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

    Science.gov (United States)

    Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

    1997-10-31

    Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

  14. Porous magnesium-based scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Yazdimamaghani, Mostafa; Razavi, Mehdi; Vashaee, Daryoosh; Moharamzadeh, Keyvan; Boccaccini, Aldo R.; Tayebi, Lobat

    2017-01-01

    Significant amount of research efforts have been dedicated to the development of scaffolds for tissue engineering. Although at present most of the studies are focused on non-load bearing scaffolds, many scaffolds have also been investigated for hard tissue repair. In particular, metallic scaffolds are being studied for hard tissue engineering due to their suitable mechanical properties. Several biocompatible metallic materials such as stainless steels, cobalt alloys, titanium alloys, tantalum, nitinol and magnesium alloys have been commonly employed as implants in orthopedic and dental treatments. They are often used to replace and regenerate the damaged bones or to provide structural support for healing bone defects. Among the common metallic biomaterials, magnesium (Mg) and a number of its alloys are effective because of their mechanical properties close to those of human bone, their natural ionic content that may have important functional roles in physiological systems, and their in vivo biodegradation characteristics in body fluids. Due to such collective properties, Mg based alloys can be employed as biocompatible, bioactive, and biodegradable scaffolds for load-bearing applications. Recently, porous Mg and Mg alloys have been specially suggested as metallic scaffolds for bone tissue engineering. With further optimization of the fabrication techniques, porous Mg is expected to make a promising hard substitute scaffold. The present review covers research conducted on the fabrication techniques, surface modifications, properties and biological characteristics of Mg alloys based scaffolds. Furthermore, the potential applications, challenges and future trends of such degradable metallic scaffolds are discussed in detail. - Highlights: • A porous 3D material provides the required pathways for cells to grow, proliferate, and differentiate • Porous magnesium and Mg alloys could be used as load-bearing scaffolds • Porous magnesium and Mg alloys are good

  15. Porous magnesium-based scaffolds for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Yazdimamaghani, Mostafa [School of Chemical Engineering, Oklahoma State University, Stillwater, OK 74078 (United States); Razavi, Mehdi [Department of Radiology, School of Medicine, Stanford University, Palo Alto, CA 94304 (United States); Vashaee, Daryoosh [Electrical and Computer Engineering Department, North Carolina State University, Raleigh, NC 27606 (United States); Moharamzadeh, Keyvan [School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield (United Kingdom); Marquette University School of Dentistry, Milwaukee, WI 53233 (United States); Boccaccini, Aldo R. [Institute of Biomaterials, University of Erlangen-Nuremberg, Cauerstrasse 6, 91058 Erlangen (Germany); Tayebi, Lobat, E-mail: lobat.tayebi@marquette.edu [Marquette University School of Dentistry, Milwaukee, WI 53233 (United States)

    2017-02-01

    Significant amount of research efforts have been dedicated to the development of scaffolds for tissue engineering. Although at present most of the studies are focused on non-load bearing scaffolds, many scaffolds have also been investigated for hard tissue repair. In particular, metallic scaffolds are being studied for hard tissue engineering due to their suitable mechanical properties. Several biocompatible metallic materials such as stainless steels, cobalt alloys, titanium alloys, tantalum, nitinol and magnesium alloys have been commonly employed as implants in orthopedic and dental treatments. They are often used to replace and regenerate the damaged bones or to provide structural support for healing bone defects. Among the common metallic biomaterials, magnesium (Mg) and a number of its alloys are effective because of their mechanical properties close to those of human bone, their natural ionic content that may have important functional roles in physiological systems, and their in vivo biodegradation characteristics in body fluids. Due to such collective properties, Mg based alloys can be employed as biocompatible, bioactive, and biodegradable scaffolds for load-bearing applications. Recently, porous Mg and Mg alloys have been specially suggested as metallic scaffolds for bone tissue engineering. With further optimization of the fabrication techniques, porous Mg is expected to make a promising hard substitute scaffold. The present review covers research conducted on the fabrication techniques, surface modifications, properties and biological characteristics of Mg alloys based scaffolds. Furthermore, the potential applications, challenges and future trends of such degradable metallic scaffolds are discussed in detail. - Highlights: • A porous 3D material provides the required pathways for cells to grow, proliferate, and differentiate • Porous magnesium and Mg alloys could be used as load-bearing scaffolds • Porous magnesium and Mg alloys are good

  16. Diastereoselective Synthesis of Novel Heterocyclic Scaffolds through Tandem Petasis 3-Component/Intramolecular Diels-Alder and ROM-RCM Reactions

    DEFF Research Database (Denmark)

    Ishøy, Mette; Petersen, Rico; Petersen, Michael Åxman

    2017-01-01

    A high-yielding, stereoselective and extraordinarily complexity generatingPetasis 3-component/intramolecular Diels-Alderreaction has been developed. In combination with ROM-RCM, rapid access to complex sp3-rich heterocyclic scaffolds amenableto subsequent functionalization and library synthesis...

  17. Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds.

    Science.gov (United States)

    Asthana, Amish; White, Charles McRae; Douglass, Megan; Kisaalita, William S

    2018-03-01

    The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-l-lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505-514, 2018. © 2018 American Institute of Chemical Engineers.

  18. The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi.

    Science.gov (United States)

    Kück, Ulrich; Beier, Anna M; Teichert, Ines

    2016-05-01

    The striatin-interacting phosphatases and kinases (STRIPAK) complex is a highly conserved eukaryotic protein complex that was recently described for diverse animal and fungal species. Here, we summarize our current knowledge about the composition and function of the STRIPAK complex from the ascomycete Sordaria macrospora, which we discovered by investigating sexually sterile mutants (pro), having a defect in fruiting body development. Mass spectrometry and yeast two-hybrid analysis defined core subunits of the STRIPAK complex, which have structural homologs in animal and other fungal organisms. These subunits (and their mammalian homologs) are PRO11 (striatin), PRO22 (STRIP1/2), SmMOB3 (Mob3), PRO45 (SLMAP), and PP2AA, the structural, and PP2Ac, the catalytic subunits of protein phosphatase 2A (PP2A). Beside fruiting body formation, the STRIPAK complex controls vegetative growth and hyphal fusion in S. macrospora. Although the contribution of single subunits to diverse cellular and developmental processes is not yet fully understood, functional analysis has already shown that mammalian homologs are able to substitute the function of distinct fungal STRIPAK subunits. This underscores the view that fungal model organisms serve as useful tools to get a molecular insight into cellular and developmental processes of eukaryotes in general. Future work will unravel the precise localization of single subunits within the cell and decipher their STRIPAK-related and STRIPAK-independent functions. Finally, evidence is accumulating that there is a crosstalk between STRIPAK and various signaling pathways, suggesting that eukaryotic development is dependent on STRIPAK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. A Bayesian Network Meta-Analysis to Synthesize the Influence of Contexts of Scaffolding Use on Cognitive Outcomes in STEM Education

    Science.gov (United States)

    Belland, Brian R.; Walker, Andrew E.; Kim, Nam Ju

    2017-01-01

    Computer-based scaffolding provides temporary support that enables students to participate in and become more proficient at complex skills like problem solving, argumentation, and evaluation. While meta-analyses have addressed between-subject differences on cognitive outcomes resulting from scaffolding, none has addressed within-subject gains. This leaves much quantitative scaffolding literature not covered by existing meta-analyses. To address this gap, this study used Bayesian network meta-analysis to synthesize within-subjects (pre–post) differences resulting from scaffolding in 56 studies. We generated the posterior distribution using 20,000 Markov Chain Monte Carlo samples. Scaffolding has a consistently strong effect across student populations, STEM (science, technology, engineering, and mathematics) disciplines, and assessment levels, and a strong effect when used with most problem-centered instructional models (exception: inquiry-based learning and modeling visualization) and educational levels (exception: secondary education). Results also indicate some promising areas for future scaffolding research, including scaffolding among students with learning disabilities, for whom the effect size was particularly large (ḡ = 3.13). PMID:29200508

  20. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  1. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

    Directory of Open Access Journals (Sweden)

    Jens Milbradt

    2018-01-01

    Full Text Available The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  2. Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

    International Nuclear Information System (INIS)

    Favreau, Catherine; Delbarre, Erwan; Courvalin, Jean-Claude; Buendia, Brigitte

    2008-01-01

    Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process

  3. Neocellularization and neovascularization of nanosized bioactive glass-coated decellularized trabecular bone scaffolds

    KAUST Repository

    Gerhardt, Lutz Christian

    2012-09-11

    In this study, the in vivo recellularization and neovascularization of nanosized bioactive glass (n-BG)-coated decellu-larized trabecular bone scaffolds were studied in a rat model and quantified using stereological analyses. Based on the highest amount of vascular endothelial growth factor (VEGF) secreted by human fibroblasts grown on n-BG coatings (0-1.245 mg/cm 2), decellularized trabecular bone samples (porosity: 43-81%) were coated with n-BG particles. Grown on n-BG particles at a coating density of 0.263 mg/cm2, human fibroblasts produced 4.3 times more VEGF than on uncoated controls. After 8 weeks of implantation in Sprague-Dawley rats, both uncoated and n-BG-coated samples were well infiltrated with newly formed tissue (47-48%) and blood vessels (3-4%). No significant differences were found in cellularization and vascularization between uncoated bone scaffolds and n-BG-coated scaffolds. This finding indicates that the decellularized bone itself may exhibit growth-promoting properties induced by the highly interconnected pore microarchitecture and/or proteins left behind on decellularized scaffolds. Even if we did not find proangiogenic effects in n-BG-coated bone scaffolds, a bioactive coating is considered to be beneficial to impart osteoinductive and osteoconductive properties to decellularized bone. n-BG-coated bone grafts have thus high clinical potential for the regeneration of complex tissue defects given their ability for recellularization and neovascularization. © 2012 Wiley Periodicals, Inc.

  4. Peer scaffolding in an EFL writing classroom: An investigation of writing accuracy and scaffolding behaviors

    Directory of Open Access Journals (Sweden)

    Parastou Gholami Pasand

    2017-09-01

    Full Text Available Considering the tenets of Sociocultural Theory with its emphasis on co-construction of knowledge, L2 writing can be regarded as a co-writing practice whereby assistance is provided to struggling writers. To date, most studies have dealt with peer scaffolding in the revision phase of writing, as such planning and drafting are remained untouched. The present study examines the impact of peer scaffolding on writing accuracy of a group of intermediate EFL learners, and explores scaffolding behaviors employed by them in planning and drafting phases of writing. To these ends, 40 freshmen majoring in English Language and Literature in the University of Guilan were randomly divided into a control group and an experimental group consisting of dyads in which a competent writer provided scaffolding to a less competent one using the process approach to writing. Results of independent samples t-tests revealed that learners in the experimental group produced more accurate essays. Microgenetic analysis of one dyad’s talks showed that scaffolding behaviors used in planning and drafting phases of writing were more or less the same as those identified in the revision phase. These findings can be used to inform peer intervention in L2 writing classes, and assist L2 learners in conducting successful peer scaffolding in the planning and drafting phases of writing.

  5. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  6. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  7. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

    Science.gov (United States)

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

    2016-08-01

    Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner

  8. 1,3,5-Triazine-based analogues of purine: from isosteres to privileged scaffolds in medicinal chemistry.

    Science.gov (United States)

    Lim, Felicia Phei Lin; Dolzhenko, Anton V

    2014-10-06

    Purines can be considered as the most ubiquitous and functional N-heterocyclic compounds in nature. Structural modifications of natural purines, particularly using isosteric ring systems, have been in the focus of many drug discovery programs. Fusion of 1,3,5-triazine ring with pyrrole, pyrazole, imidazole, 1,2,3-triazole or 1,2,4-triazole results in seven bicyclic heterocyclic systems isosteric to purine. Application of the isosterism concept for the development of new compounds with therapeutic potential in areas involving purinergic regulation or purine metabolism led to significant advances in medicinal chemistry of the azolo[1,3,5]triazines. These 1,3,5-triazine-based purine-like scaffolds significantly increase level of molecular diversity and allow covering chemical space in the important areas of medicinal chemistry. Some of these azolo[1,3,5]triazine systems have become privileged scaffolds in the development of inhibitors of various kinases, phosphodiesterase, xanthine oxidase, and thymidine phosphorylase, antagonists of adenosine and corticotropin-releasing hormone receptors, anticancer and antiviral agents. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  9. Biodegradable Polymer-Based Scaffolds for Bone Tissue Engineering

    CERN Document Server

    Sultana, Naznin

    2013-01-01

    This book addresses the principles, methods and applications of biodegradable polymer based scaffolds for bone tissue engineering. The general principle of bone tissue engineering is reviewed and the traditional and novel scaffolding materials, their properties and scaffold fabrication techniques are explored. By acting as temporary synthetic extracellular matrices for cell accommodation, proliferation, and differentiation, scaffolds play a pivotal role in tissue engineering. This book does not only provide the comprehensive summary of the current trends in scaffolding design but also presents the new trends and directions for scaffold development for the ever expanding tissue engineering applications.

  10. The long and the short of SAD-1 kinase.

    Science.gov (United States)

    Kim, Joanne S M; Hung, Wesley; Zhen, Mei

    2010-05-01

    The Ser/Thr SAD kinases are evolutionarily conserved, critical regulators of neural development. Exciting findings in recent years have significantly advanced our understanding of the mechanism through which SAD kinases regulate neural development. Mammalian SAD-A and SAD-B, activated by a master kinase LKB1, regulate microtubule dynamics and polarize neurons. In C. elegans, the sad-1 gene encodes two isoforms, namely the long and the short, which exhibit overlapping and yet distinct functions in neuronal polarity and synaptic organization. Surprisingly, our most recent findings in C. elegans revealed a SAD-1-independent LKB1 activity in neuronal polarity. We also found that the long SAD-1 isoform directly interacts with a STRADalpha pseudokinase, STRD-1, to regulate neuronal polarity and synaptic organization. We elaborate here a working model of SAD-1 in which the two isoforms dimer/oligomerize to form a functional complex, and STRD-1 clusters and localizes the SAD-1 complex to synapses. While the mechanistic difference between the vertebrate and invertebrate SAD kinases may be puzzling, a recent discovery of the functionally distinct SAD-B isoforms predicts that the difference likely arises from our incomplete understanding of the SAD kinase mechanism and may eventually be reconciled as the revelation continues.

  11. Platelet lysate embedded scaffolds for skin regeneration.

    Science.gov (United States)

    Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Cervio, Marila; Riva, Federica; Liakos, Ioannis; Athanassiou, Athanassia; Saporito, Francesca; Marini, Lara; Caramella, Carla

    2015-04-01

    The work presents the development of acellular scaffolds extemporaneously embedded with platelet lysate (PL), as an innovative approach in the field of tissue regeneration/reparation. PL embedded scaffolds should have a tridimensional architecture to support cell migration and growth, in order to restore skin integrity. For this reason, chondroitin sulfate (CS) was associated with sodium alginate (SA) to prepare highly porous systems. The developed scaffolds were characterized for chemical stability to γ-radiation, morphology, hydration and mechanical properties. Moreover, the capability of fibroblasts and endothelial cells to populate the scaffold was evaluated by means of proliferation test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and confocal laser scanning microscopy study. The scaffolds, not altered by sterilization, were characterized by limited swelling and high flexibility, by foam-like structure with bubbles that formed a high surface area and irregular texture suitable for cell adhesion. Cell growth and scaffold population were evident on the bubble surface, where the cells appeared anchored to the scaffold structure. Scaffold network based on CS and SA demonstrated to be an effective support to enhance and to allow fibroblasts and endothelial cells (human umbilical vein endothelial cells, HUVEC) adhesion and proliferation. In particular, it could be hypothesized that cell adhesion was facilitated by the synergic effect of PL and CS. Although further in vivo evaluation is needed, on the basis of in vitro results, PL embedded scaffolds seem promising systems for skin wound healing.

  12. Semiotic Scaffolding in Mathematics

    DEFF Research Database (Denmark)

    Johansen, Mikkel Willum; Misfeldt, Morten

    2015-01-01

    This paper investigates the notion of semiotic scaffolding in relation to mathematics by considering its influence on mathematical activities, and on the evolution of mathematics as a research field. We will do this by analyzing the role different representational forms play in mathematical...... cognition, and more broadly on mathematical activities. In the main part of the paper, we will present and analyze three different cases. For the first case, we investigate the semiotic scaffolding involved in pencil and paper multiplication. For the second case, we investigate how the development of new...... in both mathematical cognition and in the development of mathematics itself, but mathematical cognition cannot itself be reduced to the use of semiotic scaffolding....

  13. The Scaffolding Protein Synapse-Associated Protein 97 is Required for Enhanced Signaling Through Isotype-Switched IgG Memory B Cell Receptors

    Science.gov (United States)

    Liu, Wanli; Chen, Elizabeth; Zhao, Xing Wang; Wan, Zheng Peng; Gao, Yi Ren; Davey, Angel; Huang, Eric; Zhang, Lijia; Crocetti, Jillian; Sandoval, Gabriel; Joyce, M. Gordon; Miceli, Carrie; Lukszo, Jan; Aravind, L.; Swat, Wojciech; Brzostowski, Joseph; Pierce, Susan K.

    2012-01-01

    Memory B cells are generated during an individual's first encounter with a foreign antigen and respond to re-encounter with the same antigen through cell surface immunoglobulin G (IgG) B cell receptors (BCRs) resulting in rapid, high-titered IgG antibody responses. Despite a central role for IgG BCRs in B cell memory, our understanding of the molecular mechanism by which IgG BCRs enhance antibody responses is incomplete. Here, we showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ-binding motif, associated with synapse-associated protein 97 (SAP97), a member of the PDZ domain–containing, membrane-associated guanylate-kinase family of scaffolding molecules that play key roles in controlling receptor density and signal strength at neuronal synapses. We showed that SAP97 accumulated and bound to IgG BCRs in the immune synapses that formed in response to engagement of the B cell with antigen. Knocking down SAP97 in IgG-expressing B cells or mutating the putative PDZ-binding motif in the tail impaired immune synapse formation, the initiation of IgG BCR signaling, and downstream activation of p38 mitogen-activated protein kinase. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immune synapse. PMID:22855505

  14. Fluorescent composite scaffolds made of nanodiamonds/polycaprolactone

    Science.gov (United States)

    Cao, Li; Hou, Yanwen; Lafdi, Khalid; Urmey, Kirk

    2015-11-01

    Polycaprolactone (PCL) has been widely studied for biological applications. Biodegradable PCL fibrous scaffold can work as an appropriate substrate for tissue regeneration. In this letter, fluorescent nanodiamonds (FNDs) were prepared after surface passivation with octadecylamine. The FNDs were then mixed with PCL polymer and subsequently electrospun into FNDs/PCL fibrous scaffolds. The obtained scaffolds not only exhibited photoluminescence, but also showed reinforced mechanical strength. Toxicity study indicated FNDs/PCL scaffolds were nontoxic. This biocompatible fluorescent composite fibrous scaffold can support in vitro cell growth and also has the potential to act as an optical probe for tissue engineering application in vitro and in vivo.

  15. 3D printing of novel osteochondral scaffolds with graded microstructure

    Science.gov (United States)

    Nowicki, Margaret A.; Castro, Nathan J.; Plesniak, Michael W.; Zhang, Lijie Grace

    2016-10-01

    Osteochondral tissue has a complex graded structure where biological, physiological, and mechanical properties vary significantly over the full thickness spanning from the subchondral bone region beneath the joint surface to the hyaline cartilage region at the joint surface. This presents a significant challenge for tissue-engineered structures addressing osteochondral defects. Fused deposition modeling (FDM) 3D bioprinters present a unique solution to this problem. The objective of this study is to use FDM-based 3D bioprinting and nanocrystalline hydroxyapatite for improved bone marrow human mesenchymal stem cell (hMSC) adhesion, growth, and osteochondral differentiation. FDM printing parameters can be tuned through computer aided design and computer numerical control software to manipulate scaffold geometries in ways that are beneficial to mechanical performance without hindering cellular behavior. Additionally, the ability to fine-tune 3D printed scaffolds increases further through our investment casting procedure which facilitates the inclusion of nanoparticles with biochemical factors to further elicit desired hMSC differentiation. For this study, FDM was used to print investment-casting molds innovatively designed with varied pore distribution over the full thickness of the scaffold. The mechanical and biological impacts of the varied pore distributions were compared and evaluated to determine the benefits of this physical manipulation. The results indicate that both mechanical properties and cell performance improve in the graded pore structures when compared to homogeneously distributed porous and non-porous structures. Differentiation results indicated successful osteogenic and chondrogenic manipulation in engineered scaffolds.

  16. Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

    Science.gov (United States)

    Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

    2013-01-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

  17. Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

    2013-12-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

  18. Impedance Biosensors and Deep Crater Salivary Gland Scaffolds for Tissue Engineering

    Science.gov (United States)

    Schramm, Robert A.

    The salivary gland is a complex, branching organ whose primary biological function is the production of the fluid critical to alimentary function and the lubrication and maintenance of the oral cavity, saliva. The most frequent disruption of the salivary organ system is one in which the rate of supply of saliva into the oral cavity is diminished, and this may vary from a minor reduction, to near cessation. Regenerative medicine is a field which seeks to find ways to overcome the symptoms of organ malfunction or damage by inducing regrowth, repair and replacement of partial or whole organ function. Historically, the only methods available to medical experts were certain chemical drugs and transplantation, each of which suffers from significant risks and drawbacks. Tissue Engineering arose in the past few decades thanks to the seminal work of Robert Langer with the charter mission of finding new biomaterials and techniques to achieve these ends. The original concept of tissue engineering was the cell or tissue scaffold, which is supports the regrowth of cells by making intimate contact with adherent cells, and induces improved regrowth in vitro or in vivo by providing mechanical or chemical signaling cues. Epithelial cell types such as salivary glands have structural functional polarity at the cellular level, an apical side which faces a void, and a basal side which faces the support substrate. While 3D scaffolds such as hydrogels maximize interaction area between cells and substrate, they struggle to develop cohesive tissues beyond the scale of small cellular clusters . 2D scaffolds enforce a defined polarity by allowing cell interaction at only one side of the cell. Langer pioneered the use of polymer nanofibers as the premier synthetic 2D scaffold biomaterial, due to their exceptionally high nano-scale surface area, and collagen-imitating structure. Prior work has established PLGA nanofibers, which allow salivary cells to attach, proliferate, and generate a

  19. Novel biodegradable porous scaffold applied to skin regeneration.

    Science.gov (United States)

    Wang, Hui-Min; Chou, Yi-Ting; Wen, Zhi-Hong; Wang, Chau-Zen; Wang, Zhao-Ren; Chen, Chun-Hong; Ho, Mei-Ling

    2013-01-01

    Skin wound healing is an important lifesaving issue for massive lesions. A novel porous scaffold with collagen, hyaluronic acid and gelatin was developed for skin wound repair. The swelling ratio of this developed scaffold was assayed by water absorption capacity and showed a value of over 20 g water/g dried scaffold. The scaffold was then degraded in time- and dose-dependent manners by three enzymes: lysozyme, hyaluronidase and collagenase I. The average pore diameter of the scaffold was 132.5±8.4 µm measured from SEM images. With human skin cells growing for 7 days, the SEM images showed surface fractures on the scaffold due to enzymatic digestion, indicating the biodegradable properties of this scaffold. To simulate skin distribution, the human epidermal keratinocytes, melanocytes and dermal fibroblasts were seeded on the porous scaffold and the cross-section immunofluorescent staining demonstrated normal human skin layer distributions. The collagen amount was also quantified after skin cells seeding and presented an amount 50% higher than those seeded on culture wells. The in vivo histological results showed that the scaffold ameliorated wound healing, including decreasing neutrophil infiltrates and thickening newly generated skin compared to the group without treatments.

  20. Novel biodegradable porous scaffold applied to skin regeneration.

    Directory of Open Access Journals (Sweden)

    Hui-Min Wang

    Full Text Available Skin wound healing is an important lifesaving issue for massive lesions. A novel porous scaffold with collagen, hyaluronic acid and gelatin was developed for skin wound repair. The swelling ratio of this developed scaffold was assayed by water absorption capacity and showed a value of over 20 g water/g dried scaffold. The scaffold was then degraded in time- and dose-dependent manners by three enzymes: lysozyme, hyaluronidase and collagenase I. The average pore diameter of the scaffold was 132.5±8.4 µm measured from SEM images. With human skin cells growing for 7 days, the SEM images showed surface fractures on the scaffold due to enzymatic digestion, indicating the biodegradable properties of this scaffold. To simulate skin distribution, the human epidermal keratinocytes, melanocytes and dermal fibroblasts were seeded on the porous scaffold and the cross-section immunofluorescent staining demonstrated normal human skin layer distributions. The collagen amount was also quantified after skin cells seeding and presented an amount 50% higher than those seeded on culture wells. The in vivo histological results showed that the scaffold ameliorated wound healing, including decreasing neutrophil infiltrates and thickening newly generated skin compared to the group without treatments.

  1. Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

    Science.gov (United States)

    Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

    1992-01-01

    Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

  2. [Strategies to choose scaffold materials for tissue engineering].

    Science.gov (United States)

    Gao, Qingdong; Zhu, Xulong; Xiang, Junxi; Lü, Yi; Li, Jianhui

    2016-02-01

    Current therapies of organ failure or a wide range of tissue defect are often not ideal. Transplantation is the only effective way for long time survival. But it is hard to meet huge patients demands because of donor shortage, immune rejection and other problems. Tissue engineering could be a potential option. Choosing a suitable scaffold material is an essential part of it. According to different sources, tissue engineering scaffold materials could be divided into three types which are natural and its modified materials, artificial and composite ones. The purpose of tissue engineering scaffold is to repair the tissues or organs damage, so could reach the ideal recovery in its function and structure aspect. Therefore, tissue engineering scaffold should even be as close as much to the original tissue or organs in function and structure. We call it "organic scaffold" and this strategy might be the drastic perfect substitute for the tissues or organs in concern. Optimized organization with each kind scaffold materials could make up for biomimetic structure and function of the tissue or organs. Scaffold material surface modification, optimized preparation procedure and cytosine sustained-release microsphere addition should be considered together. This strategy is expected to open new perspectives for tissue engineering. Multidisciplinary approach including material science, molecular biology, and engineering might find the most ideal tissue engineering scaffold. Using the strategy of drawing on each other strength and optimized organization with each kind scaffold material to prepare a multifunctional biomimetic tissue engineering scaffold might be a good method for choosing tissue engineering scaffold materials. Our research group had differentiated bone marrow mesenchymal stem cells into bile canaliculi like cells. We prepared poly(L-lactic acid)/poly(ε-caprolactone) biliary stent. The scaffold's internal played a part in the long-term release of cytokines which

  3. [Mechanical properties of polylactic acid/beta-tricalcium phosphate composite scaffold with double channels based on three-dimensional printing technique].

    Science.gov (United States)

    Lian, Qin; Zhuang, Pei; Li, Changhai; Jin, Zhongmin; Li, Dichen

    2014-03-01

    controlled complex micropipes and can significantly enhance mechanical properties, which is a promising strategy to solve the contradiction of strength and high-porosity of the ceramic scaffolds for the bone tissue engineering application.

  4. Nano-ceramic composite scaffolds for bioreactor-based bone engineering.

    Science.gov (United States)

    Lv, Qing; Deng, Meng; Ulery, Bret D; Nair, Lakshmi S; Laurencin, Cato T

    2013-08-01

    Composites of biodegradable polymers and bioactive ceramics are candidates for tissue-engineered scaffolds that closely match the properties of bone. We previously developed a porous, three-dimensional poly (D,L-lactide-co-glycolide) (PLAGA)/nanohydroxyapatite (n-HA) scaffold as a potential bone tissue engineering matrix suitable for high-aspect ratio vessel (HARV) bioreactor applications. However, the physical and cellular properties of this scaffold are unknown. The present study aims to evaluate the effect of n-HA in modulating PLAGA scaffold properties and human mesenchymal stem cell (HMSC) responses in a HARV bioreactor. By comparing PLAGA/n-HA and PLAGA scaffolds, we asked whether incorporation of n-HA (1) accelerates scaffold degradation and compromises mechanical integrity; (2) promotes HMSC proliferation and differentiation; and (3) enhances HMSC mineralization when cultured in HARV bioreactors. PLAGA/n-HA scaffolds (total number = 48) were loaded into HARV bioreactors for 6 weeks and monitored for mass, molecular weight, mechanical, and morphological changes. HMSCs were seeded on PLAGA/n-HA scaffolds (total number = 38) and cultured in HARV bioreactors for 28 days. Cell migration, proliferation, osteogenic differentiation, and mineralization were characterized at four selected time points. The same amount of PLAGA scaffolds were used as controls. The incorporation of n-HA did not alter the scaffold degradation pattern. PLAGA/n-HA scaffolds maintained their mechanical integrity throughout the 6 weeks in the dynamic culture environment. HMSCs seeded on PLAGA/n-HA scaffolds showed elevated proliferation, expression of osteogenic phenotypic markers, and mineral deposition as compared with cells seeded on PLAGA scaffolds. HMSCs migrated into the scaffold center with nearly uniform cell and extracellular matrix distribution in the scaffold interior. The combination of PLAGA/n-HA scaffolds with HMSCs in HARV bioreactors may allow for the generation of engineered

  5. The effect of porosity on cell ingrowth into accurately defined, laser-made, polylactide-based 3D scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Danilevicius, Paulius; Georgiadi, Leoni [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Pateman, Christopher J.; Claeyssens, Frederik [Kroto Research Institute, Department of Materials Science and Engineering, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom); Chatzinikolaidou, Maria, E-mail: mchatzin@materials.uoc.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Department of Materials Science and Technology, University of Crete, PO Box 2208, 71303 Heraklion (Greece); Farsari, Maria, E-mail: mfarsari@iesl.forth.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece)

    2015-05-01

    Highlights: • We studied the porosity of laser-made 3D scaffolds on MC3T3-E1 pre-osteoblastic cells. • We made polylactide 3D scaffolds with pores 25–110 μm. - Abstract: The aim of this study is to demonstrate the accuracy required for the investigation of the role of solid scaffolds’ porosity in cell proliferation. We therefore present a qualitative investigation into the effect of porosity on MC3T3-E1 pre-osteoblastic cell ingrowth of three-dimensional (3D) scaffolds fabricated by direct femtosecond laser writing. The material we used is a purpose made photosensitive pre-polymer based on polylactide. We designed and fabricated complex, geometry-controlled 3D scaffolds with pore sizes ranging from 25 to 110 μm, representing porosities 70%, 82%, 86%, and 90%. The 70% porosity scaffolds did not support cell growth initially and in the long term. For the other porosities, we found a strong adhesion of the pre-osteoblastic cells from the first hours after seeding and a remarkable proliferation increase after 3 weeks and up to 8 weeks. The 86% porosity scaffolds exhibited a higher efficiency compared to 82% and 90%. In addition, bulk material degradation studies showed that the employed, highly-acrylated polylactide is degradable. These findings support the potential use of the proposed material and the scaffold fabrication technique in bone tissue engineering.

  6. A radiopaque electrospun scaffold for engineering fibrous musculoskeletal tissues: Scaffold characterization and in vivo applications.

    Science.gov (United States)

    Martin, John T; Milby, Andrew H; Ikuta, Kensuke; Poudel, Subash; Pfeifer, Christian G; Elliott, Dawn M; Smith, Harvey E; Mauck, Robert L

    2015-10-01

    Tissue engineering strategies have emerged in response to the growing prevalence of chronic musculoskeletal conditions, with many of these regenerative methods currently being evaluated in translational animal models. Engineered replacements for fibrous tissues such as the meniscus, annulus fibrosus, tendons, and ligaments are subjected to challenging physiologic loads, and are difficult to track in vivo using standard techniques. The diagnosis and treatment of musculoskeletal conditions depends heavily on radiographic assessment, and a number of currently available implants utilize radiopaque markers to facilitate in vivo imaging. In this study, we developed a nanofibrous scaffold in which individual fibers included radiopaque nanoparticles. Inclusion of radiopaque particles increased the tensile modulus of the scaffold and imparted radiation attenuation within the range of cortical bone. When scaffolds were seeded with bovine mesenchymal stem cells in vitro, there was no change in cell proliferation and no evidence of promiscuous conversion to an osteogenic phenotype. Scaffolds were implanted ex vivo in a model of a meniscal tear in a bovine joint and in vivo in a model of total disc replacement in the rat coccygeal spine (tail), and were visualized via fluoroscopy and microcomputed tomography. In the disc replacement model, histological analysis at 4 weeks showed that the scaffold was biocompatible and supported the deposition of fibrous tissue in vivo. Nanofibrous scaffolds that include radiopaque nanoparticles provide a biocompatible template with sufficient radiopacity for in vivo visualization in both small and large animal models. This radiopacity may facilitate image-guided implantation and non-invasive long-term evaluation of scaffold location and performance. The healing capacity of fibrous musculoskeletal tissues is limited, and injury or degeneration of these tissues compromises the standard of living of millions in the US. Tissue engineering repair

  7. Development of nanofibrous scaffolds containing gum tragacanth/poly (ε-caprolactone) for application as skin scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Ranjbar-Mohammadi, Marziyeh [Textile Engineering Department, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Bahrami, S. Hajir, E-mail: hajirb@aut.ac.ir [Textile Engineering Department, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Center for excellence Modern Textile Characterization, Tehran (Iran, Islamic Republic of)

    2015-03-01

    Outstanding wound healing activity of gum tragacanth (GT) and higher mechanical strength of poly (ε-caprolactone) (PCL) may produce an excellent nanofibrous patch for either skin tissue engineering or wound dressing application. PCL/GT scaffold containing different concentrations of PCL with different blend ratios of GT/PCL was produced using 90% acetic acid as solvent. The results demonstrated that the PCL/GT (3:1.5) with PCL concentration of 20% (w/v) produced nanofibers with proper morphology. Scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were utilized to characterize the nanofibers. Surface wettability, functional groups analysis, porosity and tensile properties of nanofibers were evaluated. Morphological characterization showed that the addition of GT to PCL solution results in decreasing the average diameter of the PCL/GT nanofibers. However, the hydrophilicity increased in the PCL/GT nanofibers. Slight increase in melting peaks was observed due to the blending of PCL with GT nanofibers. PCL/GT nanofibers were used for in vitro cell culture of human fibroblast cell lines AGO and NIH 3T3 fibroblast cells. MTT assay and SEM results showed that the biocomposite PCL/GT mats enhanced the fibroblast adhesion and proliferation compared to PCL scaffolds. The antibacterial activity of PCL/GT and GT nanofibers against Staphylococcus aureus and Pseudomonas aeruginosa was also examined. - Highlights: • A new skin tissue engineering scaffold from poly (ε-caprolactone) (PCL) and gum tragacanth (GT) has been developed. • These scaffolds might be an effectual simulator of the structure and composition of native skin. • Very slight increase in melting peaks was observed due to the blending of PCL with GT nanofibers. • Biodegradation, water uptake and hydrophilicity properties of these scaffolds showed that produced scaffolds were adherent. • The electrospun PCL/GT scaffold can promote the skin regeneration of full

  8. Development of nanofibrous scaffolds containing gum tragacanth/poly (ε-caprolactone) for application as skin scaffolds

    International Nuclear Information System (INIS)

    Ranjbar-Mohammadi, Marziyeh; Bahrami, S. Hajir

    2015-01-01

    Outstanding wound healing activity of gum tragacanth (GT) and higher mechanical strength of poly (ε-caprolactone) (PCL) may produce an excellent nanofibrous patch for either skin tissue engineering or wound dressing application. PCL/GT scaffold containing different concentrations of PCL with different blend ratios of GT/PCL was produced using 90% acetic acid as solvent. The results demonstrated that the PCL/GT (3:1.5) with PCL concentration of 20% (w/v) produced nanofibers with proper morphology. Scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were utilized to characterize the nanofibers. Surface wettability, functional groups analysis, porosity and tensile properties of nanofibers were evaluated. Morphological characterization showed that the addition of GT to PCL solution results in decreasing the average diameter of the PCL/GT nanofibers. However, the hydrophilicity increased in the PCL/GT nanofibers. Slight increase in melting peaks was observed due to the blending of PCL with GT nanofibers. PCL/GT nanofibers were used for in vitro cell culture of human fibroblast cell lines AGO and NIH 3T3 fibroblast cells. MTT assay and SEM results showed that the biocomposite PCL/GT mats enhanced the fibroblast adhesion and proliferation compared to PCL scaffolds. The antibacterial activity of PCL/GT and GT nanofibers against Staphylococcus aureus and Pseudomonas aeruginosa was also examined. - Highlights: • A new skin tissue engineering scaffold from poly (ε-caprolactone) (PCL) and gum tragacanth (GT) has been developed. • These scaffolds might be an effectual simulator of the structure and composition of native skin. • Very slight increase in melting peaks was observed due to the blending of PCL with GT nanofibers. • Biodegradation, water uptake and hydrophilicity properties of these scaffolds showed that produced scaffolds were adherent. • The electrospun PCL/GT scaffold can promote the skin regeneration of full

  9. Polycaprolactone nanofiber interspersed collagen type-I scaffold for bone regeneration: a unique injectable osteogenic scaffold

    International Nuclear Information System (INIS)

    Baylan, Nuray; Ditto, Maggie; Lawrence, Joseph G; Yildirim-Ayan, Eda; Bhat, Samerna; Lecka-Czernik, Beata

    2013-01-01

    There is an increasing demand for an injectable cell coupled three-dimensional (3D) scaffold to be used as bone fracture augmentation material. To address this demand, a novel injectable osteogenic scaffold called PN-COL was developed using cells, a natural polymer (collagen type-I), and a synthetic polymer (polycaprolactone (PCL)). The injectable nanofibrous PN-COL is created by interspersing PCL nanofibers within pre-osteoblast cell embedded collagen type-I. This simple yet novel and powerful approach provides a great benefit as an injectable bone scaffold over other non-living bone fracture stabilization polymers, such as polymethylmethacrylate and calcium content resin-based materials. The advantages of injectability and the biomimicry of collagen was coupled with the structural support of PCL nanofibers, to create cell encapsulated injectable 3D bone scaffolds with intricate porous internal architecture and high osteoconductivity. The effects of PCL nanofiber inclusion within the cell encapsulated collagen matrix has been evaluated for scaffold size retention and osteocompatibility, as well as for MC3T3-E1 cells osteogenic activity. The structural analysis of novel bioactive material proved that the material is chemically stable enough in an aqueous solution for an extended period of time without using crosslinking reagents, but it is also viscous enough to be injected through a syringe needle. Data from long-term in vitro proliferation and differentiation data suggests that novel PN-COL scaffolds promote the osteoblast proliferation, phenotype expression, and formation of mineralized matrix. This study demonstrates for the first time the feasibility of creating a structurally competent, injectable, cell embedded bone tissue scaffold. Furthermore, the results demonstrate the advantages of mimicking the hierarchical architecture of native bone with nano- and micro-size formation through introducing PCL nanofibers within macron-size collagen fibers and in

  10. Effect of oxidative stress on homer scaffolding proteins.

    Directory of Open Access Journals (Sweden)

    Igor Nepliouev

    Full Text Available Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G. Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.

  11. Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds

    International Nuclear Information System (INIS)

    Hockaday, L A; Kang, K H; Colangelo, N W; Cheung, P Y C; Duan, B; Wu, J; Bonassar, L J; Butcher, J T; Malone, E; Lipson, H; Girardi, L N; Chu, C C

    2012-01-01

    The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for the long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12–22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over tenfold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 min, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0 and 73.3±5.2% for 22, 17 and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6 and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment. (paper)

  12. [Three-dimensional parallel collagen scaffold promotes tendon extracellular matrix formation].

    Science.gov (United States)

    Zheng, Zefeng; Shen, Weiliang; Le, Huihui; Dai, Xuesong; Ouyang, Hongwei; Chen, Weishan

    2016-03-01

    To investigate the effects of three-dimensional parallel collagen scaffold on the cell shape, arrangement and extracellular matrix formation of tendon stem cells. Parallel collagen scaffold was fabricated by unidirectional freezing technique, while random collagen scaffold was fabricated by freeze-drying technique. The effects of two scaffolds on cell shape and extracellular matrix formation were investigated in vitro by seeding tendon stem/progenitor cells and in vivo by ectopic implantation. Parallel and random collagen scaffolds were produced successfully. Parallel collagen scaffold was more akin to tendon than random collagen scaffold. Tendon stem/progenitor cells were spindle-shaped and unified orientated in parallel collagen scaffold, while cells on random collagen scaffold had disorder orientation. Two weeks after ectopic implantation, cells had nearly the same orientation with the collagen substance. In parallel collagen scaffold, cells had parallel arrangement, and more spindly cells were observed. By contrast, cells in random collagen scaffold were disorder. Parallel collagen scaffold can induce cells to be in spindly and parallel arrangement, and promote parallel extracellular matrix formation; while random collagen scaffold can induce cells in random arrangement. The results indicate that parallel collagen scaffold is an ideal structure to promote tendon repairing.

  13. Micropore-induced capillarity enhances bone distribution in vivo in biphasic calcium phosphate scaffolds.

    Science.gov (United States)

    Rustom, Laurence E; Boudou, Thomas; Lou, Siyu; Pignot-Paintrand, Isabelle; Nemke, Brett W; Lu, Yan; Markel, Mark D; Picart, Catherine; Wagoner Johnson, Amy J

    2016-10-15

    The increasing demand for bone repair solutions calls for the development of efficacious bone scaffolds. Biphasic calcium phosphate (BCP) scaffolds with both macropores and micropores (MP) have improved healing compared to those with macropores and no micropores (NMP), but the role of micropores is unclear. Here, we evaluate capillarity induced by micropores as a mechanism that can affect bone growth in vivo. Three groups of cylindrical scaffolds were implanted in pig mandibles for three weeks: MP were implanted either dry (MP-Dry), or after submersion in phosphate buffered saline, which fills pores with fluid and therefore suppresses micropore-induced capillarity (MP-Wet); NMP were implanted dry. The amount and distribution of bone in the scaffolds were quantified using micro-computed tomography. MP-Dry had a more homogeneous bone distribution than MP-Wet, although the average bone volume fraction, BVF‾, was not significantly different for these two groups (0.45±0.03 and 0.37±0.03, respectively). There was no significant difference in the radial bone distribution of NMP and MP-Wet, but the BVF‾, of NMP was significantly lower among the three groups (0.25±0.02). These results suggest that micropore-induced capillarity enhances bone regeneration by improving the homogeneity of bone distribution in BCP scaffolds. The explicit design and use of capillarity in bone scaffolds may lead to more effective treatments of large and complex bone defects. The increasing demand for bone repair calls for more efficacious bone scaffolds and calcium phosphate-based materials are considered suitable for this application. Macropores (>100μm) are necessary for bone ingrowth and vascularization. However, studies have shown that microporosity (micropore-induced capillarity had the potential to enhance bone growth in vivo. This work illustrates the positive effects of capillarity on bone regeneration in vivo; it demonstrates that micropore-induced capillarity significantly

  14. Bioresorbable scaffolds: talking about a new interventional revolution [corrected].

    Science.gov (United States)

    Hassell, M E C J; Grundeken, M J D; Delewi, R; Wykrzykowska, J J; Piek, J J

    2013-04-01

    After the introduction of coronary balloon angioplasty, bare-metal, and drug-eluting stents, fully bioresorbable scaffolds (BRS) could be the fourth revolution in interventional cardiology. The BRS technology shares the advantages of metallic stents regarding acute gain and prevention of acute vessel occlusion by providing transient scaffolding, while potentially overcoming many of the safety concerns of drug-eluting stents. Furthermore, without a permanent metallic cage, the vessel could remodel favourably and atherosclerotic plaques could regress in the long-term. This attracted increased interest and several BRS have been developed. In this review we will describe all BRS which are thus far clinically evaluated and provide an overview of ongoing clinical studies. Although the technology seems to be very promising, more studies including patients with more complex lesions are needed to evaluate whether the BRS can be used in daily clinical practice and if it is indeed becoming a new interventional revolution.

  15. Inverse Opal Scaffolds and Their Biomedical Applications.

    Science.gov (United States)

    Zhang, Yu Shrike; Zhu, Chunlei; Xia, Younan

    2017-09-01

    Three-dimensional porous scaffolds play a pivotal role in tissue engineering and regenerative medicine by functioning as biomimetic substrates to manipulate cellular behaviors. While many techniques have been developed to fabricate porous scaffolds, most of them rely on stochastic processes that typically result in scaffolds with pores uncontrolled in terms of size, structure, and interconnectivity, greatly limiting their use in tissue regeneration. Inverse opal scaffolds, in contrast, possess uniform pores inheriting from the template comprised of a closely packed lattice of monodispersed microspheres. The key parameters of such scaffolds, including architecture, pore structure, porosity, and interconnectivity, can all be made uniform across the same sample and among different samples. In conjunction with a tight control over pore sizes, inverse opal scaffolds have found widespread use in biomedical applications. In this review, we provide a detailed discussion on this new class of advanced materials. After a brief introduction to their history and fabrication, we highlight the unique advantages of inverse opal scaffolds over their non-uniform counterparts. We then showcase their broad applications in tissue engineering and regenerative medicine, followed by a summary and perspective on future directions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Additively Manufactured Scaffolds for Bone Tissue Engineering and the Prediction of their Mechanical Behavior: A Review.

    Science.gov (United States)

    Zhang, Xiang-Yu; Fang, Gang; Zhou, Jie

    2017-01-10

    Additive manufacturing (AM), nowadays commonly known as 3D printing, is a revolutionary materials processing technology, particularly suitable for the production of low-volume parts with high shape complexities and often with multiple functions. As such, it holds great promise for the fabrication of patient-specific implants. In recent years, remarkable progress has been made in implementing AM in the bio-fabrication field. This paper presents an overview on the state-of-the-art AM technology for bone tissue engineering (BTE) scaffolds, with a particular focus on the AM scaffolds made of metallic biomaterials. It starts with a brief description of architecture design strategies to meet the biological and mechanical property requirements of scaffolds. Then, it summarizes the working principles, advantages and limitations of each of AM methods suitable for creating porous structures and manufacturing scaffolds from powdered materials. It elaborates on the finite-element (FE) analysis applied to predict the mechanical behavior of AM scaffolds, as well as the effect of the architectural design of porous structure on its mechanical properties. The review ends up with the authors' view on the current challenges and further research directions.

  17. Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering

    International Nuclear Information System (INIS)

    Ghasemi-Mobarakeh, Laleh; Prabhakaran, Molamma P.; Morshed, Mohammad; Nasr-Esfahani, Mohammad Hossein; Ramakrishna, S.

    2010-01-01

    Surface properties of scaffolds such as hydrophilicity and the presence of functional groups on the surface of scaffolds play a key role in cell adhesion, proliferation and migration. Different modification methods for hydrophilicity improvement and introduction of functional groups on the surface of scaffolds have been carried out on synthetic biodegradable polymers, for tissue engineering applications. In this study, alkaline hydrolysis of poly (ε-caprolactone) (PCL) nanofibrous scaffolds was carried out for different time periods (1 h, 4 h and 12 h) to increase the hydrophilicity of the scaffolds. The formation of reactive groups resulting from alkaline hydrolysis provides opportunities for further surface functionalization of PCL nanofibrous scaffolds. Matrigel was attached covalently on the surface of an optimized 4 h hydrolyzed PCL nanofibrous scaffolds and additionally the fabrication of blended PCL/matrigel nanofibrous scaffolds was carried out. Chemical and mechanical characterization of nanofibrous scaffolds were evaluated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle, scanning electron microscopy (SEM) and tensile measurement. In vitro cell adhesion and proliferation study was carried out after seeding nerve precursor cells (NPCs) on different scaffolds. Results of cell proliferation assay and SEM studies showed that the covalently functionalized PCL/matrigel nanofibrous scaffolds promote the proliferation and neurite outgrowth of NPCs compared to PCL and hydrolyzed PCL nanofibrous scaffolds, providing suitable substrates for nerve tissue engineering.

  18. Scaffolding vector representations for student learning inside a physics game

    Science.gov (United States)

    D'Angelo, Cynthia

    Vectors and vector addition are difficult concepts for many introductory physics students and traditional instruction does not usually sufficiently address these difficulties. Vectors play a major role in most topics in introductory physics and without a complete understanding of them many students are unable to make sense of the physics topics covered in their classes. Video games present a unique opportunity to help students develop an intuitive understanding of motion, forces, and vectors while immersed in an enjoyable and interactive environment. This study examines two dimensions of design decisions to help students learn while playing a physics-based game. The representational complexity dimension looked at two ways of presenting dynamic information about the velocity of the game object on the screen. The scaffolding context dimension looked at two different contexts for presenting vector addition problems that were related to the game. While all students made significant learning games from the pre to the post test, there were virtually no differences between students along the representational complexity dimension and small differences between students along the scaffolding context dimension. A context that directly connects to students' game playing experience was in most cases more productive to learning than an abstract context.

  19. Three-dimensional printing of porous ceramic scaffolds for bone tissue engineering.

    Science.gov (United States)

    Seitz, Hermann; Rieder, Wolfgang; Irsen, Stephan; Leukers, Barbara; Tille, Carsten

    2005-08-01

    This article reports a new process chain for custom-made three-dimensional (3D) porous ceramic scaffolds for bone replacement with fully interconnected channel network for the repair of osseous defects from trauma or disease. Rapid prototyping and especially 3D printing is well suited to generate complex-shaped porous ceramic matrices directly from powder materials. Anatomical information obtained from a patient can be used to design the implant for a target defect. In the 3D printing technique, a box filled with ceramic powder is printed with a polymer-based binder solution layer by layer. Powder is bonded in wetted regions. Unglued powder can be removed and a ceramic green body remains. We use a modified hydroxyapatite (HA) powder for the fabrication of 3D printed scaffolds due to the safety of HA as biocompatible implantable material and efficacy for bone regeneration. The printed ceramic green bodies are consolidated at a temperature of 1250 degrees C in a high temperature furnace in ambient air. The polymeric binder is pyrolysed during sintering. The resulting scaffolds can be used in tissue engineering of bone implants using patient-derived cells that are seeded onto the scaffolds. This article describes the process chain, beginning from data preparation to 3D printing tests and finally sintering of the scaffold. Prototypes were successfully manufactured and characterized. It was demonstrated that it is possible to manufacture parts with inner channels with a dimension down to 450 microm and wall structures with a thickness down to 330 microm. The mechanical strength of dense test parts is up to 22 MPa. Copyright 2005 Wiley Periodicals, Inc.

  20. Low-temperature deposition manufacturing: A novel and promising rapid prototyping technology for the fabrication of tissue-engineered scaffold.

    Science.gov (United States)

    Liu, Wei; Wang, Daming; Huang, Jianghong; Wei, You; Xiong, Jianyi; Zhu, Weimin; Duan, Li; Chen, Jielin; Sun, Rong; Wang, Daping

    2017-01-01

    Developed in recent years, low-temperature deposition manufacturing (LDM) represents one of the most promising rapid prototyping technologies. It is not only based on rapid deposition manufacturing process but also combined with phase separation process. Besides the controlled macropore size, tissue-engineered scaffold fabricated by LDM has inter-connected micropores in the deposited lines. More importantly, it is a green manufacturing process that involves non-heating liquefying of materials. It has been employed to fabricate tissue-engineered scaffolds for bone, cartilage, blood vessel and nerve tissue regenerations. It is a promising technology in the fabrication of tissue-engineered scaffold similar to ideal scaffold and the design of complex organs. In the current paper, this novel LDM technology is introduced, and its control parameters, biomedical applications and challenges are included and discussed as well. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

    Science.gov (United States)

    Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

    2017-11-01

    The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

  2. Integration of 3D Printed and Micropatterned Polycaprolactone Scaffolds for Guidance of Oriented Collagenous Tissue Formation In Vivo.

    Science.gov (United States)

    Pilipchuk, Sophia P; Monje, Alberto; Jiao, Yizu; Hao, Jie; Kruger, Laura; Flanagan, Colleen L; Hollister, Scott J; Giannobile, William V

    2016-03-01

    Scaffold design incorporating multiscale cues for clinically relevant, aligned tissue regeneration has potential to improve structural and functional integrity of multitissue interfaces. The objective of this preclinical study is to develop poly(ε-caprolactone) (PCL) scaffolds with mesoscale and microscale architectural cues specific to human ligament progenitor cells and assess their ability to form aligned bone-ligament-cementum complexes in vivo. PCL scaffolds are designed to integrate a 3D printed bone region with a micropatterned PCL thin film consisting of grooved pillars. The patterned film region is seeded with human ligament cells, fibroblasts transduced with bone morphogenetic protein-7 genes seeded within the bone region, and a tooth dentin segment positioned on the ligament region prior to subcutaneous implantation into a murine model. Results indicate increased tissue alignment in vivo using micropatterned PCL films, compared to random-porous PCL. At week 6, 30 μm groove depth significantly enhances oriented collagen fiber thickness, overall cell alignment, and nuclear elongation relative to 10 μm groove depth. This study demonstrates for the first time that scaffolds with combined hierarchical mesoscale and microscale features can align cells in vivo for oral tissue repair with potential for improving the regenerative response of other bone-ligament complexes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Purification and characterization of a thylakoid protein kinase

    International Nuclear Information System (INIS)

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs

  4. Preparation of bioactive porous HA/PCL composite scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, J.; Guo, L.Y.; Yang, X.B. [Key Laboratory of Advanced Technologies of Materials (Ministry of Education), School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Weng, J. [Key Laboratory of Advanced Technologies of Materials (Ministry of Education), School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)], E-mail: jweng@swjtu.cn

    2008-12-30

    Porous hydroxyapatite (HA) bioceramic scaffold has been widely attracted the attention to act as a three-dimensional (3D) template for cell adhesion, proliferation, differentiation and thus promoting bone and cartilage regeneration because of its osteoinduction. However, the porous bioceramic scaffold is fragile so that it is not suitable to be applied in clinic for bone repair or replacement. Therefore, it is significant to improve the mechanical property of porous HA bioceramics while the interconnected structure is maintained for tissue ingrowth in vivo. In the present research, a porous composite scaffold composed of HA scaffold and polycaprolactone (PCL) lining was fabricated by the method of polymer impregnating to produce HA scaffold coated with PCL lining. Subsequently, the composite scaffolds were deposited with biomimetic coating for improving the bioactivity. The HA/PCL composite scaffolds with improved mechanical property and bioactivity is expected to be a promising bone substitute in tissue engineering applications.

  5. Preparation of bioactive porous HA/PCL composite scaffolds

    International Nuclear Information System (INIS)

    Zhao, J.; Guo, L.Y.; Yang, X.B.; Weng, J.

    2008-01-01

    Porous hydroxyapatite (HA) bioceramic scaffold has been widely attracted the attention to act as a three-dimensional (3D) template for cell adhesion, proliferation, differentiation and thus promoting bone and cartilage regeneration because of its osteoinduction. However, the porous bioceramic scaffold is fragile so that it is not suitable to be applied in clinic for bone repair or replacement. Therefore, it is significant to improve the mechanical property of porous HA bioceramics while the interconnected structure is maintained for tissue ingrowth in vivo. In the present research, a porous composite scaffold composed of HA scaffold and polycaprolactone (PCL) lining was fabricated by the method of polymer impregnating to produce HA scaffold coated with PCL lining. Subsequently, the composite scaffolds were deposited with biomimetic coating for improving the bioactivity. The HA/PCL composite scaffolds with improved mechanical property and bioactivity is expected to be a promising bone substitute in tissue engineering applications

  6. siRNA nanoparticle functionalization of nanostructured scaffolds enables controlled multilineage differentiation of stem cells

    DEFF Research Database (Denmark)

    Andersen, Morten Ø; Nygaard, Jens V; Burns, Jorge S

    2010-01-01

    The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different ...

  7. A Simple and Efficient Method to Improve Mechanical Properties of Collagen Scaffolds by UV Irradiation

    Directory of Open Access Journals (Sweden)

    F. Khayyatan

    2010-12-01

    Full Text Available Collagen is the major protein component of cartilage, bone, skin and connective tissue and constitutes the major part of the extracellular matrix. Collagen type I has complex structural hierarchy, which consists of treepolypeptide α-chains wound together in a rod-like helical structure. Collagen is an important biomaterial, finding many applications in the field of tissue engineering. It has been processed into various shapes, such as, gel, film, sponge and fiber. It is commonly used as the scaffolding material for tissue engineering due to its many superior properties including low antigenicity and high growth promotion. Unfortunately, poor mechanical properties and rapid degradation rates of collagen scaffolds can cause instability and difficulty in handling. By crosslinking, the structural stability of the collagen and its rate of resorption can be adapted with respect to its demanding requirements. The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. In thisstudy, the effect of UV irradiation on collagen scaffolds has been carried out.Collagen scaffolds were fabricated using freeze drying method with freezing temperature of -80oC, then exposed to UV irradiation. Mean pore size of the scaffolds was obtained as 98.52±14.51 μm using scanning electron microscopy. Collagen scaffolds exposed to UV Irradiation (254 nm for 15 min showed the highest tensile strain (17.37±0.98 %, modulus (1.67±0.15 MPa and maximum load (24.47±2.38 cN values. As partial loss of the native collagen structure may influence attachment, migration, and proliferation of cells on collagen scaffolds, we detected no intact α-chains after SDS-Page chromatography. We demonstrate that UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen scaffolds without any molecular fracture.

  8. Engineering anatomically shaped vascularized bone grafts with hASCs and 3D-printed PCL scaffolds.

    Science.gov (United States)

    Temple, Joshua P; Hutton, Daphne L; Hung, Ben P; Huri, Pinar Yilgor; Cook, Colin A; Kondragunta, Renu; Jia, Xiaofeng; Grayson, Warren L

    2014-12-01

    The treatment of large craniomaxillofacial bone defects is clinically challenging due to the limited availability of transplantable autologous bone grafts and the complex geometry of the bones. The ability to regenerate new bone tissues that faithfully replicate the anatomy would revolutionize treatment options. Advances in the field of bone tissue engineering over the past few decades offer promising new treatment alternatives using biocompatible scaffold materials and autologous cells. This approach combined with recent advances in three-dimensional (3D) printing technologies may soon allow the generation of large, bioartificial bone grafts with custom, patient-specific architecture. In this study, we use a custom-built 3D printer to develop anatomically shaped polycaprolactone (PCL) scaffolds with varying internal porosities. These scaffolds are assessed for their ability to support induction of human adipose-derived stem cells (hASCs) to form vasculature and bone, two essential components of functional bone tissue. The development of functional tissues is assessed in vitro and in vivo. Finally, we demonstrate the ability to print large mandibular and maxillary bone scaffolds that replicate fine details extracted from patient's computed tomography scans. The findings of this study illustrate the capabilities and potential of 3D printed scaffolds to be used for engineering autologous, anatomically shaped, vascularized bone grafts. © 2014 Wiley Periodicals, Inc.

  9. Scaffolding Singaporean Students to Write Vividly in the Chinese ‘Mother Tongue’, Mandarin

    Directory of Open Access Journals (Sweden)

    Tzemin Chung

    2014-02-01

    Full Text Available This paper details results from a three-year study investigating how to help students in Singapore write vivid compositions in Mandarin, the Chinese ‘mother tongue’. Mastery of the mother tongue by Singaporean students has become an important government priority in recent years. The strategies employed by this study included the use of information and communications technology (ICT mediated scaffolds such as collaborative mind maps and online peer editing to help students learn micro-writing strategies. This paper outlines the process of using various scaffolds to support students to learn and apply the action chain micro-writing strategy. A class of 31 Primary 4 from a neighbourhood school participated in this study. Findings indicated that students were very enthusiastic about writing in the ICT-mediated environment. Contrary to the teacher’s initial belief, students could be scaffolded to write action chains quickly. Findings highlighted the potential of scaffolding students in learning small chunks of writing strategy in an ICT-mediated environment that enabled them to practice these strategies in their composition writing until they could master and apply them. These micro-writing strategies gradually built up to a complex set of skills, including expressive writing. Moreover, students enjoyed working in groups and editing their peers’ work online. This showed that peers could be engaged as scaffolders in the classroom to free up the teacher’ time, allowing the teacher more time to spend with students who were not performing well.

  10. A framework for classification of prokaryotic protein kinases.

    Directory of Open Access Journals (Sweden)

    Nidhi Tyagi

    Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

  11. Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

    Science.gov (United States)

    Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J; Zimmer, Danna B; Honkanen, Richard E; Wadzinski, Brian E

    2014-02-14

    Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

  12. Anisotropic silk fibroin/gelatin scaffolds from unidirectional freezing

    Energy Technology Data Exchange (ETDEWEB)

    Asuncion, Maria Christine Tankeh, E-mail: christine.asuncion@u.nus.edu [National University of Singapore, Department of Biomedical Engineering (Singapore); Goh, James Cho-Hong [National University of Singapore, Department of Biomedical Engineering (Singapore); National University of Singapore, Department of Orthopedic Surgery (Singapore); Toh, Siew-Lok [National University of Singapore, Department of Biomedical Engineering (Singapore); National University of Singapore, Department of Mechanical Engineering (Singapore)

    2016-10-01

    Recent studies have underlined the importance of matching scaffold properties to the biological milieu. Tissue, and thus scaffold, anisotropy is one such property that is important yet sometimes overlooked. Methods that have been used to achieve anisotropic scaffolds present challenges such as complicated fabrication steps, harsh processing conditions and toxic chemicals involved. In this study, unidirectional freezing was employed to fabricate anisotropic silk fibroin/gelatin scaffolds in a simple and mild manner. Morphological, mechanical, chemical and cellular compatibility properties were investigated, as well as the effect of the addition of gelatin to certain properties of the scaffold. It was shown that scaffold properties were suitable for cell proliferation and that mesenchymal stem cells were able to align themselves along the directed fibers. The fabricated scaffolds present a platform that can be used for anisotropic tissue engineering applications such as cardiac patches. - Highlights: • Silk/gelatin scaffolds with unidirectional alignment were fabricated using a simple and scalable process • Presence of gelatin in silk resulted to lesser shrinkage, better water retention and improved cell proliferation. • Mesenchymal stem cells were shown to align themselves according to the fiber alignment.

  13. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  14. β-Tricalcium phosphate/poly(glycerol sebacate) scaffolds with robust mechanical property for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Kai [The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Zhang, Jing; Ma, Xiaoyu; Ma, Yifan; Kan, Chao [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Ma, Haiyan [Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Li, Yulin, E-mail: yulinli@ecust.edu.cn [Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Yuan, Yuan, E-mail: yyuan@ecust.edu.cn [The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Liu, Changsheng, E-mail: liucs@ecust.edu.cn [The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China)

    2015-11-01

    Despite good biocompatibility and osteoconductivity, porous β-TCP scaffolds still lack the structural stability and mechanical robustness, which greatly limit their application in the field of bone regeneration. The hybridization of β-TCP with conventional synthetic biodegradable PLA and PCL only produced a limited toughening effect due to the plasticity of the polymers in nature. In this study, a β-TCP/poly(glycerol sebacate) scaffold (β-TCP/PGS) with well interconnected porous structure and robust mechanical property was prepared. Porous β-TCP scaffold was first prepared with polyurethane sponge as template and then impregnated into PGS pre-polymer solution with moderate viscosity, followed by in situ heat crosslinking and freezing–drying process. The results indicated that the freezing–drying under vacuum process could further facilitate crosslinking of PGS and formation of Ca{sup 2+}–COO{sup −} ionic complexing and thus synergistically improved the mechanical strength of the β-TCP/PGS with in situ heat crosslinking. Particularly, the β-TCP/PGS with 15% PGS content after heat crosslinking at 130 °C and freezing–drying at − 50 °C under vacuum exhibited an elongation at break of 375 ± 25% and a compressive strength of 1.73 MPa, 3.7-fold and 200-fold enhancement compared to the β-TCP, respectively. After the abrupt drop of compressive load, the β-TCP/PGS scaffolds exhibited a full recovery of their original shape. More importantly, the PGS polymer in the β-TCP/PGS scaffolds could direct the biomineralization of Ca/P from particulate shape into a nanofiber-interweaved structure. Furthermore, the β-TCP/PGS scaffolds allowed for cell penetration and proliferation, indicating a good cytobiocompatibility. It is believed that β-TCP/PGS scaffolds have great potential application in rigid tissue regeneration. - Graphical abstract: Robust β-TCP/PGS porous scaffolds are developed by incorporation of poly(glycerol sebacate) (PGS, a flexible

  15. Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

    International Nuclear Information System (INIS)

    Prabhakaran, Molamma P; Venugopal, J; Chan, Casey K; Ramakrishna, S

    2008-01-01

    The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.

  16. Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.

    Science.gov (United States)

    Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I; Smithgall, Thomas E

    2014-10-10

    HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment*

    Science.gov (United States)

    Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I.; Smithgall, Thomas E.

    2014-01-01

    HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. PMID:25122770

  18. Design of a bioresorbable polymeric scaffold for osteoblast culture

    Science.gov (United States)

    Ditaranto, Vincent M., Jr.

    Bioresorbable polymeric scaffolds were designed for the purpose of growing rat osteosarcoma cells (ROS 17/2.8) using the compression molding method. The material used in the construction of the scaffolds was a mixture of polycaprolactone (PCL), Hydroxyapatite (HA), Glycerin (GL) and salt (NaCl) for porosity. The concentration of the several materials utilized, was determined by volume. Past research at the University of Massachusetts Lowell (UML) has successfully utilized the compression molding method for the construction of scaffolds, but was unable to accomplish the goal of long term cell survival and complete cellular proliferation throughout a three dimensional scaffold. This research investigated various concentrations of the materials and molding temperatures used for the manufacture of scaffolds in order to improve the scaffold design and address those issues. The design of the scaffold using the compression molding process is detailed in the Method and Materials section of this thesis. The porogen (salt) used for porosity was suspected as a possible source of contamination causing cell apoptosis in past studies. This research addressed the issues for cell survival and proliferation throughout a three dimensional scaffold. The leaching of the salt was one major design modification. This research successfully used ultrasonic leaching in addition to the passive method. Prior to cell culture, the scaffolds were irradiated to 2.75 Mrad, with cobalt-60 gamma radionuclide. The tissue culture consisted of two trials: (1) cell culture in scaffolds cleaned with passive leaching; (2) cell culture with scaffolds cleaned with ultrasonic leaching. Cell survival and proliferation was accomplished only with the addition of ultrasonic leaching of the scaffolds. Analysis of the scaffolds included Scanning Electron Microscopy (SEM), Nikon light microscopy and x-ray mapping of the calcium, sodium and chloride ion distribution. The cells were analyzed by Environmental Scanning

  19. Multilayer porous UHMWPE scaffolds for bone defects replacement

    International Nuclear Information System (INIS)

    Maksimkin, A.V.; Senatov, F.S.; Anisimova, N.Yu.; Kiselevskiy, M.V.; Zalepugin, D.Yu.; Chernyshova, I.V.; Tilkunova, N.A.; Kaloshkin, S.D.

    2017-01-01

    Reconstruction of the structural integrity of the damaged bone tissue is an urgent problem. UHMWPE may be potentially used for the manufacture of porous implants simulating as closely as possible the porous cancellous bone tissue. But the extremely high molecular weight of the polymer does not allow using traditional methods of foaming. Porous and multilayer UHMWPE scaffolds with nonporous bulk layer and porous layer that mimics cancellous bone architecture were obtained by solid-state mixing, thermopressing and washing in subcritical water. Structural and mechanical properties of the samples were studied. Porous UHMWPE samples were also studied in vitro and in vivo. The pores of UHMWPE scaffold are open and interconnected. Volume porosity of the obtained samples was 79 ± 2%; the pore size range was 80–700 μm. Strong connection of the two layers in multilayer UHMWPE scaffolds was observed with decreased number of fusion defects. Functionality of implants based on multilayer UHMWPE scaffolds is provided by the fixation of scaffolds in the bone defect through ingrowths of the connective tissue into the pores, which ensures the maintenance of the animals' mobility - Highlights: • Porous UHMWPE scaffold mimics cancellous bone architecture, maintaining its flexibility. • Multilayer UHMWPE scaffold is able to simulate different types of bone tissue. • Fixation of scaffolds in the bone provides through ingrowths of the connective tissue into pores. • Multilayer UHMWPE scaffolds can be used for the formation of bone implants.

  20. Multilayer porous UHMWPE scaffolds for bone defects replacement

    Energy Technology Data Exchange (ETDEWEB)

    Maksimkin, A.V. [National University of Science and Technology “MISIS”, Moscow (Russian Federation); Senatov, F.S., E-mail: senatov@misis.ru [National University of Science and Technology “MISIS”, Moscow (Russian Federation); Anisimova, N.Yu.; Kiselevskiy, M.V. [National University of Science and Technology “MISIS”, Moscow (Russian Federation); N.N. Blokhin Russian Cancer Research Center, Moscow (Russian Federation); Zalepugin, D.Yu.; Chernyshova, I.V.; Tilkunova, N.A. [State Plant of Medicinal Drugs, Moscow (Russian Federation); Kaloshkin, S.D. [National University of Science and Technology “MISIS”, Moscow (Russian Federation)

    2017-04-01

    Reconstruction of the structural integrity of the damaged bone tissue is an urgent problem. UHMWPE may be potentially used for the manufacture of porous implants simulating as closely as possible the porous cancellous bone tissue. But the extremely high molecular weight of the polymer does not allow using traditional methods of foaming. Porous and multilayer UHMWPE scaffolds with nonporous bulk layer and porous layer that mimics cancellous bone architecture were obtained by solid-state mixing, thermopressing and washing in subcritical water. Structural and mechanical properties of the samples were studied. Porous UHMWPE samples were also studied in vitro and in vivo. The pores of UHMWPE scaffold are open and interconnected. Volume porosity of the obtained samples was 79 ± 2%; the pore size range was 80–700 μm. Strong connection of the two layers in multilayer UHMWPE scaffolds was observed with decreased number of fusion defects. Functionality of implants based on multilayer UHMWPE scaffolds is provided by the fixation of scaffolds in the bone defect through ingrowths of the connective tissue into the pores, which ensures the maintenance of the animals' mobility - Highlights: • Porous UHMWPE scaffold mimics cancellous bone architecture, maintaining its flexibility. • Multilayer UHMWPE scaffold is able to simulate different types of bone tissue. • Fixation of scaffolds in the bone provides through ingrowths of the connective tissue into pores. • Multilayer UHMWPE scaffolds can be used for the formation of bone implants.

  1. Highly charged cyanine fluorophores for trafficking scaffold degradation

    International Nuclear Information System (INIS)

    Owens, Eric A; Alyabyev, Sergey; Henary, Maged; Hyun, Hoon; Kim, Soon Hee; Lee, Jeong Heon; Park, GwangLi; Ashitate, Yoshitomo; Choi, Jungmun; Hong, Gloria H; Choi, Hak Soo; Lee, Sang Jin; Khang, Gilson

    2013-01-01

    Biodegradable scaffolds have been extensively used in the field of tissue engineering and regenerative medicine. However, noninvasive monitoring of in vivo scaffold degradation is still lacking. In order to develop a real-time trafficking technique, a series of meso-brominated near-infrared (NIR) fluorophores were synthesized and conjugated to biodegradable gelatin scaffolds. Since the pentamethine cyanine core is highly lipophilic, the side chain of each fluorophore was modified with either quaternary ammonium salts or sulfonate groups. The physicochemical properties such as lipophilicity and net charge of fluorophores played a key role in the fate of NIR-conjugated scaffolds in vivo after biodegradation. The positively charged fluorophore-conjugated scaffold fragments were found in salivary glands, lymph nodes, and most of the hepatobiliary excretion route. However, halogenated fluorophores intensively accumulated into lymph nodes and the liver. Interestingly, balanced-charged gelatin scaffolds were degraded into urine in a short period of time. These results demonstrate that the noninvasive optical imaging using NIR fluorophores can be useful for the translation of biodegradable scaffolds into the clinic. (paper)

  2. Bioactive gyroid scaffolds formed by sacrificial templating of nanocellulose and nanochitin hydrogels as instructive platforms for biomimetic tissue engineering.

    Science.gov (United States)

    Torres-Rendon, Jose Guillermo; Femmer, Tim; De Laporte, Laura; Tigges, Thomas; Rahimi, Khosrow; Gremse, Felix; Zafarnia, Sara; Lederle, Wiltrud; Ifuku, Shinsuke; Wessling, Matthias; Hardy, John G; Walther, Andreas

    2015-05-20

    A sacrificial templating process using lithographically printed minimal surface structures allows complex de novo geo-metries of delicate hydrogel materials. The hydrogel scaffolds based on cellulose and chitin nanofibrils show differences in terms of attachment of human mesenchymal stem cells, and allow their differentiation into osteogenic outcomes. The approach here serves as a first example toward designer hydrogel scaffolds viable for biomimetic tissue engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size.

    Science.gov (United States)

    Scaffaro, Roberto; Lopresti, Francesco; Botta, Luigi; Maio, Andrea; Sutera, Fiorenza; Mistretta, Maria Chiara; La Mantia, Francesco Paolo

    2016-10-17

    Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 °C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

  4. Teenaged Internet Tutors' Use of Scaffolding with Older Learners

    Science.gov (United States)

    Tambaum, Tiina

    2017-01-01

    This study analyses how teenaged instructors paired with older learners make use of scaffolding. Video data were categorised according to 15 types of direct scaffolding tactics, indirect scaffolding, and unused scaffolding opportunities. The results show that a teenager who is unprepared for the role of an instructor of Internet skills for older…

  5. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States); Busenlehner, Laura [Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487 (United States); Marcus, Stevan, E-mail: smarcus@bama.ua.edu [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States)

    2010-08-20

    Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  6. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

    2010-01-01

    Research highlights: → Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. → The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. → Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. → PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  7. Scaffold hopping in drug discovery using inductive logic programming.

    Science.gov (United States)

    Tsunoyama, Kazuhisa; Amini, Ata; Sternberg, Michael J E; Muggleton, Stephen H

    2008-05-01

    In chemoinformatics, searching for compounds which are structurally diverse and share a biological activity is called scaffold hopping. Scaffold hopping is important since it can be used to obtain alternative structures when the compound under development has unexpected side-effects. Pharmaceutical companies use scaffold hopping when they wish to circumvent prior patents for targets of interest. We propose a new method for scaffold hopping using inductive logic programming (ILP). ILP uses the observed spatial relationships between pharmacophore types in pretested active and inactive compounds and learns human-readable rules describing the diverse structures of active compounds. The ILP-based scaffold hopping method is compared to two previous algorithms (chemically advanced template search, CATS, and CATS3D) on 10 data sets with diverse scaffolds. The comparison shows that the ILP-based method is significantly better than random selection while the other two algorithms are not. In addition, the ILP-based method retrieves new active scaffolds which were not found by CATS and CATS3D. The results show that the ILP-based method is at least as good as the other methods in this study. ILP produces human-readable rules, which makes it possible to identify the three-dimensional features that lead to scaffold hopping. A minor variant of a rule learnt by ILP for scaffold hopping was subsequently found to cover an inhibitor identified by an independent study. This provides a successful result in a blind trial of the effectiveness of ILP to generate rules for scaffold hopping. We conclude that ILP provides a valuable new approach for scaffold hopping.

  8. Development of a novel collagen-GAG nanofibrous scaffold via electrospinning

    Energy Technology Data Exchange (ETDEWEB)

    Zhong Shaoping [Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Teo, Wee Eong [Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Zhu Xiao [Singapore Eye Research Institute, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751 (Singapore); Beuerman, Roger [Singapore Eye Research Institute, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751 (Singapore); Ramakrishna, Seeram [Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Yung, Lin Yue Lanry [Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore)]. E-mail: cheyly@nus.edu.sg

    2007-03-15

    Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen-GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen-GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen-GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen-GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.

  9. Development of a novel collagen-GAG nanofibrous scaffold via electrospinning

    International Nuclear Information System (INIS)

    Zhong Shaoping; Teo, Wee Eong; Zhu Xiao; Beuerman, Roger; Ramakrishna, Seeram; Yung, Lin Yue Lanry

    2007-01-01

    Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen-GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen-GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen-GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen-GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds

  10. Scaffolding With and Through Videos

    DEFF Research Database (Denmark)

    Otrel-Cass, Kathrin; Khoo, Elaine; Cowie, Bronwen

    2012-01-01

    In New Zealand and internationally claims are being made about the potential for information and communication technologies (ICTs) to transform teaching and learning. However, the theoretical underpinnings explaining the complex interplay between the content, pedagogy and technology a teacher needs...... to scaffold learning. It showcases the intricate interplay between teachers’ knowledge about content, digital video technology, and students’ learning needs based on a qualitative study of two science teachers and their students in a New Zealand primary school....... to consider must be expanded. This article explicates theoretical and practical ideas related to teachers’ application of their ICT technology, pedagogy, and content knowledge (TPACK) in science. The article unpacks the social and technological dimensions of teachers’ use of TPACK when they use digital videos...

  11. Strategies for osteochondral repair: Focus on scaffolds

    Directory of Open Access Journals (Sweden)

    Seog-Jin Seo

    2014-07-01

    Full Text Available Interest in osteochondral repair has been increasing with the growing number of sports-related injuries, accident traumas, and congenital diseases and disorders. Although therapeutic interventions are entering an advanced stage, current surgical procedures are still in their infancy. Unlike other tissues, the osteochondral zone shows a high level of gradient and interfacial tissue organization between bone and cartilage, and thus has unique characteristics related to the ability to resist mechanical compression and restoration. Among the possible therapies, tissue engineering of osteochondral tissues has shown considerable promise where multiple approaches of utilizing cells, scaffolds, and signaling molecules have been pursued. This review focuses particularly on the importance of scaffold design and its role in the success of osteochondral tissue engineering. Biphasic and gradient composition with proper pore configurations are the basic design consideration for scaffolds. Surface modification is an essential technique to improve the scaffold function associated with cell regulation or delivery of signaling molecules. The use of functional scaffolds with a controllable delivery strategy of multiple signaling molecules is also considered a promising therapeutic approach. In this review, we updated the recent advances in scaffolding approaches for osteochondral tissue engineering.

  12. A review: fabrication of porous polyurethane scaffolds.

    Science.gov (United States)

    Janik, H; Marzec, M

    2015-03-01

    The aim of tissue engineering is the fabrication of three-dimensional scaffolds that can be used for the reconstruction and regeneration of damaged or deformed tissues and organs. A wide variety of techniques have been developed to create either fibrous or porous scaffolds from polymers, metals, composite materials and ceramics. However, the most promising materials are biodegradable polymers due to their comprehensive mechanical properties, ability to control the rate of degradation and similarities to natural tissue structures. Polyurethanes (PUs) are attractive candidates for scaffold fabrication, since they are biocompatible, and have excellent mechanical properties and mechanical flexibility. PU can be applied to various methods of porous scaffold fabrication, among which are solvent casting/particulate leaching, thermally induced phase separation, gas foaming, emulsion freeze-drying and melt moulding. Scaffold properties obtained by these techniques, including pore size, interconnectivity and total porosity, all depend on the thermal processing parameters, and the porogen agent and solvents used. In this review, various polyurethane systems for scaffolds are discussed, as well as methods of fabrication, including the latest developments, and their advantages and disadvantages. Copyright © 2014. Published by Elsevier B.V.

  13. Scaffold library for tissue engineering: a geometric evaluation.

    Science.gov (United States)

    Chantarapanich, Nattapon; Puttawibul, Puttisak; Sucharitpwatskul, Sedthawatt; Jeamwatthanachai, Pongnarin; Inglam, Samroeng; Sitthiseripratip, Kriskrai

    2012-01-01

    Tissue engineering scaffold is a biological substitute that aims to restore, to maintain, or to improve tissue functions. Currently available manufacturing technology, that is, additive manufacturing is essentially applied to fabricate the scaffold according to the predefined computer aided design (CAD) model. To develop scaffold CAD libraries, the polyhedrons could be used in the scaffold libraries development. In this present study, one hundred and nineteen polyhedron models were evaluated according to the established criteria. The proposed criteria included considerations on geometry, manufacturing feasibility, and mechanical strength of these polyhedrons. CAD and finite element (FE) method were employed as tools in evaluation. The result of evaluation revealed that the close-cellular scaffold included truncated octahedron, rhombicuboctahedron, and rhombitruncated cuboctahedron. In addition, the suitable polyhedrons for using as open-cellular scaffold libraries included hexahedron, truncated octahedron, truncated hexahedron, cuboctahedron, rhombicuboctahedron, and rhombitruncated cuboctahedron. However, not all pore size to beam thickness ratios (PO:BT) were good for making the open-cellular scaffold. The PO:BT ratio of each library, generating the enclosed pore inside the scaffold, was excluded to avoid the impossibility of material removal after the fabrication. The close-cellular libraries presented the constant porosity which is irrespective to the different pore sizes. The relationship between PO:BT ratio and porosity of open-cellular scaffold libraries was displayed in the form of Logistic Power function. The possibility of merging two different types of libraries to produce the composite structure was geometrically evaluated in terms of the intersection index and was mechanically evaluated by means of FE analysis to observe the stress level. The couples of polyhedrons presenting low intersection index and high stress level were excluded. Good couples for

  14. Scaffold Library for Tissue Engineering: A Geometric Evaluation

    Directory of Open Access Journals (Sweden)

    Nattapon Chantarapanich

    2012-01-01

    Full Text Available Tissue engineering scaffold is a biological substitute that aims to restore, to maintain, or to improve tissue functions. Currently available manufacturing technology, that is, additive manufacturing is essentially applied to fabricate the scaffold according to the predefined computer aided design (CAD model. To develop scaffold CAD libraries, the polyhedrons could be used in the scaffold libraries development. In this present study, one hundred and nineteen polyhedron models were evaluated according to the established criteria. The proposed criteria included considerations on geometry, manufacturing feasibility, and mechanical strength of these polyhedrons. CAD and finite element (FE method were employed as tools in evaluation. The result of evaluation revealed that the close-cellular scaffold included truncated octahedron, rhombicuboctahedron, and rhombitruncated cuboctahedron. In addition, the suitable polyhedrons for using as open-cellular scaffold libraries included hexahedron, truncated octahedron, truncated hexahedron, cuboctahedron, rhombicuboctahedron, and rhombitruncated cuboctahedron. However, not all pore size to beam thickness ratios (PO : BT were good for making the open-cellular scaffold. The PO : BT ratio of each library, generating the enclosed pore inside the scaffold, was excluded to avoid the impossibility of material removal after the fabrication. The close-cellular libraries presented the constant porosity which is irrespective to the different pore sizes. The relationship between PO : BT ratio and porosity of open-cellular scaffold libraries was displayed in the form of Logistic Power function. The possibility of merging two different types of libraries to produce the composite structure was geometrically evaluated in terms of the intersection index and was mechanically evaluated by means of FE analysis to observe the stress level. The couples of polyhedrons presenting low intersection index and high stress

  15. Embroidered polymer-collagen hybrid scaffold variants for ligament tissue engineering.

    Science.gov (United States)

    Hoyer, M; Drechsel, N; Meyer, M; Meier, C; Hinüber, C; Breier, A; Hahner, J; Heinrich, G; Rentsch, C; Garbe, L-A; Ertel, W; Schulze-Tanzil, G; Lohan, A

    2014-10-01

    Embroidery techniques and patterns used for scaffold production allow the adaption of biomechanical scaffold properties. The integration of collagen into embroidered polylactide-co-caprolactone [P(LA-CL)] and polydioxanone (PDS) scaffolds could stimulate neo-tissue formation by anterior cruciate ligament (ACL) cells. Therefore, the aim of this study was to test embroidered P(LA-CL) and PDS scaffolds as hybrid scaffolds in combination with collagen hydrogel, sponge or foam for ligament tissue engineering. ACL cells were cultured on embroidered P(LA-CL) and PDS scaffolds without or with collagen supplementation. Cell adherence, vitality, morphology and ECM synthesis were analyzed. Irrespective of thread size, ACL cells seeded on P(LA-CL) scaffolds without collagen adhered and spread over the threads, whereas the cells formed clusters on PDS and larger areas remained cell-free. Using the collagen hydrogel, the scaffold colonization was limited by the gel instability. The collagen sponge layers integrated into the scaffolds were hardly penetrated by the cells. Collagen foams increased scaffold colonization in P(LA-CL) but did not facilitate direct cell-thread contacts in the PDS scaffolds. The results suggest embroidered P(LA-CL) scaffolds as a more promising basis for tissue engineering an ACL substitute than PDS due to superior cell attachment. Supplementation with a collagen foam presents a promising functionalization strategy. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

    Science.gov (United States)

    Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

    2006-09-06

    Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

  17. Scaffolds in regenerative endodontics: A review

    Science.gov (United States)

    Gathani, Kinjal M.; Raghavendra, Srinidhi Surya

    2016-01-01

    Root canal therapy has enabled us to save numerous teeth over the years. The most desired outcome of endodontic treatment would be when diseased or nonvital pulp is replaced with healthy pulp tissue that would revitalize the teeth through regenerative endodontics. ‘A search was conducted using the Pubmed and MEDLINE databases for articles with the criteria ‘Platelet rich plasma’, ‘Platelet rich fibrin’, ‘Stem cells’, ‘Natural and artificial scaffolds’ from 1982–2015’. Tissues are organized as three-dimensional structures, and appropriate scaffolding is necessary to provide a spatially correct position of cell location and regulate differentiation, proliferation, or metabolism of the stem cells. Extracellular matrix molecules control the differentiation of stem cells, and an appropriate scaffold might selectively bind and localize cells, contain growth factors, and undergo biodegradation over time. Different scaffolds facilitate the regeneration of different tissues. To ensure a successful regenerative procedure, it is essential to have a thorough and precise knowledge about the suitable scaffold for the required tissue. This article gives a review on the different scaffolds providing an insight into the new developmental approaches on the horizon. PMID:27857762

  18. Scaffolds in regenerative endodontics: A review

    Directory of Open Access Journals (Sweden)

    Kinjal M Gathani

    2016-01-01

    Full Text Available Root canal therapy has enabled us to save numerous teeth over the years. The most desired outcome of endodontic treatment would be when diseased or nonvital pulp is replaced with healthy pulp tissue that would revitalize the teeth through regenerative endodontics. ′A search was conducted using the Pubmed and MEDLINE databases for articles with the criteria ′Platelet rich plasma′, ′Platelet rich fibrin′, ′Stem cells′, ′Natural and artificial scaffolds′ from 1982-2015′. Tissues are organized as three-dimensional structures, and appropriate scaffolding is necessary to provide a spatially correct position of cell location and regulate differentiation, proliferation, or metabolism of the stem cells. Extracellular matrix molecules control the differentiation of stem cells, and an appropriate scaffold might selectively bind and localize cells, contain growth factors, and undergo biodegradation over time. Different scaffolds facilitate the regeneration of different tissues. To ensure a successful regenerative procedure, it is essential to have a thorough and precise knowledge about the suitable scaffold for the required tissue. This article gives a review on the different scaffolds providing an insight into the new developmental approaches on the horizon.

  19. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

    DEFF Research Database (Denmark)

    Serafimova, Iana M; Pufall, Miles A; Krishnan, Shyam

    2012-01-01

    Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we...... of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted...

  20. Effects of scaffold surface morphology on cell adhesion and survival rate in vitreous cryopreservation of tenocyte-scaffold constructs

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhi [State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu 610041 (China); Department of Bone and Joint Surgery, The affiliated hospital of Luzhou Medical College, Luzhou 646000 (China); Qing, Quan [Sichuan College of Traditional Chinese Medicine, Mianyang 621000 (China); Regenerative Medicine Research Center, West China Hospital of Sichuan University, Chengdu 610041 (China); Chen, Xi; Liu, Cheng-Jun; Luo, Jing-Cong [State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu 610041 (China); Hu, Jin-Lian [Institute of Textiles and Clothing, The Hong Kong Polytechnic University, Hong Kong (China); Qin, Ting-Wu, E-mail: tingwuqin@hotmail.com [State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu 610041 (China)

    2016-12-01

    Highlights: • The shapes of tenocytes varied when seeded on different surface of scaffolds. • Tenocytes were flat on smooth surface and spindle on micro-grooved surface. • Tenocytes were ellipse or spindle on porous surface. • Tenocytes got varying adhesion shape and elongation index on varying surfaces. • The tenocyte survival on porous surface was superior to the other two groups. - Abstract: The purpose of this study was to investigate the effects of scaffold surface morphology on cell adhesion and survival rate in vitreous cryopreservation of tenocyte-scaffold constructs. Tenocytes were obtained from tail tendons of rats. Polydimethylsiloxane (PDMS) was used to fabricate three types of scaffolds with varying surface morphological characteristics, i.e., smooth, micro-grooved, and porous surfaces, respectively. The tenocytes were seeded on the surfaces of the scaffolds to form tenocyte-scaffold constructs. The constructs were cryopreserved in a vitreous cryoprotectant (CPA) with a multi-step protocol. The cell adhesion to scaffolds was observed with electronic scanning microscopy (SEM). The elongation index of the living tenocytes and ratio of live/dead cell number were examined based on a live/dead dual fluorescent staining technique, and the survival rate of tenocytes was studied with flow cytometry (FC). The results showed the shapes of tenocytes varied between the different groups: flat or polygonal (on smooth surface), spindle (on micro-grooved surface), and spindle or ellipse (on porous surface). After thawing, the porous surface got the most living tenocytes and a higher survival rate, suggesting its potential application for vitreous cryopreservation of engineered tendon constructs.

  1. Synergistic Effect of Carbon Nanotubes and Graphene on Diopside Scaffolds.

    Science.gov (United States)

    Liu, Tingting; Wu, Ping; Gao, Chengde; Feng, Pei; Xiao, Tao; Deng, Youwen; Shuai, Cijun; Peng, Shuping

    2016-01-01

    A synergetic effect between carbon nanotubes (CNTs) and graphene on diopside (Di) scaffolds was demonstrated. 3D network architecture in the matrix was formed through the 1D CNTs inlaid among the 2D graphene platelets (GNPs). The mechanical properties of the CNTs/GNPs/Di scaffolds were significantly improved compared with the CNTs/Di scaffolds and GNPs/Di scaffolds. In addition, the scaffolds exhibited excellent apatite-forming ability, a modest degradation rate, and stable mechanical properties in simulated body fluid (SBF). Moreover, cell culturing tests indicated that the scaffolds supported the cells attachment and proliferation. Taken together, the CNTs/GNPs/Di scaffolds offered great potential for bone tissue engineering.

  2. The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

    Science.gov (United States)

    Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

    2017-12-15

    Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

  3. Structural Changes of Creatine Kinase upon Substrate Binding

    OpenAIRE

    Forstner, Michael; Kriechbaum, Manfred; Laggner, Peter; Wallimann, Theo

    1998-01-01

    Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzym...

  4. Fabrication and Mechanical Characterization of Hydrogel Infused Network Silk Scaffolds

    Directory of Open Access Journals (Sweden)

    Lakshminath Kundanati

    2016-09-01

    Full Text Available Development and characterization of porous scaffolds for tissue engineering and regenerative medicine is of great importance. In recent times, silk scaffolds were developed and successfully tested in tissue engineering and drug release applications. We developed a novel composite scaffold by mechanical infusion of silk hydrogel matrix into a highly porous network silk scaffold. The mechanical behaviour of these scaffolds was thoroughly examined for their possible use in load bearing applications. Firstly, unconfined compression experiments show that the denser composite scaffolds displayed significant enhancement in the elastic modulus as compared to either of the components. This effect was examined and further explained with the help of foam mechanics principles. Secondly, results from confined compression experiments that resemble loading of cartilage in confinement, showed nonlinear material responses for all scaffolds. Finally, the confined creep experiments were performed to calculate the hydraulic permeability of the scaffolds using soil mechanics principles. Our results show that composite scaffolds with some modifications can be a potential candidate for use of cartilage like applications. We hope such approaches help in developing novel scaffolds for tissue engineering by providing an understanding of the mechanics and can further be used to develop graded scaffolds by targeted infusion in specific regions.

  5. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    International Nuclear Information System (INIS)

    Chhabra, Hemlata; Gupta, Priyanka; Verma, Paul J.; Jadhav, Sameer; Bellare, Jayesh R.

    2014-01-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold

  6. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  7. β-Tricalcium phosphate/poly(glycerol sebacate) scaffolds with robust mechanical property for bone tissue engineering.

    Science.gov (United States)

    Yang, Kai; Zhang, Jing; Ma, Xiaoyu; Ma, Yifan; Kan, Chao; Ma, Haiyan; Li, Yulin; Yuan, Yuan; Liu, Changsheng

    2015-11-01

    Despite good biocompatibility and osteoconductivity, porous β-TCP scaffolds still lack the structural stability and mechanical robustness, which greatly limit their application in the field of bone regeneration. The hybridization of β-TCP with conventional synthetic biodegradable PLA and PCL only produced a limited toughening effect due to the plasticity of the polymers in nature. In this study, a β-TCP/poly(glycerol sebacate) scaffold (β-TCP/PGS) with well interconnected porous structure and robust mechanical property was prepared. Porous β-TCP scaffold was first prepared with polyurethane sponge as template and then impregnated into PGS pre-polymer solution with moderate viscosity, followed by in situ heat crosslinking and freezing-drying process. The results indicated that the freezing-drying under vacuum process could further facilitate crosslinking of PGS and formation of Ca(2+)-COO(-) ionic complexing and thus synergistically improved the mechanical strength of the β-TCP/PGS with in situ heat crosslinking. Particularly, the β-TCP/PGS with 15% PGS content after heat crosslinking at 130°C and freezing-drying at -50°C under vacuum exhibited an elongation at break of 375±25% and a compressive strength of 1.73MPa, 3.7-fold and 200-fold enhancement compared to the β-TCP, respectively. After the abrupt drop of compressive load, the β-TCP/PGS scaffolds exhibited a full recovery of their original shape. More importantly, the PGS polymer in the β-TCP/PGS scaffolds could direct the biomineralization of Ca/P from particulate shape into a nanofiber-interweaved structure. Furthermore, the β-TCP/PGS scaffolds allowed for cell penetration and proliferation, indicating a good cytobiocompatibility. It is believed that β-TCP/PGS scaffolds have great potential application in rigid tissue regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. ASTM International Workshop on Standards & Measurements for Tissue Engineering Scaffolds

    Science.gov (United States)

    Simon, Carl G.; Yaszemski, Michael J.; Ratcliffe, Anthony; Tomlins, Paul; Luginbuehl, Reto; Tesk, John A.

    2016-01-01

    The “Workshop on Standards & Measurements for Tissue Engineering Scaffolds” was held on May 21, 2013 in Indianapolis, IN and was sponsored by the ASTM International (ASTM). The purpose of the workshop was to identify the highest priority items for future standards work for scaffolds used in the development and manufacture of tissue engineered medical products (TEMPs). Eighteen speakers and 78 attendees met to assess current scaffold standards and to prioritize needs for future standards. A key finding was that the ASTM TEMPs subcommittees (F04.41-46) have many active “guide” documents for educational purposes, but that few standard “test methods” or “practices” have been published. Overwhelmingly, the most clearly identified need was standards for measuring the structure of scaffolds, followed by standards for biological characterization, including in vitro testing, animal models and cell-material interactions. The third most pressing need was to develop standards for assessing the mechanical properties of scaffolds. Additional needs included standards for assessing scaffold degradation, clinical outcomes with scaffolds, effects of sterilization on scaffolds, scaffold composition and drug release from scaffolds. Discussions also highlighted the need for additional scaffold reference materials and the need to use them for measurement traceability. Finally, dialogue emphasized the needs to promote the use of standards in scaffold fabrication, characterization, and commercialization and to assess the use and impact of standards in the TEMPs community. Many scaffold standard needs have been identified and focus should now turn to generating these standards to support the use of scaffolds in TEMPs. PMID:25220952

  9. Soy Protein Scaffold Biomaterials for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Chien, Karen B.

    Developing functional biomaterials using highly processable materials with tailorable physical and bioactive properties is an ongoing challenge in tissue engineering. Soy protein is an abundant, natural resource with potential use for regenerative medicine applications. Preliminary studies show that soy protein can be physically modified and fabricated into various biocompatible constructs. However, optimized soy protein structures for tissue regeneration (i.e. 3D porous scaffolds) have not yet been designed. Furthermore, little work has established the in vivo biocompatibility of implanted soy protein and the benefit of using soy over other proteins including FDA-approved bovine collagen. In this work, freeze-drying and 3D printing fabrication processes were developed using commercially available soy protein to create porous scaffolds that improve cell growth and infiltration compared to other soy biomaterials previously reported. Characterization of scaffold structure, porosity, and mechanical/degradation properties was performed. In addition, the behavior of human mesenchymal stem cells seeded on various designed soy scaffolds was analyzed. Biological characterization of the cell-seeded scaffolds was performed to assess feasibility for use in liver tissue regeneration. The acute and humoral response of soy scaffolds implanted in an in vivo mouse subcutaneous model was also investigated. All fabricated soy scaffolds were modified using thermal, chemical, and enzymatic crosslinking to change properties and cell growth behavior. 3D printing allowed for control of scaffold pore size and geometry. Scaffold structure, porosity, and degradation rate significantly altered the in vivo response. Freeze-dried soy scaffolds had similar biocompatibility as freeze-dried collagen scaffolds of the same protein content. However, the soy scaffolds degraded at a much faster rate, minimizing immunogenicity. Interestingly, subcutaneously implanted soy scaffolds affected blood

  10. Cell-matrix mechanical interaction in electrospun polymeric scaffolds for tissue engineering: Implications for scaffold design and performance.

    Science.gov (United States)

    Kennedy, Kelsey M; Bhaw-Luximon, Archana; Jhurry, Dhanjay

    2017-03-01

    Engineered scaffolds produced by electrospinning of biodegradable polymers offer a 3D, nanofibrous environment with controllable structural, chemical, and mechanical properties that mimic the extracellular matrix of native tissues and have shown promise for a number of tissue engineering applications. The microscale mechanical interactions between cells and electrospun matrices drive cell behaviors including migration and differentiation that are critical to promote tissue regeneration. Recent developments in understanding these mechanical interactions in electrospun environments are reviewed, with emphasis on how fiber geometry and polymer structure impact on the local mechanical properties of scaffolds, how altering the micromechanics cues cell behaviors, and how, in turn, cellular and extrinsic forces exerted on the matrix mechanically remodel an electrospun scaffold throughout tissue development. Techniques used to measure and visualize these mechanical interactions are described. We provide a critical outlook on technological gaps that must be overcome to advance the ability to design, assess, and manipulate the mechanical environment in electrospun scaffolds toward constructs that may be successfully applied in tissue engineering and regenerative medicine. Tissue engineering requires design of scaffolds that interact with cells to promote tissue development. Electrospinning is a promising technique for fabricating fibrous, biomimetic scaffolds. Effects of electrospun matrix microstructure and biochemical properties on cell behavior have been extensively reviewed previously; here, we consider cell-matrix interaction from a mechanical perspective. Micromechanical properties as a driver of cell behavior has been well established in planar substrates, but more recently, many studies have provided new insights into mechanical interaction in fibrillar, electrospun environments. This review provides readers with an overview of how electrospun scaffold mechanics and

  11. Nano/macro porous bioactive glass scaffold

    Science.gov (United States)

    Wang, Shaojie

    Bioactive glass (BG) and ceramics have been widely studied and developed as implants to replace hard tissues of the musculo-skeletal system, such as bones and teeth. Recently, instead of using bulk materials, which usually do not degrade rapidly enough and may remain in the human body for a long time, the idea of bioscaffold for tissue regeneration has generated much interest. An ideal bioscaffold is a porous material that would not only provide a three-dimensional structure for the regeneration of natural tissue, but also degrade gradually and, eventually be replaced by the natural tissue completely. Among various material choices the nano-macro dual porous BG appears as the most promising candidate for bioscaffold applications. Here macropores facilitate tissue growth while nanopores control degradation and enhance cell response. The surface area, which controls the degradation of scaffold can also be tuned by changing the nanopore size. However, fabrication of such 3D structure with desirable nano and macro pores has remained challenging. In this dissertation, sol-gel process combined with spinodal decomposition or polymer sponge replication method has been developed to fabricate the nano-macro porous BG scaffolds. Macropores up to 100microm are created by freezing polymer induced spinodal structure through sol-gel transition, while larger macropores (>200um) of predetermined size are obtained by the polymer sponge replication technique. The size of nanopores, which are inherent to the sol-gel method of glass fabrication, has been tailored using several approaches: Before gel point, small nanopores are generated using acid catalyst that leads to weakly-branched polymer-like network. On the other hand, larger nanopores are created with the base-catalyzed gel with highly-branched cluster-like structure. After the gel point, the nanostructure can be further modified by manipulating the sintering temperature and/or the ammonia concentration used in the solvent

  12. Fatigue behaviour of NiTi shape memory alloy scaffolds produced by SLM, a unit cell design comparison.

    Science.gov (United States)

    Speirs, M; Van Hooreweder, B; Van Humbeeck, J; Kruth, J-P

    2017-06-01

    Selective laser melting (SLM) is an additive manufacturing technique able to produce complex functional parts via successively melting layers of metal powder. This process grants the freedom to design highly complex scaffold components to allow bone ingrowth and aid mechanical anchorage. This paper investigates the compression fatigue behaviour of three different unit cells (octahedron, cellular gyroid and sheet gyroid) of SLM nitinol scaffolds. It was found that triply periodic minimal surfaces display superior static mechanical properties in comparison to conventional octahedron beam lattice structures at identical volume fractions. Fatigue resistance was also found to be highly geometry dependent due to the effects of AM processing techniques on the surface topography and notch sensitivity. Geometries minimising nodal points and the staircase effect displayed the greatest fatigue resistance when normalized to yield strength. Furthermore oxygen analysis showed a large oxygen uptake during SLM processing which must be altered to meet ASTM medical grade standards and may significantly reduce fatigue life. These achieved fatigue properties indicate that NiTi scaffolds produced via SLM can provide sufficient mechanical support over an implants lifetime within stress range values experienced in real life. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Binding of influenza A virus NS1 protein to the inter-SH2 domain of p85 suggests a novel mechanism for phosphoinositide 3-kinase activation.

    Science.gov (United States)

    Hale, Benjamin G; Batty, Ian H; Downes, C Peter; Randall, Richard E

    2008-01-18

    Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85beta regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85beta. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85beta is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85beta and NS1. Equivalent residues in p85alpha have previously been implicated in the basal inhibition of p110. However, such p85alpha residues were unable to substitute for those in p85beta with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85beta inter-SH2 domain.

  14. Exact approaches for scaffolding

    OpenAIRE

    Weller, Mathias; Chateau, Annie; Giroudeau, Rodolphe

    2015-01-01

    This paper presents new structural and algorithmic results around the scaffolding problem, which occurs prominently in next generation sequencing. The problem can be formalized as an optimization problem on a special graph, the "scaffold graph". We prove that the problem is polynomial if this graph is a tree by providing a dynamic programming algorithm for this case. This algorithm serves as a basis to deduce an exact algorithm for general graphs using a tree decomposition of the input. We ex...

  15. Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor.

    Science.gov (United States)

    Anandhakumar, Jayamani; Moustafa, Yara W; Chowdhary, Surabhi; Kainth, Amoldeep S; Gross, David S

    2016-07-15

    Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.; Kwezi, Lusisizwe; Wheeler, Janet I.; Gehring, Christoph A

    2012-01-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  17. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  18. Mechanical anisotropy of titanium scaffolds

    Directory of Open Access Journals (Sweden)

    Rüegg Jasmine

    2017-09-01

    Full Text Available The clinical performance of an implant, e.g. for the treatment of large bone defects, depends on the implant material, anchorage, surface topography and chemistry, but also on the mechanical properties, like the stiffness. The latter can be adapted by the porosity. Whereas foams show isotropic mechanical properties, digitally modelled scaffolds can be designed with anisotropic behaviour. In this study, we designed and produced 3D scaffolds based on an orthogonal architecture and studied its angle-dependent stiffness. The aim was to produce scaffolds with different orientations of the microarchitecture by selective laser melting and compare the angle-specific mechanical behaviour with an in-silico simulation. The anisotropic characteristics of open-porous implants and technical limitations of the production process were studied.

  19. HA/nylon 6,6 porous scaffolds fabricated by salt-leaching/solvent casting technique: effect of nano-sized filler content on scaffold properties

    Directory of Open Access Journals (Sweden)

    Mehrabanian M

    2011-08-01

    Full Text Available Mehran Mehrabanian1, Mojtaba Nasr-Esfahani21Member of Young Researchers Club, Najafabad Branch, Islamic Azad University, Isfahan, Iran; 2Department of Materials Science and Engineering, Najafabad Branch, Islamic Azad University, Isfahan, IranAbstract: Nanohydroxyapatite (n-HA/nylon 6,6 composite scaffolds were produced by means of the salt-leaching/solvent casting technique. NaCl with a distinct range size was used with the aim of optimizing the pore network. Composite powders with different n-HA contents (40%, 60% for scaffold fabrication were synthesized and tested. The composite scaffolds thus obtained were characterized for their microstructure, mechanical stability and strength, and bioactivity. The microstructure of the composite scaffolds possessed a well-developed interconnected porosity with approximate optimal pore size ranging from 200 to 500 µm, ideal for bone regeneration and vascularization. The mechanical properties of the composite scaffolds were evaluated by compressive strength and modulus tests, and the results confirmed their similarity to cortical bone. To characterize bioactivity, the composite scaffolds were immersed in simulated body fluid for different lengths of time and results monitored by scanning electron microscopy and energy dispersive X-ray microanalysis to determine formation of an apatite layer on the scaffold surface.Keywords: scaffold, nanohydroxyapatite, nylon 6,6, salt-leaching/solvent casting, bioactivity

  20. Nerve regeneration using tubular scaffolds from biodegradable polyurethane.

    Science.gov (United States)

    Hausner, T; Schmidhammer, R; Zandieh, S; Hopf, R; Schultz, A; Gogolewski, S; Hertz, H; Redl, H

    2007-01-01

    In severe nerve lesion, nerve defects and in brachial plexus reconstruction, autologous nerve grafting is the golden standard. Although, nerve grafting technique is the best available approach a major disadvantages exists: there is a limited source of autologous nerve grafts. This study presents data on the use of tubular scaffolds with uniaxial pore orientation from experimental biodegradable polyurethanes coated with fibrin sealant to regenerate a 8 mm resected segment of rat sciatic nerve. Tubular scaffolds: prepared by extrusion of the polymer solution in DMF into water coagulation bath. The polymer used for the preparation of tubular scaffolds was a biodegradable polyurethane based on hexamethylene diisocyanate, poly(epsilon-caprolactone) and dianhydro-D-sorbitol. EXPERIMENTAL MODEL: Eighteen Sprague Dawley rats underwent mid-thigh sciatic nerve transection and were randomly assigned to two experimental groups with immediate repair: (1) tubular scaffold, (2) 180 degrees rotated sciatic nerve segment (control). Serial functional measurements (toe spread test, placing tests) were performed weekly from 3rd to 12th week after nerve repair. On week 12, electrophysiological assessment was performed. Sciatic nerve and scaffold/nerve grafts were harvested for histomorphometric analysis. Collagenic connective tissue, Schwann cells and axons were evaluated in the proximal nerve stump, the scaffold/nerve graft and the distal nerve stump. The implants have uniaxially-oriented pore structure with a pore size in the range of 2 micorm (the pore wall) and 75 x 700 microm (elongated pores in the implant lumen). The skin of the tubular implants was nonporous. Animals which underwent repair with tubular scaffolds of biodegradable polyurethanes coated with diluted fibrin sealant had no significant functional differences compared with the nerve graft group. Control group resulted in a trend-wise better electrophysiological recovery but did not show statistically significant

  1. Porous 45S5 Bioglass®-based scaffolds using stereolithography: Effect of partial pre-sintering on structural and mechanical properties of scaffolds.

    Science.gov (United States)

    Thavornyutikarn, Boonlom; Tesavibul, Passakorn; Sitthiseripratip, Kriskrai; Chatarapanich, Nattapon; Feltis, Bryce; Wright, Paul F A; Turney, Terence W

    2017-06-01

    Scaffolds made from 45S5 Bioglass® ceramic (BG) show clinical potential in bone regeneration due to their excellent bioactivity and ability to bond to natural bone tissue. However, porous BG scaffolds are limited by their mechanical integrity and by the substantial volume contractions occurring upon sintering. This study examines stereolithographic (SLA) methods to fabricate mechanically robust and porous Bioglass®-based ceramic scaffolds, with regular and interconnected pore networks and using various computer-aided design architectures. It was found that a diamond-like (DM) architecture gave scaffolds the most controllable results without any observable closed porosity in the fired scaffolds. When the pore dimensions of the DM scaffolds of the same porosity (~60vol%) were decreased from 700 to 400μm, the compressive strength values increased from 3.5 to 6.7MPa. In addition, smaller dimensional shrinkage could be obtained by employing partially pre-sintered bioglass, compared to standard 45S5 Bioglass®. Scaffolds derived from pre-sintered bioglass also showed marginally improved compressive strength. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Scaffolds for peripheral nerve repair and reconstruction.

    Science.gov (United States)

    Yi, Sheng; Xu, Lai; Gu, Xiaosong

    2018-06-02

    Trauma-associated peripheral nerve defect is a widespread clinical problem. Autologous nerve grafting, the current gold standard technique for the treatment of peripheral nerve injury, has many internal disadvantages. Emerging studies showed that tissue engineered nerve graft is an effective substitute to autologous nerves. Tissue engineered nerve graft is generally composed of neural scaffolds and incorporating cells and molecules. A variety of biomaterials have been used to construct neural scaffolds, the main component of tissue engineered nerve graft. Synthetic polymers (e.g. silicone, polyglycolic acid, and poly(lactic-co-glycolic acid)) and natural materials (e.g. chitosan, silk fibroin, and extracellular matrix components) are commonly used along or together to build neural scaffolds. Many other materials, including the extracellular matrix, glass fabrics, ceramics, and metallic materials, have also been used to construct neural scaffolds. These biomaterials are fabricated to create specific structures and surface features. Seeding supporting cells and/or incorporating neurotrophic factors to neural scaffolds further improve restoration effects. Preliminary studies demonstrate that clinical applications of these neural scaffolds achieve satisfactory functional recovery. Therefore, tissue engineered nerve graft provides a good alternative to autologous nerve graft and represents a promising frontier in neural tissue engineering. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    International Nuclear Information System (INIS)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2012-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  4. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  5. SCAFFOLDING IN CONNECTIVIST MOBILE LEARNING ENVIRONMENT

    Directory of Open Access Journals (Sweden)

    Ozlem OZAN

    2013-04-01

    Full Text Available Social networks and mobile technologies are transforming learning ecology. In this changing learning environment, we find a variety of new learner needs. The aim of this study is to investigate how to provide scaffolding to the learners in connectivist mobile learning environment: Ø to learn in a networked environment, Ø to manage their networked learning process, Ø to interact in a networked society, and Ø to use the tools belonging to the network society. The researcher described how Vygotsky's “scaffolding” concept, Berge’s “learner support” strategies, and Siemens’ “connectivism” approach can be used together to satisfy mobile learners’ needs. A connectivist mobile learning environment was designed for the research, and the research was executed as a mixed-method study. Data collection tools were Facebook wall entries, personal messages, chat records; Twitter, Diigo, blog entries; emails, mobile learning management system statistics, perceived learning survey and demographic information survey. Results showed that there were four major aspects of scaffolding in connectivist mobile learning environment as type of it, provider of it, and timing of it and strategies of it. Participants preferred mostly social scaffolding, and then preferred respectively, managerial, instructional and technical scaffolding. Social scaffolding was mostly provided by peers, and managerial scaffolding was mostly provided by instructor. Use of mobile devices increased the learner motivation and interest. Some participants stated that learning was more permanent by using mobile technologies. Social networks and mobile technologies made it easier to manage the learning process and expressed a positive impact on perceived learning.

  6. Low elastic modulus titanium–nickel scaffolds for bone implants

    International Nuclear Information System (INIS)

    Li, Jing; Yang, Hailin; Wang, Huifeng; Ruan, Jianming

    2014-01-01

    The superelastic nature of repeating the human bones is crucial to the ideal artificial biomedical implants to ensure smooth load transfer and foster the ingrowth of new bone tissues. Three dimensional interconnected porous TiNi scaffolds, which have the tailorable porous structures with micro-hole, were fabricated by slurry immersing with polymer sponge and sintering method. The crystallinity and phase composition of scaffolds were studied by X-ray diffraction. The pore morphology, size and distribution in the scaffolds were characterized by scanning electron microscopy. The porosity ranged from 65 to 72%, pore size was 250–500 μm. Compressive strength and elastic modulus of the scaffolds were ∼ 73 MPa and ∼ 3GPa respectively. The above pore structural and mechanical properties are similar to those of cancellous bone. In the initial cell culture test, osteoblasts adhered well to the scaffold surface during a short time, and then grew smoothly into the interconnected pore channels. These results indicate that the porous TiNi scaffolds fabricated by this method could be bone substitute materials. - Highlights: • A novel approach for the fabrication of porous TiNi scaffolds • Macroporous structures are replicated from the polymer sponge template. • The pore characteristics and mechanical properties of TiNi scaffolds agree well with the requirement of trabecular bone. • Cytocompatibility of TiNi scaffolds is assessed, and it closely associated with pore property

  7. Development of keratin–chitosan–gelatin composite scaffold for soft tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Kakkar, Prachi [Central Leather Research Institute (Council of Scientific and Industrial Research), Adyar, Chennai 600020 (India); Verma, Sudhanshu; Manjubala, I. [Biomedical Engineering Division, School of Bio Sciences and Technology, VIT University, Vellore 632014 (India); Madhan, B., E-mail: bmadhan76@yahoo.co.in [Central Leather Research Institute (Council of Scientific and Industrial Research), Adyar, Chennai 600020 (India)

    2014-12-01

    Keratin has gained much attention in the recent past as a biomaterial for wound healing owing to its biocompatibility, biodegradability, intrinsic biological activity and presence of cellular binding motifs. In this paper, a novel biomimetic scaffold containing keratin, chitosan and gelatin was prepared by freeze drying method. The prepared keratin composite scaffold had good structural integrity. Fourier Transform Infrared (FTIR) spectroscopy showed the retention of the native structure of individual biopolymers (keratin, chitosan, and gelatin) used in the scaffold. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) results revealed a high thermal denaturation temperature of the scaffold (200–250 °C). The keratin composite scaffold exhibited tensile strength (96 kPa), compression strength (8.5 kPa) and water uptake capacity (> 1700%) comparable to that of a collagen scaffold, which was used as control. The morphology of the keratin composite scaffold observed using a Scanning Electron Microscope (SEM) exhibited good porosity and interconnectivity of pores. MTT assay using NIH 3T3 fibroblast cells demonstrated that the cell viability of the keratin composite scaffold was good. These observations suggest that the keratin–chitosan–gelatin composite scaffold is a promising alternative biomaterial for tissue engineering applications. - Highlights: • Fabrication of novel Keratin-Chitosan-Gelatin composite scaffold • Keratin composite scaffold shows excellent water uptake capacity and porosity • Keratin composite scaffold shows good thermal and physical stability • Biocompatibility of the developed scaffold is comparable to collagen scaffolds • Developed scaffold is a promising material for soft tissue engineering applications.

  8. Design and synthesis of a cyclitol-derived scaffold with axial pyridyl appendages and its encapsulation of the silver(I cation

    Directory of Open Access Journals (Sweden)

    Pierre-Marc Léo

    2010-10-01

    Full Text Available Conversion of a myo-inositol derivative into a scyllo-inositol-derived scaffold with C3v symmetry bearing three axial pyridyl appendages is presented. This pre-organized hexadentate ligand allows complexation of silver(I. The crystal structure of the complex was established.

  9. A gelatin composite scaffold strengthened by drug-loaded halloysite nanotubes.

    Science.gov (United States)

    Ji, Lijun; Qiao, Wei; Zhang, Yuheng; Wu, Huayu; Miao, Shiyong; Cheng, Zhilin; Gong, Qianming; Liang, Ji; Zhu, Aiping

    2017-09-01

    Mechanical properties and anti-infection are two of the most concerned issues for artificial bone grafting materials. Bone regeneration porous scaffolds with sustained drug release were developed by freeze-drying the mixture of nanosized drug-loaded halloysite nanotubes (HNTs) and gelatin. The scaffolds showed porous structure and excellent biocompatibility. The mechanical properties of the obtained composite scaffolds were enhanced significantly by HNTs to >300%, comparing to those of gelatin scaffold, and match to those of natural cancellous bones. The ibuprofen-loaded HNTs incorporated in the scaffolds allowed extended drug release over 100h, comparing to 8h when directly mixed the drug into the gelatin scaffold. The biological properties of the composite scaffolds were investigated by culturing MG63 cells on them. The HNTs/gelatin scaffolds with excellent mechanical properties and sustained drug release could be a promising artificial bone grating material. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); D.C. van Gent (Dik); J.H.J. Hoeijmakers (Jan)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  11. Bioactive Nano-fibrous Scaffold for Vascularized Craniofacial Bone Regeneration

    DEFF Research Database (Denmark)

    Prabha, Rahul Damodaran; Kraft, David Christian Evar; Harkness, Linda

    2018-01-01

    the limitation of cell penetration of electrospun scaffolds and improve on its osteoconductive nature, in this study, we fabricated a novel electrospun composite scaffold of polyvinyl alcohol (PVA) - poly (ε) caprolactone (PCL) - Bioceramic (HAB), namely, PVA-PCL-HAB. The scaffold prepared by dual...... electrospinning of PVA and PCL with HAB overcomes reduced cell attachment associated with hydrophobic poly (ε) caprolactone (PCL) by combination with a hydrophilic polyvinyl alcohol (PVA) and the bioceramic (HAB) can contribute to enhance osteo-conductivity. We characterized the physicochemical...... and biocompatibility properties of the new scaffold material. Our results indicate PVA-PCL-HAB scaffolds support attachment and growth of stromal stem cells; (human bone marrow skeletal (mesenchymal) stem cells (hMSC) and dental pulp stem cells (DPSC)). In addition, the scaffold supported in vitro osteogenic...

  12. Modeling material-degradation-induced elastic property of tissue engineering scaffolds.

    Science.gov (United States)

    Bawolin, N K; Li, M G; Chen, X B; Zhang, W J

    2010-11-01

    The mechanical properties of tissue engineering scaffolds play a critical role in the success of repairing damaged tissues/organs. Determining the mechanical properties has proven to be a challenging task as these properties are not constant but depend upon time as the scaffold degrades. In this study, the modeling of the time-dependent mechanical properties of a scaffold is performed based on the concept of finite element model updating. This modeling approach contains three steps: (1) development of a finite element model for the effective mechanical properties of the scaffold, (2) parametrizing the finite element model by selecting parameters associated with the scaffold microstructure and/or material properties, which vary with scaffold degradation, and (3) identifying selected parameters as functions of time based on measurements from the tests on the scaffold mechanical properties as they degrade. To validate the developed model, scaffolds were made from the biocompatible polymer polycaprolactone (PCL) mixed with hydroxylapatite (HA) nanoparticles and their mechanical properties were examined in terms of the Young modulus. Based on the bulk degradation exhibited by the PCL/HA scaffold, the molecular weight was selected for model updating. With the identified molecular weight, the finite element model developed was effective for predicting the time-dependent mechanical properties of PCL/HA scaffolds during degradation.

  13. Chitin Scaffolds in Tissue Engineering

    Science.gov (United States)

    Jayakumar, Rangasamy; Chennazhi, Krishna Prasad; Srinivasan, Sowmya; Nair, Shantikumar V.; Furuike, Tetsuya; Tamura, Hiroshi

    2011-01-01

    Tissue engineering/regeneration is based on the hypothesis that healthy stem/progenitor cells either recruited or delivered to an injured site, can eventually regenerate lost or damaged tissue. Most of the researchers working in tissue engineering and regenerative technology attempt to create tissue replacements by culturing cells onto synthetic porous three-dimensional polymeric scaffolds, which is currently regarded as an ideal approach to enhance functional tissue regeneration by creating and maintaining channels that facilitate progenitor cell migration, proliferation and differentiation. The requirements that must be satisfied by such scaffolds include providing a space with the proper size, shape and porosity for tissue development and permitting cells from the surrounding tissue to migrate into the matrix. Recently, chitin scaffolds have been widely used in tissue engineering due to their non-toxic, biodegradable and biocompatible nature. The advantage of chitin as a tissue engineering biomaterial lies in that it can be easily processed into gel and scaffold forms for a variety of biomedical applications. Moreover, chitin has been shown to enhance some biological activities such as immunological, antibacterial, drug delivery and have been shown to promote better healing at a faster rate and exhibit greater compatibility with humans. This review provides an overview of the current status of tissue engineering/regenerative medicine research using chitin scaffolds for bone, cartilage and wound healing applications. We also outline the key challenges in this field and the most likely directions for future development and we hope that this review will be helpful to the researchers working in the field of tissue engineering and regenerative medicine. PMID:21673928

  14. Patterns of Scaffolding in Computer-Mediated Collaborative Inquiry

    Science.gov (United States)

    Lakkala, Minna; Muukkonen, Hanni; Hakkarainen, Kai

    2005-01-01

    There is wide agreement on the importance of scaffolding for student learning. Yet, models of individual and face-to-face scaffolding are not necessarily applicable to educational settings in which a group of learners is pursuing a process of inquiry mediated by technology. The scaffolding needed for such a process may be examined from three…

  15. The response of tenocytes to commercial scaffolds used for rotator cuff repair

    Directory of Open Access Journals (Sweden)

    RDJ Smith

    2017-01-01

    Full Text Available Surgical repairs of rotator cuff tears have high re-tear rates and many scaffolds have been developed to augment the repair. Understanding the interaction between patients’ cells and scaffolds is important for improving scaffold performance and tendon healing. In this in vitro study, we investigated the response of patient-derived tenocytes to eight different scaffolds. Tested scaffolds included X-Repair, Poly-Tape, LARS Ligament, BioFiber (synthetic scaffolds, BioFiber-CM (biosynthetic scaffold, GraftJacket, Permacol, and Conexa (biological scaffolds. Cell attachment, proliferation, gene expression, and morphology were assessed. After one day, more cells attached to synthetic scaffolds with dense, fine and aligned fibres (X-Repair and Poly-Tape. Despite low initial cell attachment, the human dermal scaffold (GraftJacket promoted the greatest proliferation of cells over 13 days. Expression of collagen types I and III were upregulated in cells grown on non-cross-linked porcine dermis (Conexa. Interestingly, the ratio of collagen I to collagen III mRNA was lower on all dermal scaffolds compared to synthetic and biosynthetic scaffolds. These findings demonstrate significant differences in the response of patient-derived tendon cells to scaffolds that are routinely used for rotator cuff surgery. Synthetic scaffolds promoted increased cell adhesion and a tendon-like cellular phenotype, while biological scaffolds promoted cell proliferation and expression of collagen genes. However, no single scaffold was superior. Our results may help understand the way that patients’ cells interact with scaffolds and guide the development of new scaffolds in the future.

  16. Inhibitor of PI3K/Akt Signaling Pathway Small Molecule Promotes Motor Neuron Differentiation of Human Endometrial Stem Cells Cultured on Electrospun Biocomposite Polycaprolactone/Collagen Scaffolds.

    Science.gov (United States)

    Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Yazdankhah, Meysam; Ai, Jafar; Khakbiz, Mehrdad; Faghihi, Faezeh; Tajerian, Roksana; Bayat, Neda

    2017-05-01

    Small molecules as useful chemical tools can affect cell differentiation and even change cell fate. It is demonstrated that LY294002, a small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, can inhibit proliferation and promote neuronal differentiation of mesenchymal stem cells (MSCs). The purpose of this study was to investigate the differentiation effect of Ly294002 small molecule on the human endometrial stem cells (hEnSCs) into motor neuron-like cells on polycaprolactone (PCL)/collagen scaffolds. hEnSCs were cultured in a neurogenic inductive medium containing 1 μM LY294002 on the surface of PCL/collagen electrospun fibrous scaffolds. Cell attachment and viability of cells on scaffolds were characterized by scanning electron microscope (SEM) and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The expression of neuron-specific markers was assayed by real-time PCR and immunocytochemistry analysis after 15 days post induction. Results showed that attachment and differentiation of hEnSCs into motor neuron-like cells on the scaffolds with Ly294002 small molecule were higher than that of the cells on tissue culture plates as control group. In conclusion, PCL/collagen electrospun scaffolds with Ly294002 have potential for being used in neural tissue engineering because of its bioactive and three-dimensional structure which enhances viability and differentiation of hEnSCs into neurons through inhibition of the PI3K/Akt pathway. Thus, manipulation of this pathway by small molecules can enhance neural differentiation.

  17. Biomimetic composite coating on rapid prototyped scaffolds for bone tissue engineering.

    Science.gov (United States)

    Arafat, M Tarik; Lam, Christopher X F; Ekaputra, Andrew K; Wong, Siew Yee; Li, Xu; Gibson, Ian

    2011-02-01

    The objective of this present study was to improve the functional performance of rapid prototyped scaffolds for bone tissue engineering through biomimetic composite coating. Rapid prototyped poly(ε-caprolactone)/tri-calcium phosphate (PCL/TCP) scaffolds were fabricated using the screw extrusion system (SES). The fabricated PCL/TCP scaffolds were coated with a carbonated hydroxyapatite (CHA)-gelatin composite via biomimetic co-precipitation. The structure of the prepared CHA-gelatin composite coating was studied by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. Compressive mechanical testing revealed that the coating process did not have any detrimental effect on the mechanical properties of the scaffolds. The cell-scaffold interaction was studied by culturing porcine bone marrow stromal cells (BMSCs) on the scaffolds and assessing the proliferation and bone-related gene and protein expression capabilities of the cells. Confocal laser microscopy and SEM images of the cell-scaffold constructs showed a uniformly distributed cell sheet and accumulation of extracellular matrix in the interior of CHA-gelatin composite-coated PCL/TCP scaffolds. The proliferation rate of BMSCs on CHA-gelatin composite-coated PCL/TCP scaffolds was about 2.3 and 1.7 times higher than that on PCL/TCP scaffolds and CHA-coated PCL/TCP scaffolds, respectively, by day 10. Furthermore, reverse transcription polymerase chain reaction and Western blot analysis revealed that CHA-gelatin composite-coated PCL/TCP scaffolds stimulate osteogenic differentiation of BMSCs the most, compared with PCL/TCP scaffolds and CHA-coated PCL/TCP scaffolds. These results demonstrate that CHA-gelatin composite-coated rapid prototyped PCL/TCP scaffolds are promising for bone tissue engineering. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  18. Alendronate-Eluting Biphasic Calcium Phosphate (BCP Scaffolds Stimulate Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Sung Eun Kim

    2015-01-01

    Full Text Available Biphasic calcium phosphate (BCP scaffolds have been widely used in orthopedic and dental fields as osteoconductive bone substitutes. However, BCP scaffolds are not satisfactory for the stimulation of osteogenic differentiation and maturation. To enhance osteogenic differentiation, we prepared alendronate- (ALN- eluting BCP scaffolds. The coating of ALN on BCP scaffolds was confirmed by scanning electron microscopy (FE-SEM, energy-dispersive X-ray spectroscopy (EDS, and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR. An in vitro release study showed that release of ALN from ALN-eluting BCP scaffolds was sustained for up to 28 days. In vitro results revealed that MG-63 cells grown on ALN-eluting BCP scaffolds exhibited increased ALP activity and calcium deposition and upregulated gene expression of Runx2, ALP, OCN, and OPN compared with the BCP scaffold alone. Therefore, this study suggests that ALN-eluting BCP scaffolds have the potential to effectively stimulate osteogenic differentiation.

  19. Edible Scaffolds Based on Non-Mammalian Biopolymers for Myoblast Growth

    Directory of Open Access Journals (Sweden)

    Javier Enrione

    2017-12-01

    Full Text Available In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 μm diameter and biocompatibility, permitting myoblast cell adhesion (~40% and growth (~24 h duplication time. The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.

  20. A comparison of scaffold-free and scaffold-based reconstructed human skin models as alternatives to animal use.

    Science.gov (United States)

    Kinikoglu, Beste

    2017-12-01

    Tissue engineered full-thickness human skin substitutes have various applications in the clinic and in the laboratory, such as in the treatment of burns or deep skin defects, and as reconstructed human skin models in the safety testing of drugs and cosmetics and in the fundamental study of skin biology and pathology. So far, different approaches have been proposed for the generation of reconstructed skin, each with its own advantages and disadvantages. Here, the classic tissue engineering approach, based on cell-seeded polymeric scaffolds, is compared with the less-studied cell self-assembly approach, where the cells are coaxed to synthesise their own extracellular matrix (ECM). The resulting full-thickness human skin substitutes were analysed by means of histological and immunohistochemical analyses. It was found that both the scaffold-free and the scaffold-based skin equivalents successfully mimicked the functionality and morphology of native skin, with complete epidermal differentiation (as determined by the expression of filaggrin), the presence of a continuous basement membrane expressing collagen VII, and new ECM deposition by dermal fibroblasts. On the other hand, the scaffold-free model had a thicker epidermis and a significantly higher number of Ki67-positive proliferative cells, indicating a higher capacity for self-renewal, as compared to the scaffold-based model. 2017 FRAME.

  1. Semiotic Scaffolding in Living Systems

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2008-01-01

    The apparently purposeful nature of living systems is obtained through a sophisticated network of semiotic controls whereby biochemical, physiological and behavioral processes become tuned to the needs of the system. The operation of these semiotic controls takes place and is enabled across...... a diversity of levels. Such semiotic controls may be distinguished from ordinary deterministic control mechanisms through an inbuilt anticipatory capacity based on a distinct kind of causation that I call here "semiotic causation" to denote the bringing about of changes under the guidance of interpretation...... in a local .context. Anticipation through the skilled interpretation of indicators of temporal relations in the context of a particular survival project (or life strategy) guides organismic behavior towards local ends. This network of semiotic controls establishes an enormously complex semiotic scaffolding...

  2. Scaffolding knowledge building in a Web-based communication and cultural competence program for international medical graduates.

    Science.gov (United States)

    Lax, Leila R; Russell, M Lynn; Nelles, Laura J; Smith, Cathy M

    2009-10-01

    Professional behaviors, tacitly understood by Canadian-trained physicians, are difficult to teach and often create practice barriers for IMGs. The purpose of this design research study was to develop a Web-based program simulating Canadian medical literacy and culture, and to evaluate strategies of scaffolding individual knowledge building. Study 1 (N = 20) examined usability and pedagogic design. Studies 2 (N = 39) and 3 (N = 33) examined case participation patterns. Model design was validated in Study 1. Studies 2 and 3 demonstrated high levels of participation, on unprompted third tries, on knowledge tests. Recursive patterns were strongest on Reflective Exercises. Five strategies scaffolded knowledge building: (1) video simulations, (2) contextualized resources, (3) concurrent feedback, (4) Reflective Exercises, and (5) commentaries prompting "reflection on reflection." Scaffolded design supports complex knowledge building. These findings are concurrent with educational research on the importance of recursion and revision of knowledge for improvable and relational understanding.

  3. Engineered polycaprolactone–magnesium hybrid biodegradable porous scaffold for bone tissue engineering

    Directory of Open Access Journals (Sweden)

    Hoi Man Wong

    2014-10-01

    Full Text Available In this paper, we describe the fabrication of a new biodegradable porous scaffold composed of polycaprolactone (PCL and magnesium (Mg micro-particles. The compressive modulus of PCL porous scaffold was increased to at least 150% by incorporating 29% Mg particles with the porosity of 74% using Micro-CT analysis. Surprisingly, the compressive modulus of this scaffold was further increased to at least 236% when the silane-coupled Mg particles were added. In terms of cell viability, the scaffold modified with Mg particles significantly convinced the attachment and growth of osteoblasts as compared with the pure PCL scaffold. In addition, the hybrid scaffold was able to attract the formation of apatite layer over its surface after 7 days of immersion in normal culture medium, whereas it was not observed on the pure PCL scaffold. This in vitro result indicated the enhanced bioactivity of the modified scaffold. Moreover, enhanced bone forming ability was also observed in the rat model after 3 months of implantation. Though bony in-growth was found in all the implanted scaffolds. High volume of new bone formation could be found in the Mg/PCL hybrid scaffolds when compared to the pure PCL scaffold. Both pure PCL and Mg/PCL hybrid scaffolds were degraded after 3 months. However, no tissue inflammation was observed. In conclusion, these promising results suggested that the incorporation of Mg micro-particles into PCL porous scaffold could significantly enhance its mechanical and biological properties. This modified porous bio-scaffold may potentially apply in the surgical management of large bone defect fixation.

  4. ASTM international workshop on standards and measurements for tissue engineering scaffolds.

    Science.gov (United States)

    Simon, Carl G; Yaszemski, Michael J; Ratcliffe, Anthony; Tomlins, Paul; Luginbuehl, Reto; Tesk, John A

    2015-07-01

    The "Workshop on Standards & Measurements for Tissue Engineering Scaffolds" was held on May 21, 2013 in Indianapolis, IN, and was sponsored by the ASTM International (ASTM). The purpose of the workshop was to identify the highest priority items for future standards work for scaffolds used in the development and manufacture of tissue engineered medical products (TEMPs). Eighteen speakers and 78 attendees met to assess current scaffold standards and to prioritize needs for future standards. A key finding was that the ASTM TEMPs subcommittees (F04.41-46) have many active "guide" documents for educational purposes, but few standard "test methods" or "practices." Overwhelmingly, the most clearly identified need was standards for measuring the structure of scaffolds, followed by standards for biological characterization, including in vitro testing, animal models and cell-material interactions. The third most pressing need was to develop standards for assessing the mechanical properties of scaffolds. Additional needs included standards for assessing scaffold degradation, clinical outcomes with scaffolds, effects of sterilization on scaffolds, scaffold composition, and drug release from scaffolds. Discussions highlighted the need for additional scaffold reference materials and the need to use them for measurement traceability. Workshop participants emphasized the need to promote the use of standards in scaffold fabrication, characterization, and commercialization. Finally, participants noted that standards would be more broadly accepted if their impact in the TEMPs community could be quantified. Many scaffold standard needs have been identified and focus is turning to generating these standards to support the use of scaffolds in TEMPs. © 2014 Wiley Periodicals, Inc.

  5. A scaffold easy to decontaminate

    International Nuclear Information System (INIS)

    Mourek, D.

    1992-01-01

    The conventional scaffold used in the assembling work and in revisions of technological facilities at nuclear power plants has many drawbacks. The most serious of them are a high amount of radioactive waste arising from the decontamination (planing) of the floor timber and from the discarding of damaged irreparable parts, and a considerable corrosion of the carbon steel supporting structure after the decontamination. A detailed description is given of a novel scaffold assembly which can be decontaminated and which exhibits many assets, in particular a good mechanical resistance (also to bad weather), a lower weight, and the use of prepreg floor girders for the construction of service platforms or scaffold bridges which can readily be assembled from the pressed pieces in a modular way. (Z.S.). 4 figs., 4 refs

  6. Incorporation of zinc oxide nanoparticles into chitosan-collagen 3D porous scaffolds: Effect on morphology, mechanical properties and cytocompatibility of 3D porous scaffolds.

    Science.gov (United States)

    Ullah, Saleem; Zainol, Ismail; Idrus, Ruszymah Hj

    2017-11-01

    The zinc oxide nanoparticles (particles size chitosan-collagen 3D porous scaffolds and investigated the effect of zinc oxide nanoparticles incorporation on microstructure, mechanical properties, biodegradation and cytocompatibility of 3D porous scaffolds. The 0.5%, 1.0%, 2.0% and 4.0% zinc oxide nanoparticles chitosan-collagen 3D porous scaffolds were fabricated via freeze-drying technique. The zinc oxide nanoparticles incorporation effects consisting in chitosan-collagen 3D porous scaffolds were investigated by mechanical and swelling tests, and effect on the morphology of scaffolds examined microscopically. The biodegradation and cytocompatibility tests were used to investigate the effects of zinc oxide nanoparticles incorporation on the ability of scaffolds to use for tissue engineering application. The mean pore size and swelling ratio of scaffolds were decreased upon incorporation of zinc oxide nanoparticles however, the porosity, tensile modulus and biodegradation rate were increased upon incorporation of zinc oxide nanoparticles. In vitro culture of human fibroblasts and keratinocytes showed that the zinc oxide nanoparticles facilitated cell adhesion, proliferation and infiltration of chitosan-collagen 3D porous scaffolds. It was found that the zinc oxide nanoparticles incorporation enhanced porosity, tensile modulus and cytocompatibility of chitosan-collagen 3D porous scaffolds. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Novel mechanically competent polysaccharide scaffolds for bone tissue engineering

    International Nuclear Information System (INIS)

    Kumbar, S G; Toti, U S; Deng, M; James, R; Laurencin, C T; Aravamudhan, A; Harmon, M; Ramos, D M

    2011-01-01

    The success of the scaffold-based bone regeneration approach critically depends on the biomaterial's mechanical and biological properties. Cellulose and its derivatives are inherently associated with exceptional strength and biocompatibility due to their β-glycosidic linkage and extensive hydrogen bonding. This polymer class has a long medical history as a dialysis membrane, wound care system and pharmaceutical excipient. Recently cellulose-based scaffolds have been developed and evaluated for a variety of tissue engineering applications. In general porous polysaccharide scaffolds in spite of many merits lack the necessary mechanical competence needed for load-bearing applications. The present study reports the fabrication and characterization of three-dimensional (3D) porous sintered microsphere scaffolds based on cellulose derivatives using a solvent/non-solvent sintering approach for load-bearing applications. These 3D scaffolds exhibited a compressive modulus and strength in the mid-range of human trabecular bone and underwent degradation resulting in a weight loss of 10–15% after 24 weeks. A typical stress–strain curve for these scaffolds showed an initial elastic region and a less-stiff post-yield region similar to that of native bone. Human osteoblasts cultured on these scaffolds showed progressive growth with time and maintained expression of osteoblast phenotype markers. Further, the elevated expression of alkaline phosphatase and mineralization at early time points as compared to heat-sintered poly(lactic acid–glycolic acid) control scaffolds with identical pore properties affirmed the advantages of polysaccharides and their potential for scaffold-based bone regeneration.

  8. A coupled diffusion-fluid pressure model to predict cell density distribution for cells encapsulated in a porous hydrogel scaffold under mechanical loading.

    Science.gov (United States)

    Zhao, Feihu; Vaughan, Ted J; Mc Garrigle, Myles J; McNamara, Laoise M

    2017-10-01

    Tissue formation within tissue engineering (TE) scaffolds is preceded by growth of the cells throughout the scaffold volume and attachment of cells to the scaffold substrate. It is known that mechanical stimulation, in the form of fluid perfusion or mechanical strain, enhances cell differentiation and overall tissue formation. However, due to the complex multi-physics environment of cells within TE scaffolds, cell transport under mechanical stimulation is not fully understood. Therefore, in this study, we have developed a coupled multiphysics model to predict cell density distribution in a TE scaffold. In this model, cell transport is modelled as a thermal conduction process, which is driven by the pore fluid pressure under applied loading. As a case study, the model is investigated to predict the cell density patterns of pre-osteoblasts MC3T3-e1 cells under a range of different loading regimes, to obtain an understanding of desirable mechanical stimulation that will enhance cell density distribution within TE scaffolds. The results of this study have demonstrated that fluid perfusion can result in a higher cell density in the scaffold region closed to the outlet, while cell density distribution under mechanical compression was similar with static condition. More importantly, the study provides a novel computational approach to predict cell distribution in TE scaffolds under mechanical loading. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

    International Nuclear Information System (INIS)

    Lodato, D.T.; Reed, G.H.

    1987-01-01

    The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EOR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17 O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the γ-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling from 17 O regiospecifically incorporated into the γ-phosphate group of ATP. By contrast, 17 O in the α-phosphate or in the β-phosphate groups of ATP does not influence the spectrum. Experiments in 17 O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site. The structure for the enzyme-Mn(II)-oxalate-Mg(II)-ATP complex suggests a scheme for the normal reverse reaction of pyruvate kinase in which the divalent cation at the protein-based site activates the keto acid substrate through chelation and promotes phospho transfer by simultaneous coordination to the enolate oxygen and to a pendant oxygen from the γ-phosphate of ATP

  10. Engineering complex orthopaedic tissues via strategic biomimicry.

    Science.gov (United States)

    Qu, Dovina; Mosher, Christopher Z; Boushell, Margaret K; Lu, Helen H

    2015-03-01

    The primary current challenge in regenerative engineering resides in the simultaneous formation of more than one type of tissue, as well as their functional assembly into complex tissues or organ systems. Tissue-tissue synchrony is especially important in the musculoskeletal system, wherein overall organ function is enabled by the seamless integration of bone with soft tissues such as ligament, tendon, or cartilage, as well as the integration of muscle with tendon. Therefore, in lieu of a traditional single-tissue system (e.g., bone, ligament), composite tissue scaffold designs for the regeneration of functional connective tissue units (e.g., bone-ligament-bone) are being actively investigated. Closely related is the effort to re-establish tissue-tissue interfaces, which is essential for joining these tissue building blocks and facilitating host integration. Much of the research at the forefront of the field has centered on bioinspired stratified or gradient scaffold designs which aim to recapitulate the structural and compositional inhomogeneity inherent across distinct tissue regions. As such, given the complexity of these musculoskeletal tissue units, the key question is how to identify the most relevant parameters for recapitulating the native structure-function relationships in the scaffold design. Therefore, the focus of this review, in addition to presenting the state-of-the-art in complex scaffold design, is to explore how strategic biomimicry can be applied in engineering tissue connectivity. The objective of strategic biomimicry is to avoid over-engineering by establishing what needs to be learned from nature and defining the essential matrix characteristics that must be reproduced in scaffold design. Application of this engineering strategy for the regeneration of the most common musculoskeletal tissue units (e.g., bone-ligament-bone, muscle-tendon-bone, cartilage-bone) will be discussed in this review. It is anticipated that these exciting efforts will

  11. Engineering Complex Orthopaedic Tissues via Strategic Biomimicry

    Science.gov (United States)

    Qu, Dovina; Mosher, Christopher Z.; Boushell, Margaret K.; Lu, Helen H.

    2014-01-01

    The primary current challenge in regenerative engineering resides in the simultaneous formation of more than one type of tissue, as well as their functional assembly into complex tissues or organ systems. Tissue-tissue synchrony is especially important in the musculoskeletal system, whereby overall organ function is enabled by the seamless integration of bone with soft tissues such as ligament, tendon, or cartilage, as well as the integration of muscle with tendon. Therefore, in lieu of a traditional single-tissue system (e.g. bone, ligament), composite tissue scaffold designs for the regeneration of functional connective tissue units (e.g. bone-ligament-bone) are being actively investigated. Closely related is the effort to re-establish tissue-tissue interfaces, which is essential for joining these tissue building blocks and facilitating host integration. Much of the research at the forefront of the field has centered on bioinspired stratified or gradient scaffold designs which aim to recapitulate the structural and compositional inhomogeneity inherent across distinct tissue regions. As such, given the complexity of these musculoskeletal tissue units, the key question is how to identify the most relevant parameters for recapitulating the native structure-function relationships in the scaffold design. Therefore, the focus of this review, in addition to presenting the state-of-the-art in complex scaffold design, is to explore how strategic biomimicry can be applied in engineering tissue connectivity. The objective of strategic biomimicry is to avoid over-engineering by establishing what needs to be learned from nature and defining the essential matrix characteristics that must be reproduced in scaffold design. Application of this engineering strategy for the regeneration of the most common musculoskeletal tissue units (e.g. bone-ligament-bone, muscle-tendon-bone, cartilage-bone) will be discussed in this review. It is anticipated that these exciting efforts will

  12. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Science.gov (United States)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2013-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites. PMID:22116026

  13. Magnetic responsive hydroxyapatite composite scaffolds construction for bone defect reparation.

    Science.gov (United States)

    Zeng, Xiao Bo; Hu, Hao; Xie, Li Qin; Lan, Fang; Jiang, Wen; Wu, Yao; Gu, Zhong Wei

    2012-01-01

    In recent years, interest in magnetic biomimetic scaffolds for tissue engineering has increased considerably. A type of magnetic scaffold composed of magnetic nanoparticles (MNPs) and hydroxyapatite (HA) for bone repair has been developed by our research group. In this study, to investigate the influence of the MNP content (in the scaffolds) on the cell behaviors and the interactions between the magnetic scaffold and the exterior magnetic field, a series of MNP-HA magnetic scaffolds with different MNP contents (from 0.2% to 2%) were fabricated by immersing HA scaffold into MNP colloid. ROS 17/2.8 and MC3T3-E1 cells were cultured on the scaffolds in vitro, with and without an exterior magnetic field, respectively. The cell adhesion, proliferation and differentiation were evaluated via scanning electron microscopy; confocal laser scanning microscopy; and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase, and bone gla protein activity tests. The results demonstrated the positive influence of the magnetic scaffolds on cell adhesion, proliferation, and differentiation. Further, a higher amount of MNPs on the magnetic scaffolds led to more significant stimulation. The magnetic scaffold can respond to the exterior magnetic field and engender some synergistic effect to intensify the stimulating effect of a magnetic field to the proliferation and differentiation of cells.

  14. A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1.

    Science.gov (United States)

    Bar-Yosef, Hagit; Gildor, Tsvia; Ramírez-Zavala, Bernardo; Schmauch, Christian; Weissman, Ziva; Pinsky, Mariel; Naddaf, Rawi; Morschhäuser, Joachim; Arkowitz, Robert A; Kornitzer, Daniel

    2018-01-01

    The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

  15. An Insilico Design of Nanoclay Based Nanocomposites and Scaffolds in Bone Tissue Engineering

    Science.gov (United States)

    Sharma, Anurag

    A multiscale in silico approach to design polymer nanocomposites and scaffolds for bone tissue engineering applications is described in this study. This study focuses on the role of biomaterials design and selection, structural integrity and mechanical properties evolution during degradation and tissue regeneration in the successful design of polymer nanocomposite scaffolds. Polymer nanocomposite scaffolds are synthesized using aminoacid modified montmorillonite nanoclay with biomineralized hydroxyapatite and polycaprolactone (PCL/in situ HAPclay). Representative molecular models of polymer nanocomposite system are systematically developed using molecular dynamics (MD) technique and successfully validated using material characterization techniques. The constant force steered molecular dynamics (fSMD) simulation results indicate a two-phase nanomechanical behavior of the polymer nanocomposite. The MD and fSMD simulations results provide quantitative contributions of molecular interactions between different constituents of representative models and their effect on nanomechanical responses of nanoclay based polymer nanocomposite system. A finite element (FE) model of PCL/in situ HAPclay scaffold is built using micro-computed tomography images and bridging the nanomechanical properties obtained from fSMD simulations into the FE model. A new reduction factor, K is introduced into modeling results to consider the effect of wall porosity of the polymer scaffold. The effect of accelerated degradation under alkaline conditions and human osteoblast cells culture on the evolution of mechanical properties of scaffolds are studied and the damage mechanics based analytical models are developed. Finally, the novel multiscale models are developed that incorporate the complex molecular and microstructural properties, mechanical properties at nanoscale and structural levels and mechanical properties evolution during degradation and tissue formation in the polymer nanocomposite

  16. Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

    Science.gov (United States)

    Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

    2012-02-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

  17. A designated centre for people with disabilities operated by Dara Residential Services, Kildare

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

    Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  18. Biodegradable porous sheet-like scaffolds for soft-tissue engineering using a combined particulate leaching of salt particles and magnetic sugar particles.

    Science.gov (United States)

    Hu, Chengzhi; Tercero, Carlos; Ikeda, Seiichi; Nakajima, Masahiro; Tajima, Hirotaka; Shen, Yajing; Fukuda, Toshio; Arai, Fumihito

    2013-07-01

    Scaffolds serving as artificial extracellular matrixes (ECMs) play a pivotal role in the process of tissue regeneration by providing optimal cellular environments for penetration, ingrowth, and vascularization. Stacks of sheet-like scaffold can be engineered to become artificial ECMs, suggesting a great potential for achieving complex 3-D tissue regeneration to support cell survival and growth. In this study, we proposed and investigated a combined particulate leaching of magnetic sugar particles (MSPs) and salt particles for the development of a sheet-like scaffold. MSPs were fabricated by encapsulating NdFeB particles inside sugar spheres and were controlled using magnetic fields as a porogen to control pore size, pore structure and pore density while fabricating the scaffold. We studied the influence of the strength of the magnetic fields in controlling the coating thickness of the unmagnetized MSPs during the fabrication of the sheet-like scaffolds. The experimental relationship between magnetic flux density and the thickness of the MSP layer was illustrated. Furthermore, we investigated the infiltration capacity of different concentrations of poly(L-lactide-co-ɛ-caprolactone) (PLCL) as a scaffold material on MSP clusters. Following polymer casting and removal of the sugar template, spherical pores were generated inside the scaffolds. Cultivation of NIH/3T3 fibroblasts on the fabricated scaffold proves that the proposed method can be applied in the cell sheet fabrication. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells

    Directory of Open Access Journals (Sweden)

    Wu XN

    2014-08-01

    Full Text Available Xiaonan Wu,1 Leiying Miao,2,# Yingfang Yao,3 Wenlei Wu,1 Yu Liu,1 Xiaofeng Chen,1 Weibin Sun1,# 1Department of Periodontology, Hospital of Stomatology, Medical School of Nanjing University, Nanjing, People’s Republic of China; 2Department of Cariology and Endodontics, Hospital of Stomatology, Medical School of Nanjing University, Nanjing, People’s Republic of China; 3Eco-materials and Renewable Energy Research Center, Department of Materials Science and Engineering, National Laboratory of Solid State Microstructures, Nanjing University, Nanjing, People’s Republic of China #These authors contributed equally to this work Abstract: Periodontal repair is a complex process in which regeneration of alveolar bone is a vital component. The aim of this study was to develop a biodegradable scaffold with good biocompatibility and osteoinductive ability. Two types of composite fibrous scaffolds were produced by electrospinning, ie, type I collagen/poly(є-caprolactone (COL/PCL and type I collagen/poly(є-caprolactone/nanoscale hydroxyapatite (COL/PCL/nHA with an average fiber diameter of about 377 nm. After a simulated body fluid (SBF immersion test, the COL/PCL/nHA-SBF scaffold developed a rough surface because of the calcium phosphate deposited on the fibers, suggesting that the presence of nHA promoted the mineralization potential of the scaffold. Energy dispersive X-ray spectroscopy clearly showed the calcium and phosphorus content in the COL/PCL/nHA and COL/PCL/nHA-SBF scaffolds, confirming the findings of nHA and calcium phosphate precipitation on scanning electron micrographs. Water contact analysis revealed that nHA could improve the hydrophilic nature of the COL/PCL/nHA-SBF scaffold. The morphology of periodontal ligament cells cultured on COL/PCL-SBF and COL/PCL/nHA-SBF was evaluated by scanning electron microscopy. The results showed that cells adhered to either type of scaffold and were slightly spindle-shaped in the beginning, then

  20. Preparation, physicochemical properties and biocompatibility of PBLG/PLGA/bioglass composite scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ning [State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China); Qian, Junmin, E-mail: jmqian@mail.xjtu.edu.cn [State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China); Wang, Jinlei [State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China); Ji, Chuanlei [The Orthopaedic Department, XiJing Hospital Affiliated to the Fourth Military Medical University, Xi' an 710032 (China); Xu, Weijun; Wang, Hongjie [State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China)

    2017-02-01

    In this study, novel poly(γ-benzyl L-glutamate)/poly(lactic-co-glycolic acid)/bioglass (PBLG/PLGA/BG) composite scaffolds with different weight ratios were fabricated using a negative NaCl-templating method. The morphology, compression modulus and degradation kinetics of the scaffolds were characterized. The results showed that the PBLG/PLGA/BG composite scaffolds with a weight ratio of 5:5:1, namely PBLG5PLGA5BG composite scaffolds, displayed a pore size range of 50–500 μm, high compressive modulus (566.6 ± 8.8 kPa), suitable glass transition temperature (46.8 ± 0.2 °C) and low degradation rate (> 8 weeks). The in vitro biocompatibility of the scaffolds was evaluated with MC3T3-E1 cells by live-dead staining, MTT and ALP activity assays. The obtained results indicated that the PBLG5PLGA5BG composite scaffolds were more conducive to the adhesion, proliferation and osteoblastic differentiation of MC3T3-E1 cells than PBLG and PBLG/PLGA composite scaffolds. The in vivo biocompatibility of the scaffolds was evaluated in both SD rat subcutaneous model and rabbit tibia defect model. The results of H&E, Masson's trichrome and CD34 staining assays demonstrated that the PBLG5PLGA5BG composite scaffolds allowed the ingrowth of tissue and microvessels more effectively than PBLG/PLGA composite scaffolds. The results of digital radiography confirmed that the PBLG5PLGA5BG composite scaffolds significantly improved in vivo osteogenesis. Collectively, the PBLG5PLGA5BG composite scaffolds could be a promising candidate for tissue engineering applications. - Highlights: • Foamy PBLG/PLGA/bioglass composite scaffolds were fabricated by negative templating. • PBLG/PLGA/bioglass composite scaffolds displayed tunable physicochemical properties. • PBLG/PLGA/bioglass composite scaffolds had good biocompatibility in vitro and in vivo. • PBLG/PLGA/bioglass composite scaffolds could promote the healing of bone defects.

  1. Poly(Dopamine-Assisted Immobilization of Xu Duan on 3D Printed Poly(Lactic Acid Scaffolds to Up-Regulate Osteogenic and Angiogenic Markers of Bone Marrow Stem Cells

    Directory of Open Access Journals (Sweden)

    Chia-Hung Yeh

    2015-07-01

    Full Text Available Three-dimensional printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid (PLA scaffolds and use a mussel-inspired surface coating and Xu Duan (XD immobilization to regulate cell adhesion, proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBMSCs. We prepared PLA scaffolds and coated with polydopamine (PDA. The chemical composition and surface properties of PLA/PDA/XD were characterized by XPS. PLA/PDA/XD controlled hBMSCs’ responses in several ways. Firstly, adhesion and proliferation of hBMSCs cultured on PLA/PDA/XD were significantly enhanced relative to those on PLA. In addition, the focal adhesion kinase (FAK expression of cells was increased and promoted cell attachment depended on the XD content. In osteogenesis assay, the osteogenesis markers of hBMSCs cultured on PLA/PDA/XD were significantly higher than seen in those cultured on a pure PLA/PDA scaffolds. Moreover, hBMSCs cultured on PLA/PDA/XD showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hBMSCs.

  2. Poly(Dopamine)-Assisted Immobilization of Xu Duan on 3D Printed Poly(Lactic Acid) Scaffolds to Up-Regulate Osteogenic and Angiogenic Markers of Bone Marrow Stem Cells.

    Science.gov (United States)

    Yeh, Chia-Hung; Chen, Yi-Wen; Shie, Ming-You; Fang, Hsin-Yuan

    2015-07-14

    Three-dimensional printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid) (PLA) scaffolds and use a mussel-inspired surface coating and Xu Duan (XD) immobilization to regulate cell adhesion, proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBMSCs). We prepared PLA scaffolds and coated with polydopamine (PDA). The chemical composition and surface properties of PLA/PDA/XD were characterized by XPS. PLA/PDA/XD controlled hBMSCs' responses in several ways. Firstly, adhesion and proliferation of hBMSCs cultured on PLA/PDA/XD were significantly enhanced relative to those on PLA. In addition, the focal adhesion kinase (FAK) expression of cells was increased and promoted cell attachment depended on the XD content. In osteogenesis assay, the osteogenesis markers of hBMSCs cultured on PLA/PDA/XD were significantly higher than seen in those cultured on a pure PLA/PDA scaffolds. Moreover, hBMSCs cultured on PLA/PDA/XD showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hBMSCs.

  3. Role of scaffold mean pore size in meniscus regeneration.

    Science.gov (United States)

    Zhang, Zheng-Zheng; Jiang, Dong; Ding, Jian-Xun; Wang, Shao-Jie; Zhang, Lei; Zhang, Ji-Ying; Qi, Yan-Song; Chen, Xue-Si; Yu, Jia-Kuo

    2016-10-01

    Recently, meniscus tissue engineering offers a promising management for meniscus regeneration. Although rarely reported, the microarchitectures of scaffolds can deeply influence the behaviors of endogenous or exogenous stem/progenitor cells and subsequent tissue formation in meniscus tissue engineering. Herein, a series of three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds with three distinct mean pore sizes (i.e., 215, 320, and 515μm) were fabricated via fused deposition modeling. The scaffold with the mean pore size of 215μm significantly improved both the proliferation and extracellular matrix (ECM) production/deposition of mesenchymal stem cells compared to all other groups in vitro. Moreover, scaffolds with mean pore size of 215μm exhibited the greatest tensile and compressive moduli in all the acellular and cellular studies. In addition, the relatively better results of fibrocartilaginous tissue formation and chondroprotection were observed in the 215μm scaffold group after substituting the rabbit medial meniscectomy for 12weeks. Overall, the mean pore size of 3D-printed PCL scaffold could affect cell behavior, ECM production, biomechanics, and repair effect significantly. The PCL scaffold with mean pore size of 215μm presented superior results both in vitro and in vivo, which could be an alternative for meniscus tissue engineering. Meniscus tissue engineering provides a promising strategy for meniscus regeneration. In this regard, the microarchitectures (e.g., mean pore size) of scaffolds remarkably impact the behaviors of cells and subsequent tissue formation, which has been rarely reported. Herein, three three-dimensional poly(ε-caprolactone) scaffolds with different mean pore sizes (i.e., 215, 320, and 515μm) were fabricated via fused deposition modeling. The results suggested that the mean pore size significantly affected the behaviors of endogenous or exogenous stem/progenitor cells and subsequent tissue formation. This study furthers

  4. Microfibrous silver-coated polymeric scaffolds with tunable mechanical properties

    KAUST Repository

    Kalakonda, Parvathalu.; Aldhahri, Musab A.; Abdel-wahab, Mohamed Shaaban; Tamayol, Ali; Moghaddam, K. Mollazadeh; Ben Rached, Fathia; Pain, Arnab; Khademhosseini, Ali; Memic, Adnan; Chaieb, Saharoui

    2017-01-01

    Electrospun scaffolds of poly(glycerol sebacate)/poly(ε-caprolactone) (PGS/PCL) have been used for engineered tissues due to their desirable thermal and mechanical properties as well as their tunable degradability. In this paper, we fabricated micro-fibrous scaffolds from a composite of PGS/PCL using a standard electrospinning method and coated them with silver (Ag). The low temperature coating method prevented substrate melting and the Ag coating decreases the pore size and increases the diameter of fibers which resulted in enhanced thermal and mechanical properties. We further compared the mechanical properties of the composite fibrous scaffolds with different thicknesses of Ag coated scaffolds. The composite fibrous scaffold with a 275 nm Ag coating showed higher tensile modulus (E) and ultimate tensile strength (UTS) without any post-processing treatment. Lastly, potential controlled release of the Ag coating from the composite fibrous scaffolds could present interesting biomedical applications.

  5. Microfibrous silver-coated polymeric scaffolds with tunable mechanical properties

    KAUST Repository

    Kalakonda, Parvathalu.

    2017-07-07

    Electrospun scaffolds of poly(glycerol sebacate)/poly(ε-caprolactone) (PGS/PCL) have been used for engineered tissues due to their desirable thermal and mechanical properties as well as their tunable degradability. In this paper, we fabricated micro-fibrous scaffolds from a composite of PGS/PCL using a standard electrospinning method and coated them with silver (Ag). The low temperature coating method prevented substrate melting and the Ag coating decreases the pore size and increases the diameter of fibers which resulted in enhanced thermal and mechanical properties. We further compared the mechanical properties of the composite fibrous scaffolds with different thicknesses of Ag coated scaffolds. The composite fibrous scaffold with a 275 nm Ag coating showed higher tensile modulus (E) and ultimate tensile strength (UTS) without any post-processing treatment. Lastly, potential controlled release of the Ag coating from the composite fibrous scaffolds could present interesting biomedical applications.

  6. Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

    Science.gov (United States)

    Pascal, John M; Armen, Roger S

    2012-01-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

  7. Tough and flexible CNT-polymeric hybrid scaffolds for engineering cardiac constructs.

    Science.gov (United States)

    Kharaziha, Mahshid; Shin, Su Ryon; Nikkhah, Mehdi; Topkaya, Seda Nur; Masoumi, Nafiseh; Annabi, Nasim; Dokmeci, Mehmet R; Khademhosseini, Ali

    2014-08-01

    In the past few years, a considerable amount of effort has been devoted toward the development of biomimetic scaffolds for cardiac tissue engineering. However, most of the previous scaffolds have been electrically insulating or lacked the structural and mechanical robustness to engineer cardiac tissue constructs with suitable electrophysiological functions. Here, we developed tough and flexible hybrid scaffolds with enhanced electrical properties composed of carbon nanotubes (CNTs) embedded aligned poly(glycerol sebacate):gelatin (PG) electrospun nanofibers. Incorporation of varying concentrations of CNTs from 0 to 1.5% within the PG nanofibrous scaffolds (CNT-PG scaffolds) notably enhanced fiber alignment and improved the electrical conductivity and toughness of the scaffolds while maintaining the viability, retention, alignment, and contractile activities of cardiomyocytes (CMs) seeded on the scaffolds. The resulting CNT-PG scaffolds resulted in stronger spontaneous and synchronous beating behavior (3.5-fold lower excitation threshold and 2.8-fold higher maximum capture rate) compared to those cultured on PG scaffold. Overall, our findings demonstrated that aligned CNT-PG scaffold exhibited superior mechanical properties with enhanced CM beating properties. It is envisioned that the proposed hybrid scaffolds can be useful for generating cardiac tissue constructs with improved organization and maturation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Anchoring Proteins as Regulators of Signaling Pathways

    Science.gov (United States)

    Perino, Alessia; Ghigo, Alessandra; Scott, John D.; Hirsch, Emilio

    2012-01-01

    Spatial and temporal organization of signal transduction is coordinated through the segregation of signaling enzymes in selected cellular compartments. This highly evolved regulatory mechanism ensures the activation of selected enzymes only in the vicinity of their target proteins. In this context, cAMP-responsive triggering of protein kinase A is modulated by a family of scaffold proteins referred to as A-kinase anchoring proteins. A-kinase anchoring proteins form the core of multiprotein complexes and enable simultaneous but segregated cAMP signaling events to occur in defined cellular compartments. In this review we will focus on the description of A-kinase anchoring protein function in the regulation of cardiac physiopathology. PMID:22859670

  9. Quantitative and Dynamic Imaging of ATM Kinase Activity.

    Science.gov (United States)

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  10. Microporous dermal-like electrospun scaffolds promote accelerated skin regeneration.

    Science.gov (United States)

    Bonvallet, Paul P; Culpepper, Bonnie K; Bain, Jennifer L; Schultz, Matthew J; Thomas, Steven J; Bellis, Susan L

    2014-09-01

    The goal of this study was to synthesize skin substitutes that blend native extracellular matrix (ECM) molecules with synthetic polymers which have favorable mechanical properties. To this end, scaffolds were electrospun from collagen I (col) and poly(ɛ-caprolactone) (PCL), and then pores were introduced mechanically to promote fibroblast infiltration, and subsequent filling of the pores with ECM. A 70:30 col/PCL ratio was determined to provide optimal support for dermal fibroblast growth, and a pore diameter, 160 μm, was identified that enabled fibroblasts to infiltrate and fill pores with native matrix molecules, including fibronectin and collagen I. Mechanical testing of 70:30 col/PCL scaffolds with 160 μm pores revealed a tensile strength of 1.4 MPa, and the scaffolds also exhibited a low rate of contraction (pores. Keratinocytes formed a stratified layer on the surface of fibroblast-remodeled scaffolds, and staining for cytokeratin 10 revealed terminally differentiated keratinocytes at the apical surface. When implanted, 70:30 col/PCL scaffolds degraded within 3-4 weeks, an optimal time frame for degradation in vivo. Finally, 70:30 col/PCL scaffolds with or without 160 μm pores were implanted into full-thickness critical-sized skin defects. Relative to nonporous scaffolds or sham wounds, scaffolds with 160 μm pores induced accelerated wound closure, and stimulated regeneration of healthy dermal tissue, evidenced by a more normal-appearing matrix architecture, blood vessel in-growth, and hair follicle development. Collectively, these results suggest that microporous electrospun scaffolds are effective substrates for skin regeneration.

  11. PLDLA/PCL-T Scaffold for Meniscus Tissue Engineering.

    Science.gov (United States)

    Esposito, Andrea Rodrigues; Moda, Marlon; Cattani, Silvia Mara de Melo; de Santana, Gracy Mara; Barbieri, Juliana Abreu; Munhoz, Monique Moron; Cardoso, Túlio Pereira; Barbo, Maria Lourdes Peris; Russo, Teresa; D'Amora, Ugo; Gloria, Antonio; Ambrosio, Luigi; Duek, Eliana Aparecida de Rezende

    2013-04-01

    The inability of the avascular region of the meniscus to regenerate has led to the use of tissue engineering to treat meniscal injuries. The aim of this study was to evaluate the ability of fibrochondrocytes preseeded on PLDLA/PCL-T [poly(L-co-D,L-lactic acid)/poly(caprolactone-triol)] scaffolds to stimulate regeneration of the whole meniscus. Porous PLDLA/PCL-T (90/10) scaffolds were obtained by solvent casting and particulate leaching. Compressive modulus of 9.5±1.0 MPa and maximum stress of 4.7±0.9 MPa were evaluated. Fibrochondrocytes from rabbit menisci were isolated, seeded directly on the scaffolds, and cultured for 21 days. New Zealand rabbits underwent total meniscectomy, after which implants consisting of cell-free scaffolds or cell-seeded scaffolds were introduced into the medial knee meniscus; the negative control group consisted of rabbits that received no implant. Macroscopic and histological evaluations of the neomeniscus were performed 12 and 24 weeks after implantation. The polymer scaffold implants adapted well to surrounding tissues, without apparent rejection, infection, or chronic inflammatory response. Fibrocartilaginous tissue with mature collagen fibers was observed predominantly in implants with seeded scaffolds compared to cell-free implants after 24 weeks. Similar results were not observed in the control group. Articular cartilage was preserved in the polymeric implants and showed higher chondrocyte cell number than the control group. These findings show that the PLDLA/PCL-T 90/10 scaffold has potential for orthopedic applications since this material allowed the formation of fibrocartilaginous tissue, a structure of crucial importance for repairing injuries to joints, including replacement of the meniscus and the protection of articular cartilage from degeneration.

  12. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    Science.gov (United States)

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Investigation of cancer cell behavior on nanofibrous scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Szot, Christopher S.; Buchanan, Cara F. [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 (United States); Gatenholm, Paul [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 (United States); Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Goeteborg (Sweden); Rylander, Marissa Nichole [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 (United States); Freeman, Joseph W., E-mail: jwfreeman@vt.edu [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 (United States)

    2011-01-01

    Tissue engineering and the use of nanofibrous biomaterial scaffolds offer a unique perspective for studying cancer development in vitro. Current in vitro models of tumorigenesis are limited by the use of static, two-dimensional (2D) cell culture monolayers that lack the structural architecture necessary for cell-cell interaction and three-dimensional (3D) scaffolds that are too simplistic for studying basic pathological mechanisms. In this study, two nanofibrous biomaterials that mimic the structure of the extracellular matrix, bacterial cellulose and electrospun polycaprolactone (PCL)/collagen I, were investigated as potential 3D scaffolds for an in vitro cancer model. Multiple cancer cell lines were cultured on each scaffold material and monitored for cell viability, proliferation, adhesion, infiltration, and morphology. Both bacterial cellulose and electrospun PCL/collagen I, which have nano-scale structures on the order of 100-500 nm, have been used in many diverse tissue engineering applications. Cancer cell adhesion and growth were limited on bacterial cellulose, while all cellular processes were enhanced on the electrospun scaffolds. This initial analysis has demonstrated the potential of electrospun PCL/collagen I scaffolds toward the development of an improved 3D in vitro cancer model.

  14. Intrinsic Osteoinductivity of Porous Titanium Scaffold for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Maryam Tamaddon

    2017-01-01

    Full Text Available Large bone defects and nonunions are serious complications that are caused by extensive trauma or tumour. As traditional therapies fail to repair these critical-sized defects, tissue engineering scaffolds can be used to regenerate the damaged tissue. Highly porous titanium scaffolds, produced by selective laser sintering with mechanical properties in range of trabecular bone (compressive strength 35 MPa and modulus 73 MPa, can be used in these orthopaedic applications, if a stable mechanical fixation is provided. Hydroxyapatite coatings are generally considered essential and/or beneficial for bone formation; however, debonding of the coatings is one of the main concerns. We hypothesised that the titanium scaffolds have an intrinsic potential to induce bone formation without the need for a hydroxyapatite coating. In this paper, titanium scaffolds coated with hydroxyapatite using electrochemical method were fabricated and osteoinductivity of coated and noncoated scaffolds was compared in vitro. Alizarin Red quantification confirmed osteogenesis independent of coating. Bone formation and ingrowth into the titanium scaffolds were evaluated in sheep stifle joints. The examinations after 3 months revealed 70% bone ingrowth into the scaffold confirming its osteoinductive capacity. It is shown that the developed titanium scaffold has an intrinsic capacity for bone formation and is a suitable scaffold for bone tissue engineering.

  15. Structural-Geometric Functionalization of the Additively Manufactured Prototype of Biomimetic Multispiked Connecting Ti-Alloy Scaffold for Entirely Noncemented Resurfacing Arthroplasty Endoprostheses

    Directory of Open Access Journals (Sweden)

    Ryszard Uklejewski

    2017-01-01

    Full Text Available The multispiked connecting scaffold (MSC-Scaffold prototype, inspired by the biological system of anchorage of the articular cartilage in the periarticular trabecular bone by means of subchondral bone interdigitations, is the essential innovation in fixation of the bone in resurfacing arthroplasty (RA endoprostheses. The biomimetic MSC‐Scaffold, due to its complex geometric structure, can be manufactured only using additive technology, for example, selective laser melting (SLM. The major purpose of this work is determination of constructional possibilities for the structural-geometric functionalization of SLM‐manufactured MSC‐Scaffold prototype, compensating the reduced ability—due to the SLM technological limitations—to accommodate the ingrowing bone filling the interspike space of the prototype, which is important for the prototype bioengineering design. Confocal microscopy scanning of components of the SLM‐manufactured prototype of total hip resurfacing arthroplasty (THRA endoprosthesis with the MSC‐Scaffold was performed. It was followed by the geometric measurements of a variety of specimens designed as the fragments of the MSC-Scaffold of both THRA endoprosthesis components. The reduced ability to accommodate the ingrowing bone tissue in the SLM‐manufactured prototypes versus that in the corresponding CAD models has been quantitatively determined. Obtained results enabled to establish a way of compensatory structural‐geometric functionalization, allowing the MSC‐Scaffold adequate redesigning and manufacturing in additive SLM technology.

  16. PAK4 crystal structures suggest unusual kinase conformational movements.

    Science.gov (United States)

    Zhang, Eric Y; Ha, Byung Hak; Boggon, Titus J

    2018-02-01

    In order for protein kinases to exchange nucleotide they must open and close their catalytic cleft. These motions are associated with rotations of the N-lobe, predominantly around the 'hinge region'. We conducted an analysis of 28 crystal structures of the serine-threonine kinase, p21-activated kinase 4 (PAK4), including three newly determined structures in complex with staurosporine, FRAX486, and fasudil (HA-1077). We find an unusual motion between the N-lobe and C-lobe of PAK4 that manifests as a partial unwinding of helix αC. Principal component analysis of the crystal structures rationalizes these movements into three major states, and analysis of the kinase hydrophobic spines indicates concerted movements that create an accessible back pocket cavity. The conformational changes that we observe for PAK4 differ from previous descriptions of kinase motions, and although we observe these differences in crystal structures there is the possibility that the movements observed may suggest a diversity of kinase conformational changes associated with regulation. Protein kinases are key signaling proteins, and are important drug targets, therefore understanding their regulation is important for both basic research and clinical points of view. In this study, we observe unusual conformational 'hinging' for protein kinases. Hinging, the opening and closing of the kinase sub-domains to allow nucleotide binding and release, is critical for proper kinase regulation and for targeted drug discovery. We determine new crystal structures of PAK4, an important Rho-effector kinase, and conduct analyses of these and previously determined structures. We find that PAK4 crystal structures can be classified into specific conformational groups, and that these groups are associated with previously unobserved hinging motions and an unusual conformation for the kinase hydrophobic core. Our findings therefore indicate that there may be a diversity of kinase hinging motions, and that these may

  17. Scaffold diversification enhances effectiveness of a superlibrary of hyperthermophilic proteins.

    Science.gov (United States)

    Hussain, Mahmud; Gera, Nimish; Hill, Andrew B; Rao, Balaji M

    2013-01-18

    The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a "superlibrary" or "library-of-libraries" of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.

  18. Human-like collagen/nano-hydroxyapatite scaffolds for the culture of chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Liping; Duan, Zhiguang [Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Shaanxi R and D Center of Biomaterials and Fermentation Engineering, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Fan, Daidi, E-mail: fandaidi@nwu.edu.cn [Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Shaanxi R and D Center of Biomaterials and Fermentation Engineering, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Mi, Yu; Hui, Junfeng [Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Shaanxi R and D Center of Biomaterials and Fermentation Engineering, Northwest University, 229 Taibai North Road, Xi' an, Shaanxi 710069 (China); Chang, Le [School of Chemical Engineering, Northwest University, Xi' an, Shaanxi 710069 (China)

    2013-03-01

    Three dimensional (3D) biodegradable porous scaffolds play a key role in cartilage tissue repair. Freeze-drying and cross-linking techniques were used to fabricate a 3D composite scaffold that combined the excellent biological characteristics of human-like collagen (HLC) and the outstanding mechanical properties of nano-hydroxyapatite (nHA). The scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and compression tests, using Relive Registered-Sign Artificial Bone (RAB) scaffolds as a control. HLC/nHA scaffolds displayed homogeneous interconnected macroporous structure and could withstand a compression stress of 2.67 {+-} 0.37 MPa, which was higher than that of the control group. Rabbit chondrocytes were seeded on the composite porous scaffolds and cultured for 21 days. Cell/scaffold constructs were examined using SEM, histological procedures, and biochemical assays for cell proliferation and the production of glycosaminoglycans (GAGs). The results indicated that HLC/nHA porous scaffolds were capable of encouraging cell adhesion, homogeneous distribution and abundant GAG synthesis, and maintaining natural chondrocyte morphology compared to RAB scaffolds. In conclusion, the presented data warrants the further exploration of HLC/nHA scaffolds as a potential biomimetic platform for chondrocytes in cartilage tissue engineering. - Highlights: Black-Right-Pointing-Pointer Human-like collagen was first used to prepare cartilage tissue engineering scaffold. Black-Right-Pointing-Pointer Genipin, a natural biological cross-linking agent, was introduced to treat scaffold. Black-Right-Pointing-Pointer We chose market product as a control.

  19. Band 3 tyrosine kinase in avian erythrocyte plasma membrane is immunologically related to pp60c-src

    International Nuclear Information System (INIS)

    Hillsgrove, D.; Shores, C.G.; Parker, J.C.; Maness, P.F.

    1987-01-01

    The authors have identified in the plasma membrane of the chicken erythrocyte a 60-kDa tyrosine-specific protein kinase immunologically related to the transforming protein pp60 v-src of Rous sarcoma virus. The erythrocyte protein kinase phosphorylated heavy chains of tumor-bearing rabbit (TBR) antibodies reactive with pp60 c-src at tyrosine in immune complex protein kinase assays. The kinase was identified as a 60-kDa protein by [ 35 S]methionine labeling of erythrocytes and by autophosphorylation in immune complexes. The kinase migrated on two-dimensional gel electrophoresis with an apparent pI and molecular mass similar to pp60 c-src . A plasma membrane-enriched fraction isolated from chicken red cells contained the majority of the kinase activity. Incubation of the plasma membrane fraction with [ 32 P]ATP resulted in tyrosine phosphorylation of the anion transport protein band 3. Band 3 phosphorylation was blocked by TBR antibodies, indicting that the kinase recognized by pp60 c-src antibodies was responsible for band 3 phosphorylation. These results demonstrate that the avian erythrocyte plasma membrane contains a tightly bound tyrosine-specific protein kinase identical or closely related to pp60 c-src and that this kinase is responsible for band 3 phosphorylation in vitro

  20. Porous poly (lactic-co-glycolide) microsphere sintered scaffolds for tissue repair applications

    International Nuclear Information System (INIS)

    Wang Yingjun; Shi Xuetao; Ren Li; Wang Chunming; Wang Dongan

    2009-01-01

    In this paper, a new route to preparing porous poly (lactic-co-glycolide) (PLGA) scaffolds for bone tissue repair applications was developed. Novel porous PLGA scaffolds were fabricated via microsphere sintered technique and gas forming technique. Ammonium bicarbonate was used to regulate porosity of these porous scaffolds. Porosity of the scaffolds, and cell attachment, viability and proliferation on the scaffolds were evaluated. The results indicated that PLGA porous scaffolds were with the porosity from around 30% to 95% by regulating ammonium bicarbonate content from 0 to 10%. We also found that PLGA porous microsphere scaffolds benefited cell attachment and viability. Taken together, the achieved porous scaffolds have controlled porosity and also support mesenchymal stem cell proliferation, which could serve as potential scaffolds for bone repair applications.

  1. Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue engineering.

    Science.gov (United States)

    Huang, Di; Zuo, Yi; Zou, Qin; Wang, Yanying; Gao, Shibo; Wang, Xiaoyan; Liu, Haohuai; Li, Yubao

    2012-01-01

    High porosity of scaffold is always accompanied by poor mechanical property; the aim of this study was to enhance the strength and modulus of the highly porous scaffold of nanohydroxyapatite/polyamide66 (n-HA/PA66) by coating chitosan (CS) and to investigate the effect of CS content on the scaffold physical properties and cytological properties. The results show that CS coating can reinforce the scaffold effectively. The compress modulus and strength of the CS coated n-HA/PA66 scaffolds are improved to 32.71 and 2.38 MPa, respectively, being about six times and five times of those of the uncoated scaffolds. Meanwhile, the scaffolds still exhibit a highly interconnected porous structure and the porosity is approximate about 78%, slightly lower than the value (84%) of uncoated scaffold. The cytological properties of scaffolds were also studied in vitro by cocultured with osteoblast-like MG63 cells. The cytological experiments demonstrate that the reinforced scaffolds display favorable cytocompatibility and have no significant difference with the uncoated n-HA/PA66 scaffolds. The CS reinforced n-HA/PA66 scaffolds can meet the basic mechanical requirement of bone tissue engineering scaffold, presenting a potential for biomedical application in bone reconstruction and repair. Copyright © 2011 Wiley Periodicals, Inc.

  2. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  3. Automated quality characterization of 3D printed bone scaffolds

    Directory of Open Access Journals (Sweden)

    Tzu-Liang Bill Tseng

    2014-07-01

    Full Text Available Optimization of design is an important step in obtaining tissue engineering scaffolds with appropriate shapes and inner microstructures. Different shapes and sizes of scaffolds are modeled using UGS NX 6.0 software with variable pore sizes. The quality issue we are concerned is the scaffold porosity, which is mainly caused by the fabrication inaccuracies. Bone scaffolds are usually characterized using a scanning electron microscope, but this study presents a new automated inspection and classification technique. Due to many numbers and size variations for the pores, the manual inspection of the fabricated scaffolds tends to be error-prone and costly. Manual inspection also raises the chance of contamination. Thus, non-contact, precise inspection is preferred. In this study, the critical dimensions are automatically measured by the vision camera. The measured data are analyzed to classify the quality characteristics. The automated inspection and classification techniques developed in this study are expected to improve the quality of the fabricated scaffolds and reduce the overall cost of manufacturing.

  4. Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

    Science.gov (United States)

    Khandalavala, Karl; Jiang, Jiang; Shuler, Franklin D.; Xie, Jingwei

    2015-01-01

    The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion. PMID:25938562

  5. The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

    Science.gov (United States)

    Liu, Ji; Nie, Huarong; Xu, Zhengliang; Niu, Xin; Guo, Shangchun; Yin, Junhui; Guo, Fei; Li, Gang; Wang, Yang; Zhang, Changqing

    2014-01-01

    The discovery of induced pluripotent stem cells (iPSCs) rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL)/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM), the Cell Counting Kit-8 (CCK-8), histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.

  6. The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

    Directory of Open Access Journals (Sweden)

    Ji Liu

    Full Text Available The discovery of induced pluripotent stem cells (iPSCs rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM, the Cell Counting Kit-8 (CCK-8, histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.

  7. Regulation of the vertebrate cell cycle by the cdc2 protein kinase

    International Nuclear Information System (INIS)

    Draetta, G.; Brizuela, L.; Moran, B.; Beach, D.

    1988-01-01

    A homolog of the cdc2/CDC28 protein kinase of yeast is found in all vertebrate species that have been investigated. Human cdc2 exists as a complex with a 13-kD protein that is homologous to the suc1 gene product of fission yeast. In both human and fission yeast cells, the protein kinase also exists in a complex with a 62-kD polypeptide that has not been identified genetically but acts as a substrate in vitro. The authors have studied the properties of the protein kinase in rat and human cells, as well as in Xenopus eggs. They find that in baby rat kidney (BRK) cells, which are quiescent in cell culture, the cdc2 protein is not synthesized. However, synthesis is rapidly induced in response to proliferative activation by infection with adenovirus. In human HeLa cells, the protein kinase is present continuously. It behaves as a cell-cycle oscillator that is inactive in G 1 but displays maximal enzymatic activity during mitotic metaphase. These observations indicate that in a wide variety of vertebrate cells, the cdc2 protein kinase is involved in regulating mitosis. The authors' approach taken toward study of the cdc2 protein kinase highlights the possibilities that now exist for combining the advantages of ascomycete genetics with the cell-free systems of Xenopus and the biochemical advantages of tissue culture cells to investigate fundamental problems of the cell cycle

  8. Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus.

    Science.gov (United States)

    McLellan, Jason S; Correia, Bruno E; Chen, Man; Yang, Yongping; Graham, Barney S; Schief, William R; Kwong, Peter D

    2011-06-24

    Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required. Published by Elsevier Ltd.

  9. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

    Directory of Open Access Journals (Sweden)

    Sonja Rakette

    Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

  10. Teaching language teachers scaffolding professional learning

    CERN Document Server

    Maggioli, Gabriel Diaz

    2012-01-01

    Teaching Language Teachers: Scaffolding Professional Learning provides an updated view of as well as a reader-friendly introduction to the field of Teaching Teachers, with special reference to language teaching. By taking a decidedly Sociocultural perspective, the book addresses the main role of the Teacher of Teachers (ToT) as that of scaffolding the professional learning of aspiring teachers.

  11. Modified gum arabic cross-linked gelatin scaffold for biomedical applications

    International Nuclear Information System (INIS)

    Sarika, P.R.; Cinthya, Kuriakose; Jayakrishnan, A.; Anilkumar, P.R.; James, Nirmala Rachel

    2014-01-01

    The present work deals with development of modified gum arabic cross-linked gelatin scaffold for cell culture. A new biocompatible scaffold was developed by cross-linking gelatin (Gel) with gum arabic, a polysaccharide. Gum arabic was subjected to periodate oxidation to obtain gum arabic aldehyde (GAA). GAA was reacted with gelatin under appropriate pH to prepare the cross-linked hydrogel. Cross-linking occurred due to Schiff's base reaction between aldehyde groups of oxidized gum arabic and amino groups of gelatin. The scaffold prepared from the hydrogel was characterized by swelling properties, degree of cross-linking, in vitro degradation and scanning electron microscopy (SEM). Cytocompatibility evaluation using L-929 and HepG2 cells confirmed non-cytotoxic and non-adherent nature of the scaffold. These properties are essential for generating multicellular spheroids and hence the scaffold is proposed to be a suitable candidate for spheroid cell culture. - Highlights: • Gum arabic cross-linked gelatin scaffold was developed for tissue engineering. • Cross-linking was achieved by Schiff's base reaction. • The scaffold is non-cytotoxic and non adherent to fibroblast and hepatocytes. • The scaffolds are potential candidates for spheroid cell culture

  12. Modified gum arabic cross-linked gelatin scaffold for biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Sarika, P.R. [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547 (India); Cinthya, Kuriakose [Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram, Kerala 695 012 (India); Jayakrishnan, A. [Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036 (India); Anilkumar, P.R., E-mail: anilkumarpr@sctimst.ac.in [Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram, Kerala 695 012 (India); James, Nirmala Rachel, E-mail: nirmala@iist.ac.in [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547 (India)

    2014-10-01

    The present work deals with development of modified gum arabic cross-linked gelatin scaffold for cell culture. A new biocompatible scaffold was developed by cross-linking gelatin (Gel) with gum arabic, a polysaccharide. Gum arabic was subjected to periodate oxidation to obtain gum arabic aldehyde (GAA). GAA was reacted with gelatin under appropriate pH to prepare the cross-linked hydrogel. Cross-linking occurred due to Schiff's base reaction between aldehyde groups of oxidized gum arabic and amino groups of gelatin. The scaffold prepared from the hydrogel was characterized by swelling properties, degree of cross-linking, in vitro degradation and scanning electron microscopy (SEM). Cytocompatibility evaluation using L-929 and HepG2 cells confirmed non-cytotoxic and non-adherent nature of the scaffold. These properties are essential for generating multicellular spheroids and hence the scaffold is proposed to be a suitable candidate for spheroid cell culture. - Highlights: • Gum arabic cross-linked gelatin scaffold was developed for tissue engineering. • Cross-linking was achieved by Schiff's base reaction. • The scaffold is non-cytotoxic and non adherent to fibroblast and hepatocytes. • The scaffolds are potential candidates for spheroid cell culture.

  13. Apple derived cellulose scaffolds for 3D mammalian cell culture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

  14. Improved cell activity on biodegradable photopolymer scaffolds using titanate nanotube coatings

    Energy Technology Data Exchange (ETDEWEB)

    Beke, S., E-mail: szabolcs.beke@iit.it [Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy); Barenghi, R. [IEIIT, National Research Council (CNR), Via De Marini 6, 16149 Genova (Italy); Farkas, B.; Romano, I. [Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy); Kőrösi, L. [Department of Biotechnology, Nanophage Therapy Center, Enviroinvest Corporation, Kertváros u. 2, H-7632 Pécs (Hungary); Scaglione, S. [IEIIT, National Research Council (CNR), Via De Marini 6, 16149 Genova (Italy); Brandi, F. [Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy); Istituto Nazionale di Ottica, CNR, Via G. Moruzzi 1, 56124-Pisa (Italy)

    2014-11-01

    The development of bioactive materials is in the premise of tissue engineering. For several years, surface functionalization of scaffolds has been one of the most promising approaches to stimulate cellular activity and finally improve implant success. Herein, we describe the development of a bioactive composite scaffold composed of a biodegradable photopolymer scaffold and titanate nanotubes (TNTs). The biodegradable photopolymer scaffolds were fabricated by applying mask-projection excimer laser photocuring at 308 nm. TNTs were synthesized and then spin-coated on the porous scaffolds. Upon culturing fibroblast cells on scaffolds, we found that nanotubes coating affects cell viability and proliferation demonstrating that TNT coatings enhance cell growth on the scaffolds by further improving their surface topography. - Highlights: • Biodegradable scaffolds were produced by mask-assisted UV laser photocuring. • Titanate nanotube deposition was carried out without binding compounds or additives. • Titanate nanotube coatings enhanced cell viability and proliferation.

  15. Chitosan/γ-poly(glutamic acid) scaffolds with surface-modified albumin, elastin and poly-l-lysine for cartilage tissue engineering.

    Science.gov (United States)

    Kuo, Yung-Chih; Ku, Hao-Fu; Rajesh, Rajendiran

    2017-09-01

    Cartilage has limited ability to self-repair due to the absence of blood vessels and nerves. The application of biomaterial scaffolds using biomimetic extracellular matrix (ECM)-related polymers has become an effective approach to production of engineered cartilage. Chitosan/γ-poly(glutamic acid) (γ-PGA) scaffolds with different mass ratios were prepared using genipin as a cross-linker and a freeze-drying method, and their surfaces were modified with elastin, human serum albumin (HSA) and poly-l-lysine (PLL). The scaffolds were formed through a complex between NH 3 + of chitosan and COO - of γ-PGA, confirmed by Fourier transform infrared spectroscopy, and exhibited an interconnected porous morphology in field emission scanning electron microscopy analysis. The prepared chitosan/γ-PGA scaffolds, at a 3:1 ratio, obtained the required porosity (90%), pore size (≥100μm), mechanical strength (compressive strength>4MPa, Young's modulus>4MPa) and biodegradation (30-60%) for articular cartilage tissue engineering applications. Surface modification of the scaffolds showed positive indications with improved activity toward cell proliferation (deoxyribonucleic acid), cell adhesion and ECM (glycoaminoglycans and type II collagen) secretion of bovine knee chondrocytes compared with unmodified scaffolds. In caspase-3 detection, elastin had a higher inhibitory effect on chondrocyte apoptosis in vitro, followed by HSA, and then PLL. We concluded that utilizing chitosan/γ-PGA scaffolds with surface active biomolecules, including elastin, HSA and PLL, can effectively promote the growth of chondrocytes, secrete ECM and improve the regenerative ability of cartilaginous tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold.

    Science.gov (United States)

    Sha'ban, Munirah; Yoon, Sun Jung; Ko, Youn Kyung; Ha, Hyun Jung; Kim, Soon Hee; So, Jung Won; Idrus, Ruszymah Bt Hj; Khang, Gilson

    2008-01-01

    Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.

  17. Visualizing feasible operating ranges within tissue engineering systems using a "windows of operation" approach: a perfusion-scaffold bioreactor case study.

    Science.gov (United States)

    McCoy, Ryan J; O'Brien, Fergal J

    2012-12-01

    Tissue engineering approaches to developing functional substitutes are often highly complex, multivariate systems where many aspects of the biomaterials, bio-regulatory factors or cell sources may be controlled in an effort to enhance tissue formation. Furthermore, success is based on multiple performance criteria reflecting both the quantity and quality of the tissue produced. Managing the trade-offs between different performance criteria is a challenge. A "windows of operation" tool that graphically represents feasible operating spaces to achieve user-defined levels of performance has previously been described by researchers in the bio-processing industry. This paper demonstrates the value of "windows of operation" to the tissue engineering field using a perfusion-scaffold bioreactor system as a case study. In our laboratory, perfusion bioreactor systems are utilized in the context of bone tissue engineering to enhance the osteogenic differentiation of cell-seeded scaffolds. A key challenge of such perfusion bioreactor systems is to maximize the induction of osteogenesis but minimize cell detachment from the scaffold. Two key operating variables that influence these performance criteria are the mean scaffold pore size and flow-rate. Using cyclooxygenase-2 and osteopontin gene expression levels as surrogate indicators of osteogenesis, we employed the "windows of operation" methodology to rapidly identify feasible operating ranges for the mean scaffold pore size and flow-rate that achieved user-defined levels of performance for cell detachment and differentiation. Incorporation of such tools into the tissue engineer's armory will hopefully yield a greater understanding of the highly complex systems used and help aid decision making in future translation of products from the bench top to the market place. Copyright © 2012 Wiley Periodicals, Inc.

  18. Mesenchymal Stem Cells in Oriented PLGA/ACECM Composite Scaffolds Enhance Structure-Specific Regeneration of Hyaline Cartilage in a Rabbit Model.

    Science.gov (United States)

    Guo, Weimin; Zheng, Xifu; Zhang, Weiguo; Chen, Mingxue; Wang, Zhenyong; Hao, Chunxiang; Huang, Jingxiang; Yuan, Zhiguo; Zhang, Yu; Wang, Mingjie; Peng, Jiang; Wang, Aiyuan; Wang, Yu; Sui, Xiang; Xu, Wenjing; Liu, Shuyun; Lu, Shibi; Guo, Quanyi

    2018-01-01

    Articular cartilage lacks a blood supply and nerves. Hence, articular cartilage regeneration remains a major challenge in orthopedics. Decellularized extracellular matrix- (ECM-) based strategies have recently received particular attention. The structure of native cartilage exhibits complex zonal heterogeneity. Specifically, the development of a tissue-engineered scaffold mimicking the aligned structure of native cartilage would be of great utility in terms of cartilage regeneration. Previously, we fabricated oriented PLGA/ACECM (natural, nanofibrous, articular cartilage ECM) composite scaffolds. In vitro, we found that the scaffolds not only guided seeded cells to proliferate in an aligned manner but also exhibited high biomechanical strength. To detect whether oriented cartilage regeneration was possible in vivo, we used mesenchymal stem cell (MSC)/scaffold constructs to repair cartilage defects. The results showed that cartilage defects could be completely regenerated. Histologically, these became filled with hyaline cartilage and subchondral bone. Moreover, the aligned structure of cartilage was regenerated and was similar to that of native tissue. In conclusion, the MSC/scaffold constructs enhanced the structure-specific regeneration of hyaline cartilage in a rabbit model and may be a promising treatment strategy for the repair of human cartilage defects.

  19. Chitosan composite three dimensional macrospheric scaffolds for bone tissue engineering.

    Science.gov (United States)

    Vyas, Veena; Kaur, Tejinder; Thirugnanam, Arunachalam

    2017-11-01

    The present work deals with the fabrication of chitosan composite scaffolds with controllable and predictable internal architecture for bone tissue engineering. Chitosan (CS) based composites were developed by varying montmorillonite (MMT) and hydroxyapatite (HA) combinations to fabricate macrospheric three dimensional (3D) scaffolds by direct agglomeration of the sintered macrospheres. The fabricated CS, CS/MMT, CS/HA and CS/MMT/HA 3D scaffolds were characterized for their physicochemical, biological and mechanical properties. The XRD and ATR-FTIR studies confirmed the presence of the individual constituents and the molecular interaction between them, respectively. The reinforcement with HA and MMT showed reduced swelling and degradation rate. It was found that in comparison to pure CS, the CS/HA/MMT composites exhibited improved hemocompatibility and protein adsorption. The sintering of the macrospheres controlled the swelling ability of the scaffolds which played an important role in maintaining the mechanical strength of the 3D scaffolds. The CS/HA/MMT composite scaffold showed 14 folds increase in the compressive strength when compared to pure CS scaffolds. The fabricated scaffolds were also found to encourage the MG 63 cell proliferation. Hence, from the above studies it can be concluded that the CS/HA/MMT composite 3D macrospheric scaffolds have wider and more practical application in bone tissue regeneration applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Manufacture of degradable polymeric scaffolds for bone regeneration.

    Science.gov (United States)

    Ge, Zigang; Jin, Zhaoxia; Cao, Tong

    2008-06-01

    Many innovative technology platforms for promoting bone regeneration have been developed. A common theme among these is the use of scaffolds to provide mechanical support and osteoconduction. Scaffolds can be either ceramic or polymer-based, or composites of both classes of material. Both ceramics and polymers have their own merits and drawbacks, and a better solution may be to synergize the advantageous properties of both materials within composite scaffolds. In this current review, after a brief introduction of the anatomy and physiology of bone, different strategies of fabricating polymeric scaffolds for bone regeneration, including traditional and solid free-form fabrication, are critically discussed and compared, while focusing on the advantages and disadvantages of individual techniques.

  1. Manufacture of degradable polymeric scaffolds for bone regeneration

    International Nuclear Information System (INIS)

    Ge Zigang; Jin Zhaoxia; Cao Tong

    2008-01-01

    Many innovative technology platforms for promoting bone regeneration have been developed. A common theme among these is the use of scaffolds to provide mechanical support and osteoconduction. Scaffolds can be either ceramic or polymer-based, or composites of both classes of material. Both ceramics and polymers have their own merits and drawbacks, and a better solution may be to synergize the advantageous properties of both materials within composite scaffolds. In this current review, after a brief introduction of the anatomy and physiology of bone, different strategies of fabricating polymeric scaffolds for bone regeneration, including traditional and solid free-form fabrication, are critically discussed and compared, while focusing on the advantages and disadvantages of individual techniques. (topical review)

  2. Hybrid Carbon-Based Scaffolds for Applications in Soft Tissue Reconstruction

    Science.gov (United States)

    Lafdi, Khalid; Joseph, Robert M.; Tsonis, Panagiotis A.

    2012-01-01

    Current biomedical scaffolds utilized in surgery to repair soft tissues commonly fail to meet the optimal combination of biomechanical and tissue regenerative properties. Carbon is a scaffold alternative that potentially optimizes the balance between mechanical strength, durability, and function as a cell and biologics delivery vehicle that is necessary to restore tissue function while promoting tissue repair. The goals of this study were to investigate the feasibility of fabricating hybrid fibrous carbon scaffolds modified with biopolymer, polycaprolactone and to analyze their mechanical properties and ability to support cell growth and proliferation. Environmental scanning electron microscopy, micro-computed tomography, and cell adhesion and cell proliferation studies were utilized to test scaffold suitability as a cell delivery vehicle. Mechanical properties were tested to examine load failure and elastic modulus. Results were compared to an acellular dermal matrix scaffold control (GraftJacket® [GJ] Matrix), selected for its common use in surgery for the repair of soft tissues. Results indicated that carbon scaffolds exhibited similar mechanical maximums and capacity to support fibroblast adhesion and proliferation in comparison with GJ. Fibroblast adhesion and proliferation was collinear with carbon fiber orientation in regions of sparsely distributed fibers and occurred in clusters in regions of higher fiber density and low porosity. Overall, fibroblast adhesion and proliferation was greatest in lower porosity carbon scaffolds with highly aligned fibers. Stepwise multivariate regression showed that the variability in maximum load of carbon scaffolds and controls were dependent on unique and separate sets of parameters. These finding suggested that there were significant differences in the functional implications of scaffold design and material properties between carbon and dermis derived scaffolds that affect scaffold utility as a tissue replacement

  3. Biological effects of functionalizing copolymer scaffolds with nanodiamond particles.

    Science.gov (United States)

    Xing, Zhe; Pedersen, Torbjorn O; Wu, Xujun; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Kloss, Frank R; Waag, Thilo; Krueger, Anke; Steinmüller-Nethl, Doris; Mustafa, Kamal

    2013-08-01

    Significant evidence has indicated that poly(L-lactide)-co-(ɛ-caprolactone) [(poly(LLA-co-CL)] scaffolds could be one of the suitable candidates for bone tissue engineering. Oxygen-terminated nanodiamond particles (n-DP) were combined with poly(LLA-co-CL) and revealed to be positive for cell growth. In this study, we evaluated the influence of poly(LLA-co-CL) scaffolds modified by n-DP on attachment, proliferation, differentiation of bone marrow stromal cells (BMSCs) in vitro, and on bone formation using a sheep calvarial defect model. BMSCs were seeded on either poly(LLA-co-CL)- or n-DP-coated scaffolds and incubated for 1 h. Scanning electron microscopy (SEM) and fluorescence microscopy were used in addition to protein and DNA measurements to evaluate cellular attachment on the scaffolds. To determine the effect of n-DP on proliferation of BMSCs, cell/scaffold constructs were harvested after 3 days and evaluated by Bicinchoninic Acid (BCA) protein assay and SEM. In addition, the osteogenic differentiation of cells grown for 2 weeks on the various scaffolds and in a dynamic culture condition was evaluated by real-time RT-PCR. Unmodified and modified scaffolds were implanted into the calvaria of six-year-old sheep. The expression of collagen type I (COL I) and bone morphogenetic protein-2 (BMP-2) after 4 weeks as well as the formation of new bone after 12 and 24 weeks were analyzed by immunohistochemistry and histology. Scaffolds modified with n-DP supported increased cell attachment and the mRNA expression of osteopontin (OPN), bone sialoprotein (BSP), and BMP-2 were significantly increased after 2 weeks of culture. The BMSCs had spread well on the various scaffolds investigated after 3 days in the study with no significant difference in cell proliferation. Furthermore, the in vivo data revealed more positive staining of COL I and BMP-2 in relation to the n-DP-coated scaffolds after 4 weeks and presented more bone formation after 12 and 24 weeks. n

  4. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

    Kwezi, Lusisizwe

    2018-01-22

    Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

  5. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

    Kwezi, Lusisizwe; Wheeler, Janet I; Marondedze, Claudius; Gehring, Christoph A; Irving, Helen R

    2018-01-01

    Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

  6. Laser printing of cells into 3D scaffolds

    International Nuclear Information System (INIS)

    Ovsianikov, A; Gruene, M; Koch, L; Maiorana, F; Chichkov, B; Pflaum, M; Wilhelmi, M; Haverich, A

    2010-01-01

    One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.

  7. Pressure-activated microsyringe (PAM) fabrication of bioactive glass-poly(lactic-co-glycolic acid) composite scaffolds for bone tissue regeneration.

    Science.gov (United States)

    Mattioli-Belmonte, M; De Maria, C; Vitale-Brovarone, C; Baino, F; Dicarlo, M; Vozzi, G

    2017-07-01

    The aim of this work was the fabrication and characterization of bioactive glass-poly(lactic-co-glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA-CEL2 composite scaffolds were innovatively produced by a pressure-activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2-PLGA composite films (CEL2 is an experimental bioactive SiO 2 -P 2 O 5 -CaO-MgO-Na 2 O-K 2 O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10-50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone-like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast-like cells and periosteal-derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone-like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT-PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Hierarchical modeling of activation mechanisms in the ABL and EGFR kinase domains: thermodynamic and mechanistic catalysts of kinase activation by cancer mutations.

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    2009-08-01

    Full Text Available Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition

  9. Kif13b Regulates PNS and CNS Myelination through the Dlg1 Scaffold.

    Directory of Open Access Journals (Sweden)

    Roberta Noseda

    2016-04-01

    Full Text Available Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS and central nervous system (CNS myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1, a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K/v-AKT murine thymoma viral oncogene homolog (AKT pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS.

  10. Bionic Design, Materials and Performance of Bone Tissue Scaffolds

    Directory of Open Access Journals (Sweden)

    Tong Wu

    2017-10-01

    Full Text Available Design, materials, and performance are important factors in the research of bone tissue scaffolds. This work briefly describes the bone scaffolds and their anatomic structure, as well as their biological and mechanical characteristics. Furthermore, we reviewed the characteristics of metal materials, inorganic materials, organic polymer materials, and composite materials. The importance of the bionic design in preoperative diagnosis models and customized bone scaffolds was also discussed, addressing both the bionic structure design (macro and micro structure and the bionic performance design (mechanical performance and biological performance. Materials and performance are the two main problems in the development of customized bone scaffolds. Bionic design is an effective way to solve these problems, which could improve the clinical application of bone scaffolds, by creating a balance between mechanical performance and biological performance.

  11. Electrospun PVA-PCL-HAB scaffold for craniofacial bone regeneration

    DEFF Research Database (Denmark)

    Prabha, Rahul; Kraft, David Christian Evar; Melsen, Birte

    2015-01-01

    -caprolactone (PCL)- triphasic bioceramic(HAB) scaffold to biomimic native tissue and we tested its ability to support osteogenic differentiation of stromal stem cells ( MSC) and its suitability for regeneration of craniofa- cial defects. Physiochemical characterizations of the scaffold, including con- tact angle...... body fluid immersed scaffold samples. Culturing human adult dental pulp stem cells (DPSC) and human bone marrow derived MSC seeded on PVA-PCL-HAB scaffold showed enhanced cell proliferation and in vitro osteoblastic differentiation. Cell-containing scaffolds were implanted subcutaneously in immune...... deficient mice. Histologic ex- amination of retrieved implant sections stained with H&E, Col- lagenType I and Human Vimentin antibody demonstrated that the cells survived in vivo in the implants for at least 8 weeks with evidence of osteoblastic differentiation and angiogenesis within the implants. Our...

  12. In Vivo Study of Ligament-Bone Healing after Anterior Cruciate Ligament Reconstruction Using Autologous Tendons with Mesenchymal Stem Cells Affinity Peptide Conjugated Electrospun Nanofibrous Scaffold

    Directory of Open Access Journals (Sweden)

    Jingxian Zhu

    2013-01-01

    Full Text Available Electrospinning nanofibrous scaffold was commonly used in tissue regeneration recently. Nanofibers with specific topological characteristics were reported to be able to induce osteogenic differentiation of MSCs. In this in vivo study, autologous tendon grafts with lattice-like nanofibrous scaffold wrapping at two ends of autologous tendon were used to promote early stage of ligament-bone healing after rabbit ACL reconstruction. To utilize native MSCs from bone marrow, an MSCs specific affinity peptide E7 was conjugated to nanofibrous meshes. After 3 months, H-E assessment and specific staining of collagen type I, II, and III showed direct ligament-bone insertion with typical four zones (bone, calcified fibrocartilage, fibrocartilage, and ligament in bioactive scaffold reconstruction group. Diameters of bone tunnel were smaller in nanofibrous scaffold conjugated E7 peptide group than those in control group. The failure load of substitution complex also indicated a stronger ligament-bone insertion healing using bioactive scaffold. In conclusion, lattice-like nanofibrous scaffold with specific MSCs affinity peptide has great potential in promoting early stage of ligament-bone healing after ACL reconstruction.

  13. Magnetic responsive hydroxyapatite composite scaffolds construction for bone defect reparation

    Directory of Open Access Journals (Sweden)

    Zeng XB

    2012-07-01

    Full Text Available Xiao Bo Zeng, Hao Hu, Li Qin Xie, Fang Lan, Wen Jiang, Yao Wu, Zhong Wei GuNational Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan, People's Republic of ChinaIntroduction: In recent years, interest in magnetic biomimetic scaffolds for tissue engineering has increased considerably. A type of magnetic scaffold composed of magnetic nanoparticles (MNPs and hydroxyapatite (HA for bone repair has been developed by our research group.Aim and methods: In this study, to investigate the influence of the MNP content (in the scaffolds on the cell behaviors and the interactions between the magnetic scaffold and the exterior magnetic field, a series of MNP-HA magnetic scaffolds with different MNP contents (from 0.2% to 2% were fabricated by immersing HA scaffold into MNP colloid. ROS 17/2.8 and MC3T3-E1 cells were cultured on the scaffolds in vitro, with and without an exterior magnetic field, respectively. The cell adhesion, proliferation and differentiation were evaluated via scanning electron microscopy; confocal laser scanning microscopy; and 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT, alkaline phosphatase, and bone gla protein activity tests.Results: The results demonstrated the positive influence of the magnetic scaffolds on cell adhesion, proliferation, and differentiation. Further, a higher amount of MNPs on the magnetic scaffolds led to more significant stimulation.Conclusion: The magnetic scaffold can respond to the exterior magnetic field and engender some synergistic effect to intensify the stimulating effect of a magnetic field to the proliferation and differentiation of cells.Keywords: magnetic therapy, magnetic nanoparticles, bone repair, magnetic responsive

  14. Dynamic Scaffolding of Socially Regulated Learning in a Computer-Based Learning Environment

    Science.gov (United States)

    Molenaar, Inge; Roda, Claudia; van Boxtel, Carla; Sleegers, Peter

    2012-01-01

    The aim of this study is to test the effects of dynamically scaffolding social regulation of middle school students working in a computer-based learning environment. Dyads in the scaffolding condition (N=56) are supported with computer-generated scaffolds and students in the control condition (N=54) do not receive scaffolds. The scaffolds are…

  15. Development of hybrid scaffolds using ceramic and hydrogel for articular cartilage tissue regeneration.

    Science.gov (United States)

    Seol, Young-Joon; Park, Ju Young; Jeong, Wonju; Kim, Tae-Ho; Kim, Shin-Yoon; Cho, Dong-Woo

    2015-04-01

    The regeneration of articular cartilage consisting of hyaline cartilage and hydrogel scaffolds has been generally used in tissue engineering. However, success in in vivo studies has been rarely reported. The hydrogel scaffolds implanted into articular cartilage defects are mechanically unstable and it is difficult for them to integrate with the surrounding native cartilage tissue. Therefore, it is needed to regenerate cartilage and bone tissue simultaneously. We developed hybrid scaffolds with hydrogel scaffolds for cartilage tissue and with ceramic scaffolds for bone tissue. For in vivo study, hybrid scaffolds were press-fitted into osteochondral tissue defects in a rabbit knee joints and the cartilage tissue regeneration in blank, hydrogel scaffolds, and hybrid scaffolds was compared. In 12th week after implantation, the histological and immunohistochemical analyses were conducted to evaluate the cartilage tissue regeneration. In the blank and hydrogel scaffold groups, the defects were filled with fibrous tissues and the implanted hydrogel scaffolds could not maintain their initial position; in the hybrid scaffold group, newly generated cartilage tissues were morphologically similar to native cartilage tissues and were smoothly connected to the surrounding native tissues. This study demonstrates hybrid scaffolds containing hydrogel and ceramic scaffolds can provide mechanical stability to hydrogel scaffolds and enhance cartilage tissue regeneration at the defect site. © 2014 Wiley Periodicals, Inc.

  16. Gas-Foamed Scaffold Gradients for Combinatorial Screening in 3D

    Directory of Open Access Journals (Sweden)

    Joachim Kohn

    2012-03-01

    Full Text Available Current methods for screening cell-material interactions typically utilize a two-dimensional (2D culture format where cells are cultured on flat surfaces. However, there is a need for combinatorial and high-throughput screening methods to systematically screen cell-biomaterial interactions in three-dimensional (3D tissue scaffolds for tissue engineering. Previously, we developed a two-syringe pump approach for making 3D scaffold gradients for use in combinatorial screening of salt-leached scaffolds. Herein, we demonstrate that the two-syringe pump approach can also be used to create scaffold gradients using a gas-foaming approach. Macroporous foams prepared by a gas-foaming technique are commonly used for fabrication of tissue engineering scaffolds due to their high interconnectivity and good mechanical properties. Gas-foamed scaffold gradient libraries were fabricated from two biodegradable tyrosine-derived polycarbonates: poly(desaminotyrosyl-tyrosine ethyl ester carbonate (pDTEc and poly(desaminotyrosyl-tyrosine octyl ester carbonate (pDTOc. The composition of the libraries was assessed with Fourier transform infrared spectroscopy (FTIR and showed that pDTEc/pDTOc gas-foamed scaffold gradients could be repeatably fabricated. Scanning electron microscopy showed that scaffold morphology was similar between the pDTEc-rich ends and the pDTOc-rich ends of the gradient. These results introduce a method for fabricating gas-foamed polymer scaffold gradients that can be used for combinatorial screening of cell-material interactions in 3D.

  17. In vitro osteoclastogenesis on textile chitosan scaffold

    Directory of Open Access Journals (Sweden)

    C Heinemann

    2010-02-01

    Full Text Available Textile chitosan fibre scaffolds were evaluated in terms of interaction with osteoclast-like cells, derived from human primary monocytes. Part of the scaffolds was further modified by coating with fibrillar collagen type I in order to make the surface biocompatible. Monocytes were cultured directly on the scaffolds in the presence of macrophage colony stimulating factor (M-CSF and receptor activator of nuclear factor kappaB ligand (RANKL for up to 18 days. Confocal laser scanning microscopy (CLSM as well as scanning electron microscopy (SEM revealed the formation of multinuclear osteoclast-like cells on both the raw chitosan fibres and the collagen-coated scaffolds. The modified surface supported the osteoclastogenesis. Differentiation towards the osteoclastic lineage was confirmed by the microscopic detection of cathepsin K, tartrate resistant acid phosphatase (TRAP, acidic compartments using 3-(2,4-dinitroanillino-3’-amino-N-methyldipropylamine (DAMP, immunological detection of TRAP isoform 5b, and analysis of gene expression of the osteoclastic markers TRAP, cathepsin K, vitronectin receptor, and calcitonin receptor using reverse transcription-polymerase chain reaction (RT-PCR. The feature of the collagen-coated but also of the raw chitosan fibre scaffolds to support attachment and differentiation of human monocytes facilitates cell-induced material resorption – one main requirement for successful bone tissue engineering.

  18. Biocompatibility of hydroxyapatite scaffolds processed by lithography-based additive manufacturing.

    Science.gov (United States)

    Tesavibul, Passakorn; Chantaweroad, Surapol; Laohaprapanon, Apinya; Channasanon, Somruethai; Uppanan, Paweena; Tanodekaew, Siriporn; Chalermkarnnon, Prasert; Sitthiseripratip, Kriskrai

    2015-01-01

    The fabrication of hydroxyapatite scaffolds for bone tissue engineering applications by using lithography-based additive manufacturing techniques has been introduced due to the abilities to control porous structures with suitable resolutions. In this research, the use of hydroxyapatite cellular structures, which are processed by lithography-based additive manufacturing machine, as a bone tissue engineering scaffold was investigated. The utilization of digital light processing system for additive manufacturing machine in laboratory scale was performed in order to fabricate the hydroxyapatite scaffold, of which biocompatibilities were eventually evaluated by direct contact and cell-culturing tests. In addition, the density and compressive strength of the scaffolds were also characterized. The results show that the hydroxyapatite scaffold at 77% of porosity with 91% of theoretical density and 0.36 MPa of the compressive strength are able to be processed. In comparison with a conventionally sintered hydroxyapatite, the scaffold did not present any cytotoxic signs while the viability of cells at 95.1% was reported. After 14 days of cell-culturing tests, the scaffold was able to be attached by pre-osteoblasts (MC3T3-E1) leading to cell proliferation and differentiation. The hydroxyapatite scaffold for bone tissue engineering was able to be processed by the lithography-based additive manufacturing machine while the biocompatibilities were also confirmed.

  19. Enhanced chondrogenesis of human nasal septum derived progenitors on nanofibrous scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Shafiee, Abbas [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), Brisbane, QLD (Australia); Seyedjafari, Ehsan [Department of Biotechnology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Sadat Taherzadeh, Elham [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Dinarvand, Peyman [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO (United States); Soleimani, Masoud [Hematology Department, Faculty of Medical Science, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ai, Jafar, E-mail: jafar_ai@tums.ac.ir [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Brain and Spinal Injury Research Center, Imam Hospital, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2014-07-01

    Topographical cues can be exploited to regulate stem cell attachment, proliferation, differentiation and function in vitro and in vivo. In this study, we aimed to investigate the influence of different nanofibrous topographies on the chondrogenic differentiation potential of nasal septum derived progenitors (NSP) in vitro. Aligned and randomly oriented Ploy (L-lactide) (PLLA)/Polycaprolactone (PCL) hybrid scaffolds were fabricated via electrospinning. First, scaffolds were fully characterized, and then NSP were seeded on them to study their capacity to support stem cell attachment, proliferation and chondrogenic differentiation. Compared to randomly oriented nanofibers, aligned scaffolds showed a high degree of nanofiber alignment with much better tensile strength properties. Both scaffolds supported NSP adhesion, proliferation and chondrogenic differentiation. Despite the higher rate of cell proliferation on random scaffolds, a better chondrogenic differentiation was observed on aligned nanofibers as deduced from higher expression of chondrogenic markers such as collagen type II and aggrecan on aligned scaffolds. These findings demonstrate that electrospun constructs maintain NSP proliferation and differentiation, and that the aligned nanofibrous scaffolds can significantly enhance chondrogenic differentiation of nasal septum derived progenitors. - Highlights: • Electrospun nanofiber scaffolds with different topographies were fabricated. • Aligned nanofiber scaffolds had better tensile strength properties. • Nasal septum derived progenitors were cultured on nanofibrous scaffolds. • Both topographies support proliferation and chondrogenic differentiation. • Better chondrogenic differentiation was observed on aligned nanofibers.

  20. Enhanced chondrogenesis of human nasal septum derived progenitors on nanofibrous scaffolds

    International Nuclear Information System (INIS)

    Shafiee, Abbas; Seyedjafari, Ehsan; Sadat Taherzadeh, Elham; Dinarvand, Peyman; Soleimani, Masoud; Ai, Jafar

    2014-01-01

    Topographical cues can be exploited to regulate stem cell attachment, proliferation, differentiation and function in vitro and in vivo. In this study, we aimed to investigate the influence of different nanofibrous topographies on the chondrogenic differentiation potential of nasal septum derived progenitors (NSP) in vitro. Aligned and randomly oriented Ploy (L-lactide) (PLLA)/Polycaprolactone (PCL) hybrid scaffolds were fabricated via electrospinning. First, scaffolds were fully characterized, and then NSP were seeded on them to study their capacity to support stem cell attachment, proliferation and chondrogenic differentiation. Compared to randomly oriented nanofibers, aligned scaffolds showed a high degree of nanofiber alignment with much better tensile strength properties. Both scaffolds supported NSP adhesion, proliferation and chondrogenic differentiation. Despite the higher rate of cell proliferation on random scaffolds, a better chondrogenic differentiation was observed on aligned nanofibers as deduced from higher expression of chondrogenic markers such as collagen type II and aggrecan on aligned scaffolds. These findings demonstrate that electrospun constructs maintain NSP proliferation and differentiation, and that the aligned nanofibrous scaffolds can significantly enhance chondrogenic differentiation of nasal septum derived progenitors. - Highlights: • Electrospun nanofiber scaffolds with different topographies were fabricated. • Aligned nanofiber scaffolds had better tensile strength properties. • Nasal septum derived progenitors were cultured on nanofibrous scaffolds. • Both topographies support proliferation and chondrogenic differentiation. • Better chondrogenic differentiation was observed on aligned nanofibers