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Sample records for heterologous cross-seeding mimics

  1. Vasculitis mimics.

    Science.gov (United States)

    Molloy, Eamonn S; Langford, Carol A

    2008-01-01

    There are many disorders that may closely resemble the clinical, radiologic and/or pathologic features of the primary vasculitides. In this review, we focus on recently described and under-recognized syndromes that may mimic vasculitis. Hereditary causes of large-artery aneurysms such as Marfan's syndrome have long been recognized; recent years have seen a greater understanding of the genetics of Marfan's and other such disorders, including Loeys-Dietz syndrome and Ehler-Danlos syndrome type IV. Under-recognized mimics of medium-vessel vasculitis include segmental arterial mediolysis and Grange syndrome. A large number of entities can mimic small-vessel vasculitis. Recent descriptions of antibodies to human neutrophil elastase have provided insight into the occurrence of antineutrophil cytoplasmic antibodies in cocaine-induced midline destructive lesions. The differential diagnosis of cerebral vasculitis can be particularly difficult. Reversible cerebral vasoconstriction syndromes represent an important class of entities that can readily mimic cerebral vasculitis but have a very different management approach and outcome. The diagnosis of vasculitis requires careful assessment of all available clinical, laboratory, radiologic and pathologic information, and consideration of many competing differential diagnoses. Awareness of noninflammatory mimics of vasculitis is essential to avoid unnecessary and potentially harmful treatment with immunosuppressive agents.

  2. Myositis Mimics.

    Science.gov (United States)

    Michelle, E Harlan; Mammen, Andrew L

    2015-10-01

    Patients with autoimmune myositis typically present with muscle weakness, elevated serum levels of muscle enzymes, and abnormal muscle biopsies. However, patients with other acquired myopathies or genetic muscle diseases may have remarkably similar presentations. Making the correct diagnosis of another muscle disease can prevent these patients from being exposed to the risks of immunosuppressive medications, which benefit those with myositis, but not those with other types of muscle disease. Here, we review some of the most common acquired and inherited muscle diseases that can mimic autoimmune myositis, including inclusion body myositis, limb girdle muscular dystrophies, metabolic myopathies, mitochondrial myopathies, and endocrine myopathies. We emphasize aspects of the medical history, physical exam, laboratory evaluation, and muscle biopsy analysis that can help clinicians distinguish myositis mimics from true autoimmune myositis.

  3. Cross-seeding of prions by aggregated α-synuclein leads to transmissible spongiform encephalopathy.

    Directory of Open Access Journals (Sweden)

    Elizaveta Katorcha

    2017-08-01

    Full Text Available Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.

  4. Mimics of scleroderma

    Directory of Open Access Journals (Sweden)

    Kaveri K Nalianda

    2017-01-01

    Full Text Available Systemic sclerosis is a rare autoimmune connective tissue disorder characterised typically by tightening and tethering of skin. However, several other disorders are also characterised by hardening and thickening of skin. These mimics can be potentially confused with systemic sclerosis, leading to a misdiagnosis. This review describes the aetiopathogenesis, clinical features and treatment of Morphea (localised scleroderma, Scleredema, Scleromyxoedema, Eosinophilic fasciitis, Nephrogenic Systemic Fibrosis, Diabetic Cheiroarthropathy, chronic GVHD, POEMS syndrome and drug induced scleroderma like illness. A careful and thorough clinical assessment is essential in order to differentiate these mimics from each other and from systemic sclerosis, establish the diagnosis, and initiate appropriate treatment.

  5. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

    2017-01-01

    of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

  6. Gene-specific cell labeling using MiMIC transposons.

    Science.gov (United States)

    Gnerer, Joshua P; Venken, Koen J T; Dierick, Herman A

    2015-04-30

    Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Amyotrophic lateral sclerosis mimic syndromes.

    Science.gov (United States)

    Ghasemi, Majid

    2016-04-03

    Amyotrophic lateral sclerosis (ALS) misdiagnosis has many broad implications for the patient and the neurologist. Potentially curative treatments exist for certain ALS mimic syndromes, but delay in starting these therapies may have an unfavorable effect on outcome. Hence, it is important to exclude similar conditions. In this review, we discuss some of the important mimics of ALS.

  8. Enzyme Mimics: Advances and Applications.

    Science.gov (United States)

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

    2016-06-13

    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Mimic of OSU research reactor

    International Nuclear Information System (INIS)

    Lu, Hong; Miller, D.W.

    1991-01-01

    The Ohio State University research reactor (OSURR) is undergoing improvements in its research and educational capabilities. A computer-based digital data acquisition system, including a reactor system mimic, will be installed as part of these improvements. The system will monitor the reactor system parameters available to the reactor operator either in digital parameters available to the reactor operator either in digital or analog form. The system includes two computers. All the signals are sent to computer 1, which processes the data and sends the data through a serial port to computer 2 with a video graphics array VGA monitor, which is utilized to display the mimic system of the reactor

  10. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin

    2017-08-10

    The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

  11. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin; Kuwahara, Hiroyuki; Alazmi, Meshari Saud; Cui, Xuefeng

    2017-01-01

    suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived

  12. Heterologous Protein Expression by Lactococcus lactis

    NARCIS (Netherlands)

    Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

    2012-01-01

    This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a

  13. Synthetic mimics of antimicrobial peptides.

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    Som, Abhigyan; Vemparala, Satyavani; Ivanov, Ivaylo; Tew, Gregory N

    2008-01-01

    Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.

  14. Tiny plastic lung mimics human pulmonary function

    Science.gov (United States)

    Careers Inclusion & Diversity Work-Life Balance Career Resources Apply for a Job Postdocs Students Goals Recycling Green Purchasing Pollution Prevention Reusing Water Resources Environmental Management Releases - 2016 » April » Tiny plastic lung mimics human pulmonary function Tiny plastic lung mimics

  15. Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus▿

    Science.gov (United States)

    Stevens, D. Cole; Henry, Michael R.; Murphy, Kimberly A.; Boddy, Christopher N.

    2010-01-01

    New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters. PMID:20208031

  16. Carbocyclic Carbohydrate Mimics as Potential Glycosidase Inhibitors

    DEFF Research Database (Denmark)

    Fanefjord, Mette; Lundt, Inge

    It has been proven that aminocyclopentanols having the aminogroup adjacent to a carbon sidechain could be potential anomer-selective glycosidase inhibitors [1]. A successful pathway for synthesising mimics to L-carbohydrates 2, by introducing nitrogen to the C6 position in compound 1, has been...... developed in our group. A similar strategy has been used for synthesising mimics of D-carbohydrates. The α,β-unsaturated lactone 3 was cyclised to compound 4 which was further transformed into 5. The nitrogen functionality in compound 7 is introduced by an Overman rearrangement of 6 and the hydroxyl...... functionalities was introduced by either epoxidation or dihydroxylation of 7. Finally, reduction of the lactone ring led to the sugar mimics 8. The synthesis of several isomers of 8 will be presented. [1] a) Kleban, M. ; Hilgers, P. ; Greul, J.N. ; Kugler, R.D. ; Li, J. ; Picasso, S. ; Vogel, P. ; Jäger, V. Chem...

  17. Heterologous expression of cellobiohydrolases in filamentous fungi

    DEFF Research Database (Denmark)

    Zoglowek, Marta; Lübeck, Peter S.; Ahring, Birgitte K.

    2015-01-01

    Cellobiohydrolases are among the most important enzymes functioning in the hydrolysis of crystalline cellulose, significantly contributing to the efficient biorefining of recalcitrant lignocellulosic biomass into biofuels and bio-based products. Filamentous fungi are recognized as both well...... into valuable products. However, due to low cellobiohydrolase activities, certain fungi might be deficient with regard to enzymes of value for cellulose conversion, and improving cellobiohydrolase expression in filamentous fungi has proven to be challenging. In this review, we examine the effects of altering...... promoters, signal peptides, culture conditions and host post-translational modifications. For heterologous cellobiohydrolase production in filamentous fungi to become an industrially feasible process, the construction of site-integrating plasmids, development of protease-deficient strains and glycosylation...

  18. Heterologous gene expression in filamentous fungi.

    Science.gov (United States)

    Su, Xiaoyun; Schmitz, George; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. MIMIC Methods for Assessing Differential Item Functioning in Polytomous Items

    Science.gov (United States)

    Wang, Wen-Chung; Shih, Ching-Lin

    2010-01-01

    Three multiple indicators-multiple causes (MIMIC) methods, namely, the standard MIMIC method (M-ST), the MIMIC method with scale purification (M-SP), and the MIMIC method with a pure anchor (M-PA), were developed to assess differential item functioning (DIF) in polytomous items. In a series of simulations, it appeared that all three methods…

  20. Explosives mimic for testing, training, and monitoring

    Science.gov (United States)

    Reynolds, John G.; Durban, Matthew M.; Gash, Alexander E.; Grapes, Michael D.; Kelley, Ryan S.; Sullivan, Kyle T.

    2018-02-13

    Additive Manufacturing (AM) is used to make mimics for explosives. The process uses mixtures of explosives and matrices commonly used in AM. The explosives are formulated into a mixture with the matrix and printed using AM techniques and equipment. The explosive concentrations are kept less than 10% by wt. of the mixture to conform to requirements of shipping and handling.

  1. Hybrid mimics and hybrid vigor in Arabidopsis

    Science.gov (United States)

    Wang, Li; Greaves, Ian K.; Groszmann, Michael; Wu, Li Min; Dennis, Elizabeth S.; Peacock, W. James

    2015-01-01

    F1 hybrids can outperform their parents in yield and vegetative biomass, features of hybrid vigor that form the basis of the hybrid seed industry. The yield advantage of the F1 is lost in the F2 and subsequent generations. In Arabidopsis, from F2 plants that have a F1-like phenotype, we have by recurrent selection produced pure breeding F5/F6 lines, hybrid mimics, in which the characteristics of the F1 hybrid are stabilized. These hybrid mimic lines, like the F1 hybrid, have larger leaves than the parent plant, and the leaves have increased photosynthetic cell numbers, and in some lines, increased size of cells, suggesting an increased supply of photosynthate. A comparison of the differentially expressed genes in the F1 hybrid with those of eight hybrid mimic lines identified metabolic pathways altered in both; these pathways include down-regulation of defense response pathways and altered abiotic response pathways. F6 hybrid mimic lines are mostly homozygous at each locus in the genome and yet retain the large F1-like phenotype. Many alleles in the F6 plants, when they are homozygous, have expression levels different to the level in the parent. We consider this altered expression to be a consequence of transregulation of genes from one parent by genes from the other parent. Transregulation could also arise from epigenetic modifications in the F1. The pure breeding hybrid mimics have been valuable in probing the mechanisms of hybrid vigor and may also prove to be useful hybrid vigor equivalents in agriculture. PMID:26283378

  2. Three new shuttle vectors for heterologous expression in Zymomonas mobilis

    Directory of Open Access Journals (Sweden)

    Qinghua Cao

    2016-01-01

    Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

  3. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    DEFF Research Database (Denmark)

    Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars

    2008-01-01

    Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal...

  4. Biochemical mechanisms determine the functional compatibility of heterologous genes

    DEFF Research Database (Denmark)

    Porse, Andreas; Schou, Thea S.; Munck, Christian

    2018-01-01

    -gene libraries have suggested that sequence composition is a strong barrier for the successful integration of heterologous genes. Here we sample 200 diverse genes, representing >80% of sequenced antibiotic resistance genes, to interrogate the factors governing genetic compatibility in new hosts. In contrast...... factors governing the functionality and fitness of antibiotic resistance genes. These findings emphasize the importance of biochemical mechanism for heterologous gene compatibility, and suggest physiological constraints as a pivotal feature orienting the evolution of antibiotic resistance....

  5. Histologic Mimics of Basal Cell Carcinoma.

    Science.gov (United States)

    Stanoszek, Lauren M; Wang, Grace Y; Harms, Paul W

    2017-11-01

    - Basal cell carcinoma (BCC) is the most common human malignant neoplasm and is a frequently encountered diagnosis in dermatopathology. Although BCC may be locally destructive, it rarely metastasizes. Many diagnostic entities display morphologic and immunophenotypic overlap with BCC, including nonneoplastic processes, such as follicular induction over dermatofibroma; benign follicular tumors, such as trichoblastoma, trichoepithelioma, or basaloid follicular hamartoma; and malignant tumors, such as sebaceous carcinoma or Merkel cell carcinoma. Thus, misdiagnosis has significant potential to result in overtreatment or undertreatment. - To review key features distinguishing BCC from histologic mimics, including current evidence regarding immunohistochemical markers useful for that distinction. - Review of pertinent literature on BCC immunohistochemistry and differential diagnosis. - In most cases, BCC can be reliably diagnosed by histopathologic features. Immunohistochemistry may provide useful ancillary data in certain cases. Awareness of potential mimics is critical to avoid misdiagnosis and resulting inappropriate management.

  6. Heterologous aggregates promote de novo prion appearance via more than one mechanism.

    Directory of Open Access Journals (Sweden)

    Fatih Arslan

    2015-01-01

    Full Text Available Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q/asparagine (N-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN

  7. Stereocontrolled Synthesis of Methyl Silanediol Peptide Mimics

    DEFF Research Database (Denmark)

    Nielsen, Lone; Lindsay, Karl; Faber, Jesper

    2007-01-01

     The treatment of chiral sulfinimines with (methyldiphenylsilyl)lithium gives R-(methyldiphenylsilyl)-sulfinamides with excellent diastereoselectivity, and in good yield. The presence of α-protons on the imines is also well tolerated. The sulfinamide auxiliary is easily removed via treatment with...... corresponding bis-TMS siloxane via protection with TMSCl, and converted back to the desired silanediol via hydrolysis with aqueous KOH. Efforts to apply this approach to biologically relevant silanediol peptide mimics, with a view to protease inhibition, are described....

  8. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe

    2013-01-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  9. DNA mimic proteins: functions, structures, and bioinformatic analysis.

    Science.gov (United States)

    Wang, Hao-Ching; Ho, Chun-Han; Hsu, Kai-Cheng; Yang, Jinn-Moon; Wang, Andrew H-J

    2014-05-13

    DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.

  10. Production of a heterologous proteinase A by Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    Møller, Kasper; Tidemand, L.D.; Winther, J.R.

    2001-01-01

    In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a refer......In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter...

  11. Upper respiratory tract infection, heterologous immunisation and meningococcal disease

    NARCIS (Netherlands)

    Scholten, R. J.; Bijlmer, H. A.; Tobi, H.; Dankert, J.; Bouter, L. M.

    1999-01-01

    To test the hypothesis that an episode of upper respiratory tract infection or heterologous immunisation is a predisposing factor for the occurrence of meningococcal disease, data from 377 cases of meningococcal disease and their household contacts (n = 1124) were analysed by conditional logistic

  12. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    , (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression...

  13. Reexpression of Prior Stroke Symptoms in Adults: When is a Mimic a Mimic?

    Science.gov (United States)

    Siegler, James E; George, Alexander J; Martin-Schild, Sheryl

    2017-09-01

    A "stroke mimic" refers to any clinical condition that causes neurological symptoms clinically indistinguishable from a cerebral lesion that affects a vascular distribution, but is not caused by ischemia. One subtype of stroke mimic, termed stroke reexpression, is a form of mimicry in which previously recovered or improved stroke symptoms recur in the setting of a neurological disturbance (seizure, hypoperfusion state) or a systemic disturbance (toxic, metabolic, infectious). Many reports of stroke reexpression exist in the literature and are well known to clinicians, but there has been no consensus regarding terminology that has been published to date. The purpose of this review is to summarize several examples of stroke reexpression and propose simple, useful criteria for this clinical condition.

  14. Mycosis Fungoides mimic chronic eczema? Observation

    Directory of Open Access Journals (Sweden)

    Jacek Andruszkiewicz

    2013-07-01

    Full Text Available Mycosis fungoides (MF is the most common type of cutaneous T-cell lymphoma [1]. Because of its great variety of clinical features and nonspecific histological findings (especially in early stages has been named the "great imitator "and can induce many wrong diagnosis [2,3]. Mycosis fungoides (MF, is an epidermotropic lymphoma included as an indolent form in the recent WHO/EORTC classification. From a clinical point of view, the classic disease progression usually is slow and takes over years or even decades, and characterized by the evolution from patches to more infiltrated plaques and eventually to tumours or erythroderma. However, the analysis of the MF disease course has been greatly impaired by the rarity of the disease, thus data about the time course of disease progression and pattern of relapse during time are not well known [4,5]. Therefore very often Mycosis fungoides is misdiagnosed as chronic eczema [6]. MF can also mimic: vitiligo [6], alopecia-Areata [7], ecchymosis [8].

  15. Heterologous production of peptides in plants: fusion proteins and beyond.

    Science.gov (United States)

    Viana, Juliane Flávia Cançado; Dias, Simoni Campos; Franco, Octávio Luiz; Lacorte, Cristiano

    2013-11-01

    Recombinant DNA technology has allowed the ectopic production of proteins and peptides of different organisms leading to biopharmaceutical production in large cultures of bacterial, yeasts and mammalian cells. Otherwise, the expression of recombinant proteins and peptides in plants is an attractive alternative presenting several advantages over the commonly used expression systems including reduced production costs, easy scale-up and reduced risks of pathogen contamination. Different types of proteins and peptides have been expressed in plants, including antibodies, antigens, and proteins and peptides of medical, veterinary and industrial applications. However, apart from providing a proof of concept, the use of plants as platforms for heterologous protein and peptide production still depends on key steps towards optimization including the enhancement of expression levels, manipulation of post-transcriptional modifications and improvements in purification methods. In this review, strategies to increase heterologous protein and peptide stability and accumulation are discussed, focusing on the expression of peptides through the use of gene fusions.

  16. The heterologous expression strategies of antimicrobial peptides in microbial systems.

    Science.gov (United States)

    Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

    2017-12-01

    Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    Science.gov (United States)

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

  18. Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

    Science.gov (United States)

    Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

    2016-08-11

    Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

  19. Radioimmunoassay with heterologous antibody (hetero-antibody RIA)

    International Nuclear Information System (INIS)

    Iwasawa, Atsushi; Hayashi, Hiroaki; Itoh, Zen; Wakabayashi, Katsumi

    1991-01-01

    To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a 'hetero-antibody' RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCGβ or anti-ovine LHβ was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration. (author)

  20. Conformational Compatibility Is Essential for Heterologous Aggregation of α-Synuclein

    NARCIS (Netherlands)

    Sidhu, Arshdeep; Segers-Nolten, Ine; Subramaniam, Vinod

    2016-01-01

    Under aggregation-prone conditions, soluble amyloidogenic protein monomers can self-assemble into fibrils or they can fibrillize on preformed fibrillar seeds (seeded aggregation). Seeded aggregations are known to propagate the morphology of the seeds in the event of cross-seeding. However, not all

  1. Mimic sensor to monitor condition of human health; Mimic sensor wo riyoshita taicho monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Y. [Mechanical Engineering Lab., Tokyo (Japan)

    2000-04-01

    In the aging society where the birth rate decreases and the number of nuclear families increases, it is very important to inquire after the aged or physically handicapped people, to monitor their physical conditions, and to take steps to keep them healthy. As for the in-home physical measurement for the aged or physically handicapped people and the work of health management for them based on such measurement, it is feared that under the present conditions the invalid themselves or their family members or nurses will inevitably have to bear the burden and that nobody can deny the difficulty of continuing such nursing care. If daily physical condition measurement and related data collection are automatically carried out, however, interested people' burden will lessen and in-home heath management will become actually feasible. In this paper, a mimic sensor for realizing such a situation is described, which will measure physical conditions without interfering with the daily life of interested people. Serving as the mimic sensor is a blood flow sensor embedded in a telephone receiver, and changes in the blood flow during telephone conversation and changes in the gaps between peeks will be monitored. The feasibility is shown of continual collection of information necessary for the measurement of physical conditions of the aged or physically handicapped persons. (NEDO)

  2. Surgically Confirmed Intra- and Extratesticular Hematoma Clinically Mimicing Epididymo-Orchitis and Radiologically Mimicing Traumatic Torsion

    International Nuclear Information System (INIS)

    Akay, Sinan; Kaygisiz, Mustafa; Oztas, Muharrem; Turgut, Mustafa Suphi

    2015-01-01

    Scrotal blunt trauma may result in injuries, such as testicular rupture, dislocation, torsion, hematoma, spermatic cord injury or contusion, and epididymal, scrotal, and urethral injuries. Ultrasonography (US) has a crucial role in the evaluation of those kinds of pathologies. Early diagnosis of testicular rupture may lead to the salvaging of the testicle by prompt surgical exploration within 72 h. A 21-year-old male with right scrotal swelling and pain complaints was admitted to another hospital one month ago. Epididymo-orchitis was diagnosed and the patient was given medical treatment. The patient was admitted to our emergency service with ongoing complaints. The patient stated that the pain and swelling suddenly developed after prolonged marching. On US, a large hematoma was detected between the leaves of the tunica vaginalis, and rupture from the lower pole was diagnosed. On color Doppler US, no vascularity was observed in the heterogeneous testicular parenchyma. Traumatic torsion was primarily suggested. Intraoperatively, an extratesticular hematoma, hematocele, and rupture were confirmed but torsion was not found. Because the hematoma entirely replaced the normal parenchyma, orchiectomy was performed. Testicular rupture may occur even after moderate sportive action such as prolonged marching. If any delay occurs in diagnosis and/or operation, the coagulum may replace the entire parenchyma. We think that US and clinical findings may sometimes mislead the physicians and cause misdiagnoses, such as inflammation or torsion. In this paper, we present a complex delayed diagnosed example case of testicular rupture clinically mimicing epididymo-orchitis and radiologically mimicing traumatic testicular torsion

  3. Stroke mimic diagnoses presenting to a hyperacute stroke unit.

    Science.gov (United States)

    Dawson, Ang; Cloud, Geoffrey C; Pereira, Anthony C; Moynihan, Barry J

    2016-10-01

    Stroke services have been centralised in several countries in recent years. Diagnosing acute stroke is challenging and a high proportion of patients admitted to stroke units are diagnosed as a non-stroke condition (stroke mimics). This study aims to describe the stroke mimic patient group, including their impact on stroke services. We analysed routine clinical data from 2,305 consecutive admissions to a stroke unit at St George's Hospital, London. Mimic groupings were derived from 335 individual codes into 17 groupings. From 2,305 admissions, 555 stroke mimic diagnoses were identified (24.2%) and 72% of stroke mimics had at least one stroke risk factor. Common mimic diagnoses were headache, seizure and syncope. Medically unexplained symptoms and decompensation of underlying conditions were also common. Median length of stay was 1 day; a diagnosis of dementia (p=0.028) or needing MRI (p=0.006) was associated with a longer stay. Despite emergency department assessment by specialist clinicians and computed tomography brain, one in four suspected stroke patients admitted to hospital had a non-stroke diagnosis. Stroke mimics represent a heterogeneous patient group with significant impacts on stroke services. Co-location of stroke and acute neurology services may offer advantages where service reorganisation is being considered. © Royal College of Physicians 2016. All rights reserved.

  4. Membrane-Mimic Nanoparticles for Drug and Gene Delivery

    KAUST Repository

    Alamoudi, Kholod

    2017-01-01

    -mimic nanoparticles are considered highly attractive materials for in vivo and in vitro applications. Synthetic membrane vesicles (liposomes) and nanoconstructs built with native cancer cellular membrane are excellent scaffolds to improve cellular delivery. Liposomes

  5. Glutathione transferase mimics : Micellar catalysis of an enzymic reaction

    NARCIS (Netherlands)

    Lindkvist, Björn; Weinander, Rolf; Engman, Lars; Koetse, Marc; Engberts, Jan B.F.N.; Morgenstern, Ralf

    1997-01-01

    Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as alpha,beta-unsaturated carbonyls. The nucleophilic aromatic

  6. Decontamination Of Bacterial Spores by a Peptide-Mimic

    National Research Council Canada - National Science Library

    Nagarajan, R; Muller, Wayne S; Ashley, Rebekah; Mello, Charlene M

    2006-01-01

    .... In this work, we demonstrate that a peptide-mimic (cationic, amphiphilic) chemical agent, dodecylamine is capable of performing the dual functions of germinating the dormant spore as well as deactivating...

  7. Process mining in oncology using the MIMIC-III dataset

    Science.gov (United States)

    Prima Kurniati, Angelina; Hall, Geoff; Hogg, David; Johnson, Owen

    2018-03-01

    Process mining is a data analytics approach to discover and analyse process models based on the real activities captured in information systems. There is a growing body of literature on process mining in healthcare, including oncology, the study of cancer. In earlier work we found 37 peer-reviewed papers describing process mining research in oncology with a regular complaint being the limited availability and accessibility of datasets with suitable information for process mining. Publicly available datasets are one option and this paper describes the potential to use MIMIC-III, for process mining in oncology. MIMIC-III is a large open access dataset of de-identified patient records. There are 134 publications listed as using the MIMIC dataset, but none of them have used process mining. The MIMIC-III dataset has 16 event tables which are potentially useful for process mining and this paper demonstrates the opportunities to use MIMIC-III for process mining in oncology. Our research applied the L* lifecycle method to provide a worked example showing how process mining can be used to analyse cancer pathways. The results and data quality limitations are discussed along with opportunities for further work and reflection on the value of MIMIC-III for reproducible process mining research.

  8. Validating the TeleStroke Mimic Score: A Prediction Rule for Identifying Stroke Mimics Evaluated Over Telestroke Networks.

    Science.gov (United States)

    Ali, Syed F; Hubert, Gordian J; Switzer, Jeffrey A; Majersik, Jennifer J; Backhaus, Roland; Shepard, L Wylie; Vedala, Kishore; Schwamm, Lee H

    2018-03-01

    Up to 30% of acute stroke evaluations are deemed stroke mimics, and these are common in telestroke as well. We recently published a risk prediction score for use during telestroke encounters to differentiate stroke mimics from ischemic cerebrovascular disease derived and validated in the Partners TeleStroke Network. Using data from 3 distinct US and European telestroke networks, we sought to externally validate the TeleStroke Mimic (TM) score in a broader population. We evaluated the TM score in 1930 telestroke consults from the University of Utah, Georgia Regents University, and the German TeleMedical Project for Integrative Stroke Care Network. We report the area under the curve in receiver-operating characteristic curve analysis with 95% confidence interval for our previously derived TM score in which lower TM scores correspond with a higher likelihood of being a stroke mimic. Based on final diagnosis at the end of the telestroke consultation, there were 630 of 1930 (32.6%) stroke mimics in the external validation cohort. All 6 variables included in the score were significantly different between patients with ischemic cerebrovascular disease versus stroke mimics. The TM score performed well (area under curve, 0.72; 95% confidence interval, 0.70-0.73; P mimic during telestroke consultation in these diverse cohorts was similar to its performance in our original cohort. Predictive decision-support tools like the TM score may help highlight key clinical differences between mimics and patients with stroke during complex, time-critical telestroke evaluations. © 2018 American Heart Association, Inc.

  9. Identification of stroke mimics among clinically diagnosed acute strokes.

    Science.gov (United States)

    Tuntiyatorn, Lojana; Saksornchai, Pichaya; Tunlayadechanont, Supoch

    2013-09-01

    Stroke is a clinically syndrome of a sudden onset of neurological deficit in a vascular cause. Stroke mimics is the non-vascular disorders with stroke-like clinical symptoms. It is important to distinguish true stroke from mimics since treatment plan may differ To determine the incidence of the stroke mimics and identify their etiologies. All non-contrast head CT of the patients with clinically diagnosed stroke who immediately received imaging upon arrival at the emergency department of the university hospital were retrospectively reviewed in 12-month period between January 1 and December 31, 2008. Medical records, laboratory results, MRI, and 6-month clinical follow-up records were reviewed for final diagnosis. Seven hundred four patients were included in this study, including 363 (51.5%) men and 341 (48.5%) women with range in age from 24 to 108 years. Amongst those, 417 (59.2%) were ischemic stroke, 80 (11.40%) were hemorrhagic stroke, 186 (26.4%) were stroke-mimics, and 21 (3%) were inconclusive. The etiologies among stroke-mimics were metabolic/intoxication (35, 18.8%), sepsis (28, 15.0%), seizure (21, 11.3%), syncope (20, 10.8%), subdural hemorrhage (14, 7.5%), vertigo (11, 6.0%), brain tumor (10, 5.30%), central nervous system infection (5, 2.7%), others (26, 14.0%), and unspecified (16, 8.6%). Incidence rates and etiologies of the stroke mimics were similar to the western reports. However the frequency of each mimic was not.

  10. Adaptation in the innate immune system and heterologous innate immunity.

    Science.gov (United States)

    Martin, Stefan F

    2014-11-01

    The innate immune system recognizes deviation from homeostasis caused by infectious or non-infectious assaults. The threshold for its activation seems to be established by a calibration process that includes sensing of microbial molecular patterns from commensal bacteria and of endogenous signals. It is becoming increasingly clear that adaptive features, a hallmark of the adaptive immune system, can also be identified in the innate immune system. Such adaptations can result in the manifestation of a primed state of immune and tissue cells with a decreased activation threshold. This keeps the system poised to react quickly. Moreover, the fact that the innate immune system recognizes a wide variety of danger signals via pattern recognition receptors that often activate the same signaling pathways allows for heterologous innate immune stimulation. This implies that, for example, the innate immune response to an infection can be modified by co-infections or other innate stimuli. This "design feature" of the innate immune system has many implications for our understanding of individual susceptibility to diseases or responsiveness to therapies and vaccinations. In this article, adaptive features of the innate immune system as well as heterologous innate immunity and their implications are discussed.

  11. Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

    Directory of Open Access Journals (Sweden)

    Hui Xu

    2015-01-01

    Full Text Available The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA and Csac_1078 (celB from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA and TM0070 (xynB, resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization.

  12. Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

    Science.gov (United States)

    Heshof, Ruud; van Schayck, J Paul; Tamayo-Ramos, Juan Antonio; de Graaff, Leo H

    2014-01-01

    Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

  13. Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

    Science.gov (United States)

    Naqvi, Tatheer; Cheesman, Matthew J; Williams, Michelle R; Campbell, Peter M; Ahmed, Safia; Russell, Robyn J; Scott, Colin; Oakeshott, John G

    2009-10-26

    The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

  14. A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination.

    Science.gov (United States)

    León-Ortiz, Ana María; Panier, Stephanie; Sarek, Grzegorz; Vannier, Jean-Baptiste; Patel, Harshil; Campbell, Peter J; Boulton, Simon J

    2018-01-18

    Erroneous DNA repair by heterologous recombination (Ht-REC) is a potential threat to genome stability, but evidence supporting its prevalence is lacking. Here we demonstrate that recombination is possible between heterologous sequences and that it is a source of chromosomal alterations in mitotic and meiotic cells. Mechanistically, we find that the RTEL1 and HIM-6/BLM helicases and the BRCA1 homolog BRC-1 counteract Ht-REC in Caenorhabditis elegans, whereas mismatch repair does not. Instead, MSH-2/6 drives Ht-REC events in rtel-1 and brc-1 mutants and excessive crossovers in rtel-1 mutant meioses. Loss of vertebrate Rtel1 also causes a variety of unusually large and complex structural variations, including chromothripsis, breakage-fusion-bridge events, and tandem duplications with distant intra-chromosomal insertions, whose structure are consistent with a role for RTEL1 in preventing Ht-REC during break-induced replication. Our data establish Ht-REC as an unappreciated source of genome instability that underpins a novel class of complex genome rearrangements that likely arise during replication stress. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

    Science.gov (United States)

    Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

    2017-01-01

    Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

  16. Molecular mimics of the tumour antigen MUC1.

    Directory of Open Access Journals (Sweden)

    Tharappel C James

    Full Text Available A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as "self", and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as 'proof of principle' we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1 from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.

  17. Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

    Science.gov (United States)

    Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

    2017-09-18

    Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

  18. Branchial cleft anomalies and their mimics: computed tomographic evaluation

    International Nuclear Information System (INIS)

    Harnsberger, H.R.; Mancuso, A.A.; Muraki, A.S.; Byrd, S.E.; Dillon, W.P.; Johnson, L.P.; Hanafee, W.N.

    1984-01-01

    A review was made of the clinical records and radiographic examinations of 38 patients with neck lesions clinically suspected of being branchial cleft anomalies. The impact of computed tomography in this sometimes confusing clinical picture was assessed and CT criteria for diagnosing branchial cleft anomalies (BCAs) and differentiating them from their mimics were identified. Seventeen branchial cleft anomalies and 21 BCA mimics were evaluated. A definitive CT diagnosis of second branchial cleft cysts based on characteristic morphology, location, and displacement of surrounding structures was possible in 80% of cases. CT was found to be the best radiographic examination in making a definitive diagnosis of BCA if a neck mass was present

  19. Overcoming tumor resistance by heterologous adeno-poxvirus combination therapy

    Directory of Open Access Journals (Sweden)

    Markus Vähä-Koskela

    2014-01-01

    Full Text Available Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.

  20. Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bergman, Alexandra; Siewers, Verena; Nielsen, Jens

    2016-01-01

    Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does...... not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from...... C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some...

  1. Heterologous embryo transfer: Magisterial answers and metaphysical questions.

    Science.gov (United States)

    Accad, Michel

    2014-02-01

    The debate regarding the morality of heterologous embryo transfer (HET) as a solution for the fate of cryopreserved embryos remains active. This paper endeavors to show that the magisterial instructions on bioethical issues can only lead to the conclusion that HET is always morally illicit. I begin by showing that the text of Dignitas personae recognizes HET as a procedure accomplishing a procreative function, and I indicate that it is through gestation that this procreative function occurs. I further show that the previous Instruction, Donum vitae, implicitly points to an ontological or spiritual consideration at play during gestation. This consideration is likely related to the procreative function identified in Dignitas personae. Finally, I place these two textual arguments in the context of the debate concerning HET and conclude that metaphysical questions must be clarified in order for the immorality of HET to be understood from a suitable anthropological perspective and gain more widespread acceptance.

  2. Heterologous transporter expression for improved fatty alcohol secretion in yeast

    DEFF Research Database (Denmark)

    Hu, Yating; Zhu, Zhiwei; Nielsen, Jens

    2017-01-01

    The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production...... transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl......-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known...

  3. Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques.

    Directory of Open Access Journals (Sweden)

    Matthew T Aliota

    2016-12-01

    Full Text Available Zika virus (ZIKV; Flaviviridae, Flavivirus was declared a public health emergency of international concern by the World Health Organization (WHO in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV.Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form.An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of

  4. Heterologous Protection against Malaria after Immunization with Plasmodium falciparum Sporozoites.

    Directory of Open Access Journals (Sweden)

    Remko Schats

    Full Text Available Sterile protection in >90% of volunteers against homologous Plasmodium falciparum infection has been achieved only using the controlled human malaria infection (CHMI model. This efficient model involves whole parasite immunizations under chloroquine prophylaxis (CPS-immunization, requiring only 30-45 mosquitoes bites infected with P. falciparum-sporozoites. Given the large diversity of P. falciparum parasites, it is essential to assess protection against heterologous parasite strains.In an open-label follow-up study, 16 volunteers previously CPS-immunized and challenged with P. falciparum NF54 (West-Africa in a dose de-escalation and challenge trial were re-challenged with clone NF135.C10 (Cambodia at 14 months after the last immunization (NCT01660854.Two out of thirteen NF54 protected volunteers previously fully protected against NF54 were also fully protected against NF135.C10, while 11/13 showed a delayed patency (median prepatent period of 10.5 days (range 9.0-15.5 versus 8.5 days in 5 malaria-naïve controls (p = 0.0005. Analysis of patency by qPCR indicated a 91 to >99% estimated reduction of liver parasite load in 7/11 partially protected subjects. Three volunteers previously not protected against NF54, were also not protected against NF135.C10.This study shows that CPS-immunization can induce heterologous protection for a period of more than one year, which is a further impetus for clinical development of whole parasite vaccines.Clinicaltrials.gov NCT01660854.

  5. Synthesis of aminocyclopentanols: a-D-galacto configured sugar mimics

    DEFF Research Database (Denmark)

    Bøjstrup, Marie; Lundt, Inge

    2005-01-01

    Four aminocyclopentanols, as mimics of putative intermediates in hydrolysis of a-D-galactosides, have been synthesized through a number of stereoselective transformations, using the cis-fused cyclopentane-1,4-lactone (1R, 5S, 7R, 8R)-7,8-dihydroxy-2-oxabicyclo[3.3.0]oct-3-one 1 as a chiral buildi...

  6. Arrhythmogenic right ventricular cardiomyopathy mimics: role of cardiovascular magnetic resonance

    Science.gov (United States)

    2013-01-01

    Background Cardiovascular magnetic resonance (CMR) is commonly used in patients with suspected arrhythmogenic right ventricular cardiomyopathy (ARVC) based on ECG, echocardiogram and Holter. However, various diseases may present with clinical characteristics resembling ARVC causing diagnostic dilemmas. The aim of this study was to explore the role of CMR in the differential diagnosis of patients with suspected ARVC. Methods 657 CMR referrals suspicious for ARVC in a single tertiary referral centre were analysed. Standardized CMR imaging protocols for ARVC were performed. Potential ARVC mimics were grouped into: 1) displacement of the heart, 2) right ventricular overload, and 3) non ARVC-like cardiac scarring. For each, a judgment of clinical impact was made. Results Twenty patients (3.0%) fulfilled imaging ARVC criteria. Thirty (4.6%) had a potential ARVC mimic, of which 25 (3.8%) were considered clinically important: cardiac displacement (n=17), RV overload (n=7) and non-ARVC like myocardial scarring (n=4). One patient had two mimics; one patient had dual pathology with important mimic and ARVC. RV overload and scarring conditions were always thought clinically important whilst the importance of cardiac displacement depended on the degree of displacement from severe (partial absence of pericardium) to epiphenomenon (minor kyphoscoliosis). Conclusions Some patients referred for CMR with suspected ARVC fulfil ARVC imaging criteria (3%) but more have otherwise unrecognised diseases (4.6%) mimicking potentially ARVC. Clinical assessment should reflect this, emphasising the assessment and/or exclusion of potential mimics in parallel with the detection of ARVC major and minor criteria. PMID:23398958

  7. Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.

    2015-01-01

    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential

  8. Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin

    2014-01-01

    Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as stre...

  9. Targeting a heterologous protein to multiple plant organelles via rationally designed 5? mRNA tags

    NARCIS (Netherlands)

    Voges, M.J.; Silver, P.A.; Way, J.C.; Mattozzi, M.D.

    2013-01-01

    Background Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments. Results We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in

  10. Transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans

    CSIR Research Space (South Africa)

    Crampton, Michael C

    2010-07-01

    Full Text Available This presentation focused on the transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans. It concludes to a successful implementation of a high throughput mRNA sandwich hybridisation...

  11. Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

    Science.gov (United States)

    Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2009-11-01

    Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

  12. Spontaneous Spinal Epidural Hematoma as a Potentially Important Stroke Mimic

    Directory of Open Access Journals (Sweden)

    Tetsu Akimoto

    2014-01-01

    Full Text Available Hemiparesis develops in response to a wide range of neurological disorders, such as stroke, neoplasms and several inflammatory processes. Occasionally, it may also occur due to a lesion located in the high cervical spinal cord. In this concise review, we describe the features of spontaneous spinal epidural hematoma, which should be included in the large list of stroke mimics. Various concerns regarding the diagnostic and therapeutic conundrums relating to the condition are also discussed.

  13. Constraining cyclic peptides to mimic protein structure motifs

    DEFF Research Database (Denmark)

    Hill, Timothy A.; Shepherd, Nicholas E.; Diness, Frederik

    2014-01-01

    peptides can have protein-like biological activities and potencies, enabling their uses as biological probes and leads to therapeutics, diagnostics and vaccines. This Review highlights examples of cyclic peptides that mimic three-dimensional structures of strand, turn or helical segments of peptides...... and proteins, and identifies some additional restraints incorporated into natural product cyclic peptides and synthetic macrocyclic pepti-domimetics that refine peptide structure and confer biological properties....

  14. The ability of lizards to identify an artificial Batesian mimic.

    Science.gov (United States)

    Beneš, Josef; Veselý, Petr

    2017-08-01

    Birds are usually considered the main predators shaping the evolution of aposematic signals and mimicry. Nevertheless, some lizards also represent predominately visually oriented predators, so they may also play an important role in the evolution of aposematism. Despite this fact, experimental evidence regarding the responses of lizards to aposematic prey is very poor compared to such evidence in birds. Lizards possess very similar sensory and cognitive abilities to those of birds and their response to aposematic prey may thus be affected by very similar processes. We investigated the reactions of a lizard, the Gran Canaria skink (Chalcides sexlineatus), to an aposematic prey and its artificial Batesian mimic. Further, we attempted to ascertain whether the lizard's food experience has any effect on its ability to recognise an artificial Batesian mimic, by using two groups of predators differing in their prior experience with the prey from which the mimic was fabricated. The red firebug (Pyrrhocoris apterus) was used as an aposematic model, and the Guyana spotted roach (Blaptica dubia) as the palatable prey from which the mimic was fabricated. The appearance of the roach was modified by a paper sticker placed on its back. The skinks showed a strong aversion towards the model firebug. They also avoided attacking the cockroaches with the firebug pattern sticker. This suggests that a visual rather than a chemical signal is responsible for this aversion. The protection provided by the firebug sticker was even effective when the skinks were familiar with unmodified cockroaches (previous food experience). Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Use of heterologous expressed polyketide synthase and small molecule foldases to make aromatic and cyclic compounds

    DEFF Research Database (Denmark)

    2016-01-01

    A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell.......A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell....

  16. From fresh heterologous oocyte donation to autologous oocyte banking.

    Science.gov (United States)

    Stoop, D

    2012-01-01

    Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock.

  17. Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host

    Science.gov (United States)

    Coleman, Jeffrey J.; Muhammed, Maged; Kasperkovitz, Pia V.; Vyas, Jatin M.; Mylonakis, Eleftherios

    2011-01-01

    Members of the fungal genus Fusarium are capable of manifesting in a multitude of clinical infections, most commonly in immunocompromised patients. In order to better understand the interaction between the fungus and host, we have developed the larvae of the greater wax moth, Galleria mellonella, as a heterologous host for fusaria. When conidia are injected into the hemocoel of this Lepidopteran system, both clinical and environmental isolates of the fungus are able to kill the larvae at 37°C, although killing occurs more rapidly when incubated at 30°C. This killing was dependent on several other factors besides temperature, including the Fusarium strain, the number of conidia injected, and the conidia morphology, where macroconidia are more virulent than their microconidia counterpart. There was a correlation in the killing rate of Fusarium spp. when evaluated in G. mellonella and a murine model. In vivo studies indicated G. mellonella hemocytes were capable of initially phagocytosing both conidial morphologies. The G. mellonella system was also used to evaluate antifungal agents, and amphotericin B was able to confer a significant increase in survival to Fusarium infected-larvae. The G. mellonella-Fusarium pathogenicity system revealed that virulence of Fusarium spp. is similar, regardless of the origin of the isolate, and that mammalian endothermy is a major deterrent for Fusarium infection and therefore provides a suitable alternative to mammalian models to investigate the interaction between the host and this increasingly important fungal pathogen. PMID:22115447

  18. Characterization of a Lactococcus lactis promoter for heterologous protein production

    Directory of Open Access Journals (Sweden)

    Christian E. Ogaugwu

    2018-03-01

    Full Text Available Constitutively active promoter elements for heterologous protein production in Lactococcus lactis are scarce. Here, the promoter of the PTS-IIC gene cluster from L. lactis NZ3900 is described. This promoter was cloned upstream of an enhanced green fluorescent protein, GFPmut3a, and transformed into L. lactis. Transformants produced up to 13.5 μg of GFPmut3a per milliliter of log phase cells. Addition of cellobiose further increased the production of GFPmut3a by up to two-fold when compared to glucose. Analysis of mutations at two specific positions in the PTS-IIC promoter showed that a ‘T’ to ‘G’ mutation within the −35 element resulted in constitutive expression in glucose, while a ‘C’ at nucleotide 7 in the putative cre site enhanced promoter activity in cellobiose. Finally, this PTS-IIC promoter is capable of mediating protein expression in Bacillus subtilis and Escherichia coli Nissle 1917, suggesting the potential for future biotechnological applications of this element and its derivatives.

  19. A Salmonella nanoparticle mimic overcomes multidrug resistance in tumours.

    Science.gov (United States)

    Mercado-Lubo, Regino; Zhang, Yuanwei; Zhao, Liang; Rossi, Kyle; Wu, Xiang; Zou, Yekui; Castillo, Antonio; Leonard, Jack; Bortell, Rita; Greiner, Dale L; Shultz, Leonard D; Han, Gang; McCormick, Beth A

    2016-07-25

    Salmonella enterica serotype Typhimurium is a food-borne pathogen that also selectively grows in tumours and functionally decreases P-glycoprotein (P-gp), a multidrug resistance transporter. Here we report that the Salmonella type III secretion effector, SipA, is responsible for P-gp modulation through a pathway involving caspase-3. Mimicking the ability of Salmonella to reverse multidrug resistance, we constructed a gold nanoparticle system packaged with a SipA corona, and found this bacterial mimic not only accumulates in tumours but also reduces P-gp at a SipA dose significantly lower than free SipA. Moreover, the Salmonella nanoparticle mimic suppresses tumour growth with a concomitant reduction in P-gp when used with an existing chemotherapeutic drug (that is, doxorubicin). On the basis of our finding that the SipA Salmonella effector is fundamental for functionally decreasing P-gp, we engineered a nanoparticle mimic that both overcomes multidrug resistance in cancer cells and increases tumour sensitivity to conventional chemotherapeutics.

  20. An in silico platform for the design of heterologous pathways in nonnative metabolite production

    Directory of Open Access Journals (Sweden)

    Chatsurachai Sunisa

    2012-05-01

    Full Text Available Abstract Background Microorganisms are used as cell factories to produce valuable compounds in pharmaceuticals, biofuels, and other industrial processes. Incorporating heterologous metabolic pathways into well-characterized hosts is a major strategy for obtaining these target metabolites and improving productivity. However, selecting appropriate heterologous metabolic pathways for a host microorganism remains difficult owing to the complexity of metabolic networks. Hence, metabolic network design could benefit greatly from the availability of an in silico platform for heterologous pathway searching. Results We developed an algorithm for finding feasible heterologous pathways by which nonnative target metabolites are produced by host microorganisms, using Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae as templates. Using this algorithm, we screened heterologous pathways for the production of all possible nonnative target metabolites contained within databases. We then assessed the feasibility of the target productions using flux balance analysis, by which we could identify target metabolites associated with maximum cellular growth rate. Conclusions This in silico platform, designed for targeted searching of heterologous metabolic reactions, provides essential information for cell factory improvement.

  1. The frequency, characteristics and aetiology of stroke mimic presentations: a narrative review.

    Science.gov (United States)

    McClelland, Graham; Rodgers, Helen; Flynn, Darren; Price, Christopher I

    2018-05-01

    A significant proportion of patients with acute stroke symptoms have an alternative 'mimic' diagnosis. A narrative review was carried out to explore the frequency, characteristics and aetiology of stroke mimics. Prehospital and thrombolysis-treated patients were described separately. Overall, 9972 studies were identified from the initial search and 79 studies were included with a median stroke mimic rate of 19% (range: 1-64%). The prehospital median was 27% (range: 4-43%) and the thrombolysis median 10% (range: 1-25%). Seizures, migraines and psychiatric disorders are the most frequently reported causes of stroke mimics. Several characteristics are consistently associated with stroke mimics; however, they do not fully exclude the possibility of stroke. Nineteen per cent of suspected stroke patients had a mimic condition. Stroke mimics were more common with younger age and female sex. The range of mimic diagnoses, a lack of clear differentiating characteristics and the short treatment window for ischaemic stroke create challenges for early identification.

  2. Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

    Science.gov (United States)

    Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

    2015-01-01

    Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple

  3. Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2010-03-01

    Full Text Available Abstract Background Baker's yeast (Saccharomyces cerevisiae has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose. Results In this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose. Conclusions Kinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native S. cerevisiae transporters increased significantly during cultivation in xylose and

  4. The MIMIC Method with Scale Purification for Detecting Differential Item Functioning

    Science.gov (United States)

    Wang, Wen-Chung; Shih, Ching-Lin; Yang, Chih-Chien

    2009-01-01

    This study implements a scale purification procedure onto the standard MIMIC method for differential item functioning (DIF) detection and assesses its performance through a series of simulations. It is found that the MIMIC method with scale purification (denoted as M-SP) outperforms the standard MIMIC method (denoted as M-ST) in controlling…

  5. The Mediated MIMIC Model for Understanding the Underlying Mechanism of DIF

    Science.gov (United States)

    Cheng, Ying; Shao, Can; Lathrop, Quinn N.

    2016-01-01

    Due to its flexibility, the multiple-indicator, multiple-causes (MIMIC) model has become an increasingly popular method for the detection of differential item functioning (DIF). In this article, we propose the mediated MIMIC model method to uncover the underlying mechanism of DIF. This method extends the usual MIMIC model by including one variable…

  6. The MIMIC Code Repository: enabling reproducibility in critical care research.

    Science.gov (United States)

    Johnson, Alistair Ew; Stone, David J; Celi, Leo A; Pollard, Tom J

    2018-01-01

    Lack of reproducibility in medical studies is a barrier to the generation of a robust knowledge base to support clinical decision-making. In this paper we outline the Medical Information Mart for Intensive Care (MIMIC) Code Repository, a centralized code base for generating reproducible studies on an openly available critical care dataset. Code is provided to load the data into a relational structure, create extractions of the data, and reproduce entire analysis plans including research studies. Concepts extracted include severity of illness scores, comorbid status, administrative definitions of sepsis, physiologic criteria for sepsis, organ failure scores, treatment administration, and more. Executable documents are used for tutorials and reproduce published studies end-to-end, providing a template for future researchers to replicate. The repository's issue tracker enables community discussion about the data and concepts, allowing users to collaboratively improve the resource. The centralized repository provides a platform for users of the data to interact directly with the data generators, facilitating greater understanding of the data. It also provides a location for the community to collaborate on necessary concepts for research progress and share them with a larger audience. Consistent application of the same code for underlying concepts is a key step in ensuring that research studies on the MIMIC database are comparable and reproducible. By providing open source code alongside the freely accessible MIMIC-III database, we enable end-to-end reproducible analysis of electronic health records. © The Author 2017. Published by Oxford University Press on behalf of the American Medical Informatics Association.

  7. A Less Known Stroke Mimic: Posterior Reversible Encephalopathy Syndrome

    Directory of Open Access Journals (Sweden)

    Keneilwe Malomo

    2016-04-01

    Full Text Available Posterior reversible encephalopathy syndrome (PRES is a clinico-neuro-radiological diagnosis, which can complicate a wide range of conditions. Clinical features include generalised and/or focal neurological deficits. These features are also present in neurovascular disorders, such as stroke. Currently, emphasis in the management of hyperacute stroke is thrombolysis, and it is important to bear in mind stroke mimics as a possible cause of clinical features. The Authors present the case of a 66-year-old man, who presented with acute focal neurological deficit. His brain imaging and history were consistent with PRES.

  8. Approaches to mimic the metallic sheen in beetles

    DEFF Research Database (Denmark)

    Lenau, Torben Anker; Aggerbeck, Martin; Nielsen, Steffen

    2009-01-01

    layers have high and low refractive index. The other is the Bouligand structure where birefringent chiral nanofibres are organised in spiral structures. The paper describes work done to explore different approaches to mimic these structures using polymer based materials and production methods...... that are suitable for more complex double curved geometry. One approach is to use alternating layers of 2 different polymers applied by dipping and another is applying cholesteric liquid crystals in paint. However, none of them can yet make the desired metal-looking free-form surfaces....

  9. Posterior Reversible Encephalopathy Syndrome Presenting as Stroke Mimic

    Directory of Open Access Journals (Sweden)

    Daniel Frick

    2017-05-01

    Full Text Available We present the case of a 33-year-old male with end stage renal disease presenting to the emergency department (ED with headache, dizziness, and unilateral weakness. Initial concern was for ischemic or hemorrhagic stroke. Magnetic resonance imaging confirmed posterior reversible encephalopathy syndrome (PRES. The patient was treated appropriately and made a full neurologic recovery. PRES is an under-recognized diagnosis in the ED. As a stroke mimic, PRES can lead the clinician on an incorrect diagnostic pathway with potential for iatrogenic harm.

  10. Heterologous Infection of Pregnant Mice Induces Low Birth Weight and Modifies Offspring Susceptibility to Malaria.

    Directory of Open Access Journals (Sweden)

    Ankur Sharma

    Full Text Available Pregnancy malaria (PM is associated with poor pregnancy outcomes, and can arise due to relapse, recrudescence or a re-infection with heterologous parasites. We have used the Plasmodium chabaudi model of pregnancy malaria in C57BL/6 mice to examine recrudescence and heterologous infection using CB and AS parasite strains. After an initial course of patent parasitemia and first recrudescence, CB but not AS parasites were observed to recrudesce again in most animals that became pregnant. Pregnancy exacerbated heterologous CB infection of AS-experienced mice, leading to mortality and impaired post-natal growth of pups. Parasites were detected in placental blood without evidence of sequestration, unlike P. falciparum but similar to other malaria species that infect pregnant women. Inflammatory cytokine levels were elevated in pregnant females during malaria, and associated with intensity of infection and with poor outcomes. Pups born to dams during heterologous infection were more resistant to malaria infections at 6-7 weeks of age, compared to pups born to malaria-experienced but uninfected dams or to malaria-naïve dams. In summary, our mouse model reproduces several features of human PM, including recrudescences, heterologous infections, poor pregnancy outcomes associated with inflammatory cytokines, and modulation of offspring susceptibility to malaria. This model should be further studied to explore mechanisms underlying PM pathogenesis.

  11. Wolff-Parkinson-White Syndrome Mimics a Conduction Disease

    Directory of Open Access Journals (Sweden)

    S. Marrakchi

    2014-01-01

    Full Text Available Background. It is important to recognise Wolff-Parkinson-White (WPW syndrome in electrocardiograms (ECG, as it may mimic ischaemic heart disease, ventricular hypertrophy, and bundle branch block. Recognising WPW syndrome allows for risk stratification, the identification of associated conditions, and the institution of appropriate management. Objective. The present case showed that electrophysiological study is indicated in patients with abnormal ECG and syncope. Case Report. A 40-year-old man with Wolff-Parkinson-White syndrome was presented to emergency with syncope. A baseline ECG was a complete right branch block and posterior left hemiblock. He was admitted to the cardiac care unit for pacemaker implantation. The atypical figure of complete right branch block and posterior left hemiblock was thought to be a “false positive” of conduction abnormality. But the long anterograde refractory period of the both accessory pathway and atrioventricular conduction may cause difficulty in diagnosing Wolff-Parkinson-White syndrome, Conclusion. A Wolff-Parkinson-White Syndrome may mimic a conduction disease. No reliable algorithm exists for making an ECG diagnosis of a preexcitation syndrome with conduction disorders. This can lead to diagnostic and therapeutic dilemmas in the context of syncope.

  12. Wolff-Parkinson-white syndrome mimics a conduction disease.

    Science.gov (United States)

    Marrakchi, S; Kammoun, I; Kachboura, S

    2014-01-01

    Background. It is important to recognise Wolff-Parkinson-White (WPW) syndrome in electrocardiograms (ECG), as it may mimic ischaemic heart disease, ventricular hypertrophy, and bundle branch block. Recognising WPW syndrome allows for risk stratification, the identification of associated conditions, and the institution of appropriate management. Objective. The present case showed that electrophysiological study is indicated in patients with abnormal ECG and syncope. Case Report. A 40-year-old man with Wolff-Parkinson-White syndrome was presented to emergency with syncope. A baseline ECG was a complete right branch block and posterior left hemiblock. He was admitted to the cardiac care unit for pacemaker implantation. The atypical figure of complete right branch block and posterior left hemiblock was thought to be a "false positive" of conduction abnormality. But the long anterograde refractory period of the both accessory pathway and atrioventricular conduction may cause difficulty in diagnosing Wolff-Parkinson-White syndrome, Conclusion. A Wolff-Parkinson-White Syndrome may mimic a conduction disease. No reliable algorithm exists for making an ECG diagnosis of a preexcitation syndrome with conduction disorders. This can lead to diagnostic and therapeutic dilemmas in the context of syncope.

  13. Novel β-amyloid aggregation inhibitors possessing a turn mimic.

    Science.gov (United States)

    Hamada, Yoshio; Miyamoto, Naoko; Kiso, Yoshiaki

    2015-04-01

    Amyloid β peptide, the main component of senile plaques found in the brain of Alzheimer disease (AD) patients, is a molecular target for AD therapeutic intervention. A number of potential AD therapeutics have been reported, including inhibitors of β-secretase, γ-secretase, and Aβ aggregation, and anti-amyloid agents, such as neprilysin, insulin degrading enzyme (IDE), and Aβ antibodies. Recently, we reported potent small-sized β-secretase (BACE1) inhibitors, which could serve as anti-AD drugs. However AD is a progressive disorder, where dementia symptoms gradually worsen over several decades, and therefore may require many years to get cured. One possible way to achieve a greater therapeutic effect is through simultaneous administration of multiple drugs, similar to those used in Highly Active Anti-Retroviral Therapy (HAART) used to treat AIDS. In order to overcome AD, we took a drug discovery approach to evaluate, novel β-amyloid aggregation inhibitors. Previously, we reported that a tong-type compound possessing a turn mimic as the inhibitor of HIV-1 protease dimerization. Oligomerized amyloid β peptides contain a turn structure within the molecule. Here, we designed and synthesized novel β-amyloid aggregation inhibitors with a turn-mimic template, based on the turn conformer of the oligomerized amyloid β peptides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Egel-Mitani; Andersen; Diers; Hach; Thim; Hastrup; Vad

    2000-06-01

    Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

  15. Uptake and Persistence of Homologous and Heterologous Zooxanthellae in the Temperate Sea Anemone Cereus pedunculatus (Pennant).

    Science.gov (United States)

    Davy, S K; Lucas, I A N; Turner, J R

    1997-04-01

    The uptake and persistence of symbiotic dinoflagellates (zooxanthellae) were measured in the temperate sea anemone Cereus pedunculatus (Pennant). Aposymbiotic specimens of C. pedunculatus were inoculated with zooxanthellae freshly isolated from a range of temperate and subtropical Anthozoa. Each inoculate consisted of zooxanthellae from a single host species and was either homologous (zooxanthellae from a host of the same species as the one being inoculated) or heterologous (from a host of a different species than the one being inoculated). The densities of zooxanthellae in host tissues were determined at regular intervals. C. pedunculatus took up homologous and heterologous zooxanthellae to similar degrees, except for zooxanthellae from the temperate Anthopleura ballii, which were taken up to a lesser extent. The densities of all zooxanthellae declined between 4 hours and 4 days after uptake, indicating that zooxanthellae were expelled, digested, or both during this period. The densities of all zooxanthellae increased between 2 and 8 weeks after inoculation, indicating zooxanthella growth. Over the entire 8-week period after uptake, densities of homologous zooxanthellae were always greater than those of heterologous zooxanthellae. Between 8 and 36 weeks after infection, densities of homologous zooxanthellae declined markedly and densities of some heterologous zooxanthellae increased further, resulting in homologous and heterologous zooxanthella densities being the same at 36 weeks. These densities were the same as those in naturally infected C. pedunculatus of similar size. The results suggest that zooxanthellae from a range of host species and environments can establish symbioses with C. pedunculatus and that, over long periods under laboratory conditions, heterologous zooxanthellae may populate C. pedunculatus to the same extent as homologous zooxanthellae.

  16. METHOD FOR THE PRODUCTION OF HETEROLOGOUS POLYPEPTIDES IN TRANSFORMED YEAST CELLS

    DEFF Research Database (Denmark)

    2000-01-01

    The invention describes industrial fermentation of a $i(Saccharomyces) yeast species for production of a heterologous product encoded by a plasmid or DNA contained in said $i(Saccharomyces) yeast species with method utilizes the substrate more efficiently and without fermentative metabolism...... resulting in formation of ethanol and other unwanted primary products of fermentative activity whereby high yields of the heterologous product are obtained. The $i(Saccharomyces) yeast species is preferably a Crabtree negative $i(Saccharomyces species) in particular $i(Saccharomyces kluyveri)....

  17. Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

    Science.gov (United States)

    Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

    2015-03-27

    Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

  18. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics

    Science.gov (United States)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2010-03-01

    Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

  19. Colonoscopic tattoo dye spillage mimics endometriosis on laparoscopy.

    Science.gov (United States)

    Stemmer, Shlomo M; Shurshalina, Anna

    2014-01-01

    Rare adverse effects of India ink injection spillage during colonoscopy have been reported. We present a case report in which prior colonoscopic India ink tattooing was found to mimic intraperitoneal endometriosis in a 48 year-old woman undergoing laparoscopic sterilization. Multiple black lesions suspicious for endometriosis involving the anterior and posterior cul-de-sac, left ovary, and omentum were found. A pathological assessment showed peritoneal tissue with focal dark pigment associated with mild chronic inflammation and deposition of tattoo pigment; there was no evidence of endometriosis in the specimens. Surgical recognition of tattoo ink spillage in the peritoneum is very important to prevent misinterpretation of peritoneal findings. Copyright © 2014 AAGL. Published by Elsevier Inc. All rights reserved.

  20. Piezoelectric materials mimic the function of the cochlear sensory epithelium.

    Science.gov (United States)

    Inaoka, Takatoshi; Shintaku, Hirofumi; Nakagawa, Takayuki; Kawano, Satoyuki; Ogita, Hideaki; Sakamoto, Tatsunori; Hamanishi, Shinji; Wada, Hiroshi; Ito, Juichi

    2011-11-08

    Cochlear hair cells convert sound vibration into electrical potential, and loss of these cells diminishes auditory function. In response to mechanical stimuli, piezoelectric materials generate electricity, suggesting that they could be used in place of hair cells to create an artificial cochlear epithelium. Here, we report that a piezoelectric membrane generated electrical potentials in response to sound stimuli that were able to induce auditory brainstem responses in deafened guinea pigs, indicating its capacity to mimic basilar membrane function. In addition, sound stimuli were transmitted through the external auditory canal to a piezoelectric membrane implanted in the cochlea, inducing it to vibrate. The application of sound to the middle ear ossicle induced voltage output from the implanted piezoelectric membrane. These findings establish the fundamental principles for the development of hearing devices using piezoelectric materials, although there are many problems to be overcome before practical application.

  1. [Granulomatous lobular mastitis: a benign abnormality that mimics malignancy].

    Science.gov (United States)

    Vingerhoedt, N M; Janssen, S; Mravunac, M; Wauters, C A P; Strobbe, L J A

    2008-05-03

    A palpable abnormality of the breast was found in three women, one aged 57 and two aged 41. The first two patients predominantly showed the characteristics of a purulent inflammation, and on mammogram the third patient appeared to have mastitis carcinomatosa. Histopathological investigation revealed a lobular, non-caseating granulomatous inflammation. They were treated with prednisone and the first and third patients also received azathioprine. After some time, the condition recurred in the contralateral breast in the second and third patients. Once again, medicinal treatment was given. When a palpable tumour of the breast is found the primary goal is to exclude malignancy. Granulomatous lobular mastitis is a rare and benign tumour of the breast that clinically mimics carcinoma. Often, conventional imaging does not lead to the diagnosis. A histological needle biopsy is the best way to reach a diagnosis. Immunosuppressive therapy is effective and is preferred over surgery.

  2. Antiproliferative efficacy of curcumin mimics through microtubule destabilization.

    Science.gov (United States)

    Khwaja, Sadiya; Fatima, Kaneez; Hasanain, Mohammad; Behera, Chittaranjan; Kour, Avneet; Singh, Arjun; Luqman, Suaib; Sarkar, Jayanta; Chanda, Debabrata; Shanker, Karuna; Gupta, A K; Mondhe, D M; Negi, Arvind S

    2018-05-10

    Curcumin possesses an attractive chemical structure with highly conjugated diferuloylmethane core. Curcumin mimics have been designed and prepared with an additional bridged phenyl ring in conjugation. Fourteen diverse analogues were evaluated against a panel of human cancer cell lines. The best analogue of the series i.e. compound 6a exhibited potent cytotoxicity against A431, epidermoid carcinoma cell line (IC 50  = 1.5 μM) and DLD1, colorectal adenocarcinoma cell line (IC 50  = 6.9 μM). In tubulin kinetics experiment, compound 6a destabilized polymerisation process (IC 50  = 4.68 μM). In cell cycle analysis, compound 6a exerted G2/M phase arrest in A431 cells and induced apoptosis. In Ehrlich Ascites Carcinoma in Swiss-albino mice, compound 6a showed 78.6% tumour reduction at 80 mg/kg dose and 57% solid tumour reduction at 150 mg/kg dose. Further, in acute-oral toxicity experiment in rodent model, compound 6a was given in three different oral doses to Swiss albino mice. There were non-significant changes in various biochemical parameters and major body organs studied, including their absolute and relative weights. It was tolerable up to 300 mg/kg dose in Swiss-albino mice. The present study shows that the novel curcumin mimic 6a is a safe and efficacious anticancer compound. However, it needs to be optimized for better efficacy. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  3. Past Life and Future Effects—How Heterologous Infections Alter Immunity to Influenza Viruses

    Directory of Open Access Journals (Sweden)

    Aisha Souquette

    2018-05-01

    Full Text Available Influenza virus frequently mutates due to its error-prone polymerase. This feature contributes to influenza virus’s ability to evade pre-existing immunity, leading to annual epidemics and periodic pandemics. T cell memory plays a key protective role in the face of an antigenically distinct influenza virus strain because T cell targets are often derived from conserved internal proteins, whereas humoral immunity targets are often sites of increased mutation rates that are tolerated by the virus. Most studies of influenza T cell memory are conducted in naive, specific pathogen free mice and do not account for repetitive influenza infection throughout a lifetime, sequential acute heterologous infections between influenza infections, or heterologous chronic co-infections. By contrast to these mouse models, humans often experience numerous influenza infections, encounter heterologous acute infections between influenza infections, and are infected with at least one chronic virus. In this review, we discuss recent advances in understanding the effects of heterologous infections on the establishment and maintenance of CD8+ T cell immunological memory. Understanding the various factors that affect immune memory can provide insights into the development of more effective vaccines and increase reproducibility of translational studies between animal models and clinical results.

  4. Heterologous Production and Yield Improvement of Epothilones in Burkholderiales Strain DSM 7029.

    Science.gov (United States)

    Bian, Xiaoying; Tang, Biao; Yu, Yucong; Tu, Qiang; Gross, Frank; Wang, Hailong; Li, Aiying; Fu, Jun; Shen, Yuemao; Li, Yue-Zhong; Stewart, A Francis; Zhao, Guoping; Ding, Xiaoming; Müller, Rolf; Zhang, Youming

    2017-07-21

    The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 μg L -1 . These results show that strain DSM 7029 has the potential to produce epothilones with reasonable titers and might be a broadly applicable host for the heterologous expression of other myxobacterial polyketide synthases and nonribosomal peptide synthetases, expediting the process of genome mining.

  5. A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

    DEFF Research Database (Denmark)

    Jørgensen, Mikael Skaanning; Skovlund, Dominique Aubert; Johannesen, Pia Francke

    2014-01-01

    ABSTRACT: BACKGROUND: The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools...

  6. Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers

    NARCIS (Netherlands)

    Bovy, A.G.; Angenent, G.C.; Dons, H.J.M.; Altvorst, van A.

    1999-01-01

    The Arabidopsis thaliana etr1-1 allele, capable of conferring ethylene insensitivity in a heterologous host, was introduced into transgenic carnation plants. This gene was expressed under control of either its own promoter, the constitutive CaMV 35S promoter or the flower-specific petunia FBP1

  7. Heterologous humoral immune response in patients treated with human growth hormone from different sources

    International Nuclear Information System (INIS)

    Cardoso, A.I.; Llera, A.S.; Iacono, R.F.

    1993-01-01

    The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [ 125 I]hGH or [ 125 I]bGH as tracers. 57 hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[ 125 I]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response. 16 refs., 4 figs., 1 tab

  8. Heterologous humoral immune response in patients treated with human growth hormone from different sources

    Energy Technology Data Exchange (ETDEWEB)

    Cardoso, A.I.; Llera, A.S.; Iacono, R.F. (and others) (Inst. de Estudios de la Inmunidad Humoral, Buenos Aires (Argentina))

    1993-07-01

    The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [[sup 125]I]hGH or [[sup 125]I]bGH as tracers. 57 hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[[sup 125]I]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response. 16 refs., 4 figs., 1 tab.

  9. Heterologous Prime-Boost Immunizations with a Virosomal and an Alphavirus Replicon Vaccine

    NARCIS (Netherlands)

    Walczak, Mateusz; de Mare, Arjan; Riezebos-Brilman, Annelies; Regts, Joke; Hoogeboom, Baukje-Nynke; Visser, Jeroen T.; Fiedler, Marc; Jansen-Duerr, Pidder; van der Zee, Ate G. J.; Nijman, Hans W.; Wilschut, Jan; Daemen, Toos

    2011-01-01

    Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of

  10. Large screen mimic display design research for advanced main control room in nuclear power plant

    International Nuclear Information System (INIS)

    Zheng Mingguang; Yang Yanhua; Xu Jijun; Zhang Qinshun; Ning Zhonghe

    2002-01-01

    Firstly the evolution of mimic diagrams or displays used in the main control room of nuclear power plant was introduced. The active functions of mimic diagrams were analyzed on the release of operator psychological burden and pressure, the assistance of operator for the information searching, status understanding, manual actuation, correct decision making as well as the safe and reliable operation of the nuclear power plant. The importance and necessity to use the (large screen) mimic diagrams in advanced main control room of nuclear power plant, the design principle, design details and verification measures of large screen mimic display are also described

  11. Accessing the public MIMIC-II intensive care relational database for clinical research.

    Science.gov (United States)

    Scott, Daniel J; Lee, Joon; Silva, Ikaro; Park, Shinhyuk; Moody, George B; Celi, Leo A; Mark, Roger G

    2013-01-10

    The Multiparameter Intelligent Monitoring in Intensive Care II (MIMIC-II) database is a free, public resource for intensive care research. The database was officially released in 2006, and has attracted a growing number of researchers in academia and industry. We present the two major software tools that facilitate accessing the relational database: the web-based QueryBuilder and a downloadable virtual machine (VM) image. QueryBuilder and the MIMIC-II VM have been developed successfully and are freely available to MIMIC-II users. Simple example SQL queries and the resulting data are presented. Clinical studies pertaining to acute kidney injury and prediction of fluid requirements in the intensive care unit are shown as typical examples of research performed with MIMIC-II. In addition, MIMIC-II has also provided data for annual PhysioNet/Computing in Cardiology Challenges, including the 2012 Challenge "Predicting mortality of ICU Patients". QueryBuilder is a web-based tool that provides easy access to MIMIC-II. For more computationally intensive queries, one can locally install a complete copy of MIMIC-II in a VM. Both publicly available tools provide the MIMIC-II research community with convenient querying interfaces and complement the value of the MIMIC-II relational database.

  12. IgG4-Associated Cholangitis Can Mimic Hilar Cholangiocarcinoma.

    Science.gov (United States)

    Zaydfudim, Victor M; Wang, Andrew Y; de Lange, Eduard E; Zhao, Zimin; Moskaluk, Christopher A; Bauer, Todd W; Adams, Reid B

    2015-07-01

    IgG4-associated cholangitis can mimic hilar cholangiocarcinoma. Previously reported patients with IgG4-associated cholangitis mimicking cholangiocarcinoma had elevated serum IgG4 levels and long-segment biliary strictures. However, in the absence of other diagnostic criteria for malignancy, IgG4-associated cholangitis should remain a consideration among patients with normal serum IgG4 and a hilar mass suspicious for cholangiocarcinoma. The presence of a hilar mass and a malignant-appearing biliary stricture in two patients with normal serum IgG4 prompted further evaluation and subsequent concomitant liver and bile duct resection and reconstruction. The diagnosis of IgG4-associated cholangitis was established during the pathologic evaluation of the resected specimens. IgG4-associated cholangitis is a known imitator of hilar cholangiocarcinoma and should be considered in the differential diagnosis even among serologically IgG4-negative patients with a hilar mass prior to operative resection.

  13. Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction

    Directory of Open Access Journals (Sweden)

    Lukas P. Feilen

    2017-05-01

    Full Text Available Physiological function and pathology of the Alzheimer’s disease causing amyloid precursor protein (APP are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs. The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

  14. Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

    Science.gov (United States)

    Feilen, Lukas P; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

    2017-01-01

    Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

  15. Recent developments in skin mimic systems to predict transdermal permeation.

    Science.gov (United States)

    Waters, Laura J

    2015-01-01

    In recent years there has been a drive to create experimental techniques that can facilitate the accurate and precise prediction of transdermal permeation without the use of in vivo studies. This review considers why permeation data is essential, provides a brief summary as to how skin acts as a natural barrier to permeation and discusses why in vivo studies are undesirable. This is followed by an in-depth discussion on the extensive range of alternative methods that have been developed in recent years. All of the major 'skin mimic systems' are considered including: in vitro models using synthetic membranes, mathematical models including quantitative structure-permeability relationships (QSPRs), human skin equivalents and chromatographic based methods. All of these model based systems are ideally trying to achieve the same end-point, namely a reliable in vitro-in vivo correlation, i.e. matching non-in vivo obtained data with that from human clinical trials. It is only by achieving this aim, that any new method of obtaining permeation data can be acknowledged as a potential replacement for animal studies, for the determination of transdermal permeation. In this review, the relevance and potential applicability of the various models systems will also be discussed.

  16. Nanorod mediated collagen scaffolds as extra cellular matrix mimics

    International Nuclear Information System (INIS)

    Vedhanayagam, Mohan; Nair, Balachandran Unni; Sreeram, Kalarical Janardhanan; Mohan, Ranganathan

    2015-01-01

    Creating collagen scaffolds that mimic extracellular matrices without using toxic exogenous materials remains a big challenge. A new strategy to create scaffolds through end-to-end crosslinking through functionalized nanorods leading to well-designed architecture is presented here. Self-assembled scaffolds with a denaturation temperature of 110 °C, porosity of 70%, pore size of 0.32 μm and Young’s modulus of 231 MPa were developed largely driven by imine bonding between 3-mercapto-1-propanal (MPA) functionalized ZnO nanorods and collagen. The mechanical properties obtained were much higher than that of native collagen, collagen—MPA, collagen—3-mercapto-1-propanol (3MPOH) or collagen- 3-MPOH-ZnO, clearly bringing out the relevance of nanorod mediated assembly of fibrous networks. This new strategy has led to scaffolds with mechanical properties much higher than earlier reports and can provide support for cell growth and facilitation of cell attachment. (paper)

  17. Chimpanzees and humans mimic pupil-size of conspecifics.

    Directory of Open Access Journals (Sweden)

    Mariska E Kret

    Full Text Available Group-living typically provides benefits to individual group members but also confers costs. To avoid incredulity and betrayal and allow trust and cooperation, individuals must understand the intentions and emotions of their group members. Humans attend to other's eyes and from gaze and pupil-size cues, infer information about the state of mind of the observed. In humans, pupil-size tends to mimic that of the observed. Here we tested whether pupil-mimicry exists in our closest relative, the chimpanzee (P. troglodytes. We conjectured that if pupil-mimicry has adaptive value, e.g. to promote swift communication of inner states and facilitate shared understanding and coordination, pupil-mimicry should emerge within but not across species. Pupillometry data was collected from human and chimpanzee subjects while they observed images of the eyes of both species with dilating/constricting pupils. Both species showed enhanced pupil-mimicry with members of their own species, with effects being strongest in humans and chimpanzee mothers. Pupil-mimicry may be deeply-rooted, but probably gained importance from the point in human evolution where the morphology of our eyes became more prominent. Humans' white sclera surrounding the iris, and the fine muscles around their eyes facilitate non-verbal communication via eye signals.

  18. Thrombolytic treatment to stroke mimic patients via telestroke.

    Science.gov (United States)

    Asaithambi, Ganesh; Castle, Amy L; Sperl, Michael A; Ravichandran, Jayashree; Gupta, Aditi; Ho, Bridget M; Hanson, Sandra K

    2017-02-01

    The safety and outcomes of intravenous thrombolysis (IVT) to stroke patients via telestroke (TS) is similar to those presenting to stroke centers. Little is known on the accuracy of TS diagnosis among those receiving IVT. We sought to compare the rate of patients receiving IVT with diagnosis of ischemic stroke as opposed to stroke mimic (SM) in our TS network to those who presented to our comprehensive stroke center (CSC). Consecutive patients receiving IVT between August 2014 and June 2015 were identified at our CSC and TS network. We compared rates of SM, post-IVT symptomatic intracerebral hemorrhage (sICH), in-hospital mortality, and discharge destination. We evaluated 131 receiving IVT were included in the analysis. Rates of SM receiving IVT were similar (CSC 12% versus 7% TS, p=0.33). Four stroke patients experienced sICH or in-hospital mortality; neither were found among SM patients. Discharge destination was similar between stroke and SM patients (p=0.9). SM patients had higher diagnoses of migraine (p=0.05) and psychiatric illness (p<0.01). The accuracy of diagnosing stroke in IVT-eligible patients evaluated via TS is similar to evaluations at our CSC. Continued efforts should be made to minimize exposure of SM patients to IVT in both settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. A MIMIC approach to modeling the underground economy in Taiwan

    Science.gov (United States)

    Wang, David Han-Min; Lin, Jer-Yan; Yu, Tiffany Hui-Kuang

    2006-11-01

    The size of underground economy (UE) expansion usually increases the tax gap, impose a burden on the economy, and results in tax distortions. This study uses the MIMIC approach to model the causal variables and indicating variables to estimate the UE in Taiwan. We also focus on testing the data for non-stationarity and perform diagnostic tests. By using annual time-series data for Taiwan from 1961 to 2003, it is found that the estimated size of the UE varies from 11.0% to 13.1% before 1988, and from 10.6% to 11.8% from 1989 onwards. That the size of the UE experienced a substantial downward shift in 1989 indicates that there was a structural break. The UE is significantly and positively affected by such casual variables as the logarithm of real government consumption and currency inflation, but is negatively affected by the tax burden at 5% significant level. Unemployment rate and crime rate are not significantly correlated with the UE in this study.

  20. Penerapan Model MIMIC untuk Menguji Konsistensi Hasil Pengukuran melalui Skala

    Directory of Open Access Journals (Sweden)

    Wahyu Widhiarso

    2012-04-01

    Full Text Available This study aimed to demonstrate the use of multiple indicators and multiple (MIMIC model in testing the consistency of the measurement results when it's applied to individuals with different characteristics. Self-Esteem and Student Motivation Scale were employed to measure participant attributes. Impact of three student characteristics (gender, intelligence and school location on both measurement model were examined. Total of 2981 students from 30 cities of 15 provinces were participated in this study. Data analysis was utilized using confirmatory factor analysis under structural equation modeling (SEM approach. Results showed that measuring self-esteem was prone to be contaminated by individual heterogeneity. However, contrary results was found on motivation measure. Excluding three student characteristics as covariance in the model have increased model fit indices in the self-esteem measurement model, but not in the self-esteem. The presence of susceptibility scale to different characteristics indicated that this scale needs to be further modified. Therefore, when it is applied to heterogeneous population, the measurement results will produce consistently results.

  1. A web-based data visualization tool for the MIMIC-II database.

    Science.gov (United States)

    Lee, Joon; Ribey, Evan; Wallace, James R

    2016-02-04

    Although MIMIC-II, a public intensive care database, has been recognized as an invaluable resource for many medical researchers worldwide, becoming a proficient MIMIC-II researcher requires knowledge of SQL programming and an understanding of the MIMIC-II database schema. These are challenging requirements especially for health researchers and clinicians who may have limited computer proficiency. In order to overcome this challenge, our objective was to create an interactive, web-based MIMIC-II data visualization tool that first-time MIMIC-II users can easily use to explore the database. The tool offers two main features: Explore and Compare. The Explore feature enables the user to select a patient cohort within MIMIC-II and visualize the distributions of various administrative, demographic, and clinical variables within the selected cohort. The Compare feature enables the user to select two patient cohorts and visually compare them with respect to a variety of variables. The tool is also helpful to experienced MIMIC-II researchers who can use it to substantially accelerate the cumbersome and time-consuming steps of writing SQL queries and manually visualizing extracted data. Any interested researcher can use the MIMIC-II data visualization tool for free to quickly and conveniently conduct a preliminary investigation on MIMIC-II with a few mouse clicks. Researchers can also use the tool to learn the characteristics of the MIMIC-II patients. Since it is still impossible to conduct multivariable regression inside the tool, future work includes adding analytics capabilities. Also, the next version of the tool will aim to utilize MIMIC-III which contains more data.

  2. A lesion mimic phenotype in tomato obtained by isolating and silencing an Lls1 homologue

    NARCIS (Netherlands)

    Spassieva, S; Hille, J

    Lesion mimic phenotypes serve as a tool to study the regulation of cell death in plants. In order to obtain a tomato lesion mimic phenotype, we used the conservation of the lethal leaf spot 1 (Lls1) genes between plant species. The tomato Lls1 homologue was cloned, sequenced and analyzed. It showed

  3. Biological Mimics: A New Paradigm in the Detection of Toxic Compounds

    Science.gov (United States)

    Monty, Chelsea Nicole

    2009-01-01

    The purpose of this thesis is to introduce a new idea: using biological mimics in the detection of toxic compounds. Biological mimics imitate the active site of a given enzyme or have catalytic chemistry similar to enzymes and can be used in place of biological molecules to provide longer stability and simpler operation. In the following text the…

  4. Touch the Page and Mimic Me: Evaluation of a Talking-Pen Learning Tool

    Science.gov (United States)

    Odakura, Abigail

    2013-01-01

    The purpose of this paper is to evaluate the talking-pen device called the Mimic Me, an educational technology product of a large English conversation school in Japan, as an educational learning tool. The product will be reviewed in terms of its developmental appropriateness for the target audience based on current research. Although the Mimic Me…

  5. Inhibition of duck hepatitis B virus replication by mimic peptides in vitro.

    Science.gov (United States)

    Jia, Hongyu; Liu, Changhong; Yang, Ying; Zhu, Haihong; Chen, Feng; Liu, Jihong; Zhou, Linfu

    2015-11-01

    The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (Pmimic peptide 7 was lower than that in the other groups (Pmimic peptide 7 was significantly lower than that in the other groups (PMimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro .

  6. Primary study on lesion mimic mutants of rice (oryza sativa L.)

    International Nuclear Information System (INIS)

    Hao Zhongna; Zhang Hongzhi; Tao Rongixang

    2007-01-01

    Nineteen lesion mimic mutants (xsl1-19) of japonica rice Xiushui11 were obtained by γ-rays irradiation treatment. All mutants belonged to whole life lesion mimic. Lesion mimic of mutants didn't largen after tillering stage, leaves didn't wither, and no effect on the plants exsert spikes and seed. When the highest temperature in day exceeded 32 degree C in seedling stage, lesion mimic of all mutant expect xsl19 disappeared. Under 32 degree C, lesion mimic would appear gradually, and symptoms weren't inhibited by high temperature after 5 leaf stage. The plant heights of all lesion mimic mutants were 47.56-63.54 cm in the tillering stage, and that of CK was 83.75 cm; but the dwarf phenomenon of mutants only appeared before tillering stage, and didn't affect plant heights finally; the heading dates of mutants were the same to the CK, the ear length of all mutants were 9.43-15.19 cm, and that of CK was 16.41 cm; the total grain quantity per spike of all mutants were 88.17-165.33, and those of xsl19 and CK were 49.50 and 76.17. The results showed all lesion mimic mutants except xsl19 had short spikes and total grain quantity per spike increasing. All lesion mimic mutants were susceptible to Magnaporthe grisea, and they had no relationship with resistance. (authors)

  7. Testing Measurement Invariance Using MIMIC: Likelihood Ratio Test with a Critical Value Adjustment

    Science.gov (United States)

    Kim, Eun Sook; Yoon, Myeongsun; Lee, Taehun

    2012-01-01

    Multiple-indicators multiple-causes (MIMIC) modeling is often used to test a latent group mean difference while assuming the equivalence of factor loadings and intercepts over groups. However, this study demonstrated that MIMIC was insensitive to the presence of factor loading noninvariance, which implies that factor loading invariance should be…

  8. Nuclear power station: validation of a method for designing operation mimic diagrams

    International Nuclear Information System (INIS)

    Colard, M.I.; De Vlaminck, M.; Javaux, D.

    1995-01-01

    This is the first time in a Belgium nuclear power station that the design of mimic diagrams has involved biotechnologists. The methodology is based upon task analysis, the formalism of which has been evaluated. It is now integrated in the Dimos assisted supervision system to produce the final design of mimic diagram images. (authors). 3 figs., 3 refs

  9. Hyperechoic caudate nuclei: a potential mimic of germinal matrix hemorrhage

    International Nuclear Information System (INIS)

    Schlesinger, A.E.; Shackelford, G.D.; Adcock, L.M.

    1998-01-01

    Background. We have encountered bilateral hyperechoic foci in the region of the germinal matrix on cranial sonograms in neonates that have an appearance similar to germinal matrix hemorrhage (GMH), but are unusual either due to the age of the patient at presentation or to the evolution of the foci on follow-up. We believe that these findings represent hyperechoic caudate nuclei (HCN) rather than GMH. Objective. To demonstrate that bilateral HCN can be seen on cranial sonography in neonates and can mimic bilateral GMH. Materials and methods. The cranial sonograms were reviewed in nine neonates (three term and six premature) who had HCN identified on at least one sonographic examination. CT (two patients) and MR (one patient) studies were also reviewed, as well as the neuropathological examination in one patient who died and had an autopsy. The patients' medical records were reviewed to identify any clinical markers for significant risk of perinatal ischemia. Results. There was clinical evidence for risk of ischemia in five of the nine neonates. All nine patients had bilateral HCN on the initial or follow-up studies. Small cysts were seen sonographically in two patients. CT was normal in one patient and revealed a small unilateral focus of increased attenuation in one infant (very small compared to the bilateral HCN). MR was normal in one patient. Histopathological examination of the brain was normal in the one patient who died and had an autopsy. Conclusion. Hyperechoic caudate nuclei can occur in neonates either as a normal finding, or possibly related to ischemia, and should not always be attributed to GMH. (orig.)

  10. Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

    Science.gov (United States)

    Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

    2017-05-31

    Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

  11. Evaluation of the antiproliferative, proapoptotic, and antiangiogenic effects of a double-stranded RNA mimic complexed with polycations in an experimental mouse model of leiomyoma.

    Science.gov (United States)

    García-Pascual, Carmen Maria; Ferrero, Hortensia; Juarez, Irene; Martínez, Jessica; Villanueva, Ana; Pozuelo-Rubio, Mercedes; Soengas, Marisol; Tormo, Damiá; Simón, Carlos; Gómez, Raúl; Pellicer, Antonio

    2016-02-01

    To assess the antiproliferative, proapoptotic, and antiangiogenic effects of the double-stranded RNA mimic polyinosine-polycytidylic acid (pIC) complexed with polyethylenimine [pIC(PEI)] in xenografted human leiomyomas. Heterologous leiomyoma mouse model. University-affiliated infertility center. Ovariectomized and hormone-replaced nude mice (n = 16) who received human leiomyoma fragment transplantation. Leiomyoma fragments placed in the peritoneum of 5-week-old nude female mice and treated with the vehicle (n = 8) or 0.6 mg/kg [pIC(PEI)] (n = 8) for 4 weeks. The size of the leiomyoma implants, and cellular proliferation (Ki67), vascularization (PECAM), and apoptosis (OH-ends) assessed by quantitative immunohistochemical/immunofluorescent analysis of the recovered implants. No significant differences were observed in the size of the leiomyoma implants between groups. Vascularization and proliferation were significantly decreased, and apoptosis was increased in the [pIC(PEI)]-treated group versus control. We hypothesize that the antiangiogenic and apoptotic effects exerted by [pIC(PEI)] might lead to a decrease in lesion size in this animal model if the compound is administered for longer periods of time. This study provides promising data on [pIC(PEI)] as a potential novel therapeutic agent against human leiomyoma. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  12. A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

    Science.gov (United States)

    Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

    2017-08-01

    To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

  13. Heterologous surface display on lactic acid bacteria: non-GMO alternative?

    Science.gov (United States)

    Zadravec, Petra; Štrukelj, Borut; Berlec, Aleš

    2015-01-01

    Lactic acid bacteria (LAB) are food-grade hosts for surface display with potential applications in food and therapy. Alternative approaches to surface display on LAB would avoid the use of recombinant DNA technology and genetically-modified organism (GMO)-related regulatory requirements. Non-covalent surface display of proteins can be achieved by fusing them to various cell-wall binding domains, of which the Lysine motif domain (LysM) is particularly well studied. Fusion proteins have been isolated from recombinant bacteria or from their growth medium and displayed on unmodified bacteria, enabling heterologous surface display. This was demonstrated on non-viable cells devoid of protein content, termed bacteria-like particles, and on various species of genus Lactobacillus. Of the latter, Lactobacillus salivarius ATCC 11741 was recently shown to be particularly amenable for LysM-mediated display. Possible regulatory implications of heterologous surface display are discussed, particularly those relevant for the European Union.

  14. Stroke mimics under the drip-and-ship paradigm.

    Science.gov (United States)

    Mehta, Sonal; Vora, Nirav; Edgell, Randall C; Allam, Hesham; Alawi, Aws; Koehne, Jennifer; Kumar, Abhay; Feen, Eliahu; Cruz-Flores, Salvador; Alshekhlee, Amer

    2014-01-01

    Recent reports suggested better outcomes associated with the drip-and-ship paradigm for acute ischemic stroke (AIS) treated with thrombolysis. We hypothesized that a higher rate of stroke mimics (SM) among AIS treated in nonspecialized stroke centers that are transferred to comprehensive centers is responsible for such outcomes. Consecutive patients treated with thrombolysis according to the admission criteria were reviewed in a single comprehensive stroke center over 1 academic year (July 1, 2011 to June 30, 2012). Information on the basic demographic, hospital complications, psychiatric diagnoses, and discharge disposition was collected. We identified those patients who were treated at a facility and then transferred to the tertiary center (ie, drip-and-ship paradigm). In addition to comparative and adjusted analysis to identify predictors for SM, a stratified analysis by the drip-and-ship status was performed. One hundred twenty patients were treated with thrombolysis for AIS included in this analysis; 20 (16.7%) were discharged with the final diagnosis of SM; 14 of those had conversion syndrome and 6 patients had other syndromes (seizures, migraine, and hypoglycemia). Patients with SM were younger (55.6 ± 15.0 versus 69.4 ± 14.9, P = .0003) and more likely to harbor psychiatric diagnoses (45% versus 9%; P ≤ .0001). Eighteen of 20 SM patients (90%) had the drip-and-ship treatment paradigm compared with 65% of those with AIS (P = .02). None of the SM had hemorrhagic complications, and all were discharged to home. Predictors of SM on adjusted analysis included the drip-and-ship paradigm (odds ratio [OR] 12.8, 95% confidence interval [CI] 1.78, 92.1) and history of any psychiatric illness (OR 12.08; 95% CI 3.14, 46.4). Eighteen of 83 drip-and-ship patients (21.7%) were diagnosed with SM compared with 2 of 37 patients (5.4%) presented directly to the hub hospital (P = .02). The drip-and-ship paradigm and any psychiatric history predict the diagnosis of

  15. Accumulation of murine amyloid-β mimics early Alzheimer's disease.

    Science.gov (United States)

    Krohn, Markus; Bracke, Alexander; Avchalumov, Yosef; Schumacher, Toni; Hofrichter, Jacqueline; Paarmann, Kristin; Fröhlich, Christina; Lange, Cathleen; Brüning, Thomas; von Bohlen Und Halbach, Oliver; Pahnke, Jens

    2015-08-01

    Amyloidosis mouse models of Alzheimer's disease are generally established by transgenic approaches leading to an overexpression of mutated human genes that are known to be involved in the generation of amyloid-β in Alzheimer's families. Although these models made substantial contributions to the current knowledge about the 'amyloid hypothesis' of Alzheimer's disease, the overproduction of amyloid-β peptides mimics only inherited (familiar) Alzheimer's disease, which accounts for patients with Alzheimer's disease. The inherited form is even regarded a 'rare' disease according to the regulations for funding of the European Union (www.erare.eu). Here, we show that mice that are double-deficient for neprilysin (encoded by Mme), one major amyloid-β-degrading enzyme, and the ABC transporter ABCC1, a major contributor to amyloid-β clearance from the brain, develop various aspects of sporadic Alzheimer's disease mimicking the clinical stage of mild cognitive impairment. Using behavioural tests, electrophysiology and morphological analyses, we compared different ABC transporter-deficient animals and found that alterations are most prominent in neprilysin × ABCC1 double-deficient mice. We show that these mice have a reduced probability to survive, show increased anxiety in new environments, and have a reduced working memory performance. Furthermore, we detected morphological changes in the hippocampus and amygdala, e.g. astrogliosis and reduced numbers of synapses, leading to defective long-term potentiation in functional measurements. Compared to human, murine amyloid-β is poorly aggregating, due to changes in three amino acids at N-terminal positions 5, 10, and 13. Interestingly, our findings account for the action of early occurring amyloid-β species/aggregates, i.e. monomers and small amyloid-β oligomers. Thus, neprilysin × ABCC1 double-deficient mice present a new model for early effects of amyloid-β-related mild cognitive impairment that allows investigations

  16. Membrane-Mimic Nanoparticles for Drug and Gene Delivery

    KAUST Repository

    Alamoudi, Kholod

    2017-12-01

    Nanoscale organic particles have gained a prominent role in drug and gene delivery field. As the nature of the nanoparticle’s (NPs) surface plays a major role in their targeting efficiency, bioavailability, and cytotoxicity, membrane-mimic nanoparticles are considered highly attractive materials for in vivo and in vitro applications. Synthetic membrane vesicles (liposomes) and nanoconstructs built with native cancer cellular membrane are excellent scaffolds to improve cellular delivery. Liposomes have been extensively used due to their high loading capacity, biocompatibility and biodegradability. However, modifications with stimuli responsive materials are highly needed to improve their stability and turn them active participants in controlled delivery. Towards a nature inspired approach, reconstructed bilayers from cell membrane are a good candidate to enhance NP’s targeting ability and biocompatibility. The primary focus of this research is to develop smart responsive (lipid) membrane coated NPs with surface modifications for controlled and targeted drug and/or gene delivery for application in cancer therapy. Three approaches have been developed, namely i) liposomes as thermoresponsive nanocarriers for the delivery of genetic material; ii) magnetically photosensitive liposome hybrids and iii) biomimetic periodic mesoporous organo silica engineered for better a biocompatibility and targeting capabilities. In the first project synthetic liposomes were loaded with ammonium bicarbonate salt (ABC) and siRNA. The combination of lipids chosen and the relative ratios allowed the rapid release of the genetic material to the multi drug resistant cancer cells studied, upon external heat trigger. This design has improved the gene silencing efficiency via successful endosomal escape. In the second project, SPIO@Au nanoparticles were imbedded in the lipid bilayer to produce a photo/thermal responsive carrier that could be also used in cell imaging besides gene transfection

  17. Urothelial carcinoma of urinary bladder with exclusive heterologous component of epithelioid rhabdomyosarcoma at metastatic site.

    Science.gov (United States)

    Agarwal, Poojan; Pasricha, Sunil; Gupta, Gurudutt; Sharma, Anila; Mehta, Anurag

    2018-01-01

    Urothelial carcinoma of urinary bladder with divergent differentiation into rhabdomyosarcoma (RMS) is an extremely uncommon aggressive phenomenon. We present a case of a 74-year-old male with bladder carcinoma which metastasized to the abdominal wall as epithelioid RMS. To the best knowledge of our literature searches, an oligometastasis of exclusive heterologous component has not been described before. The clinical, radiological, and immunohistochemistry profile of the patient supported the monoclonal nature of the tumor.

  18. Construction of a system for heterologous production of carbonic anhydrase from Plasmodium falciparum in Pichia pastoris

    OpenAIRE

    Gullberg, Erik

    2008-01-01

    Malaria is one of the biggest current global health problems, and with the increasing occurance of drug resistant Plasmodium falciparum strains, there is an urgent need for new antimalarial drugs. Given the important role of carbonic anhydrase in Plasmodium falciparum (PfCA), it is a potential novel drug target. Heterologous expression of malaria proteins is problematic due to the unusual codon usage of the Plasmodium genome, so to overcome this problem a synthetic PfCA gene was designed, opt...

  19. The K Domain Mediates Homologous and Heterologous Interactions Between FLC and SVP Proteins of Brassica juncea

    Directory of Open Access Journals (Sweden)

    Ma Guanpeng

    2015-07-01

    Full Text Available The transcription factors FLOWERING LOCUS C (FLC and SHORT VEGETATIVE PHASE (SVP can interact to form homologous and heterologous protein complexes that regulate flowering time in Brassica juncea Coss. (Mustard.Previous studies showed that protein interactions were mediated by the K domain, which contains the subdomains K1, K2 and K3. However, it remains unknown how the subdomains mediate the interactions between FLC and SVP. In the present study, we constructed several mutants of subdomains K1–K3 and investigated the mechanisms involved in the heterologous interaction of BjFLC/BjSVP and in the homologous interaction of BjFLC/BjFLC or BjSVP/BjSVP. Yeast two-hybrid and β-Galactosidase activity assays showed that the 19 amino acids of the K1 subdomain in BjSVP and the 17 amino acids of the K1 subdomain in BjFLC were functional subdomains that interact with each other to mediate hetero-dimerization. The heterologous interaction was enhanced by the K2 subdomain of BjSVP protein, but weakened by its interhelical domain L2. The heterologous interaction was also enhanced by the K2 subdomain of BjFLC protein, but weakened by its K3 subdomain. The homologous interaction of BjSVP was mediated by the full K-domain. However, the homologous interaction of BjFLC was regulated only by its K1 and weakened by its K2 and K3 subdomains. The results provided new insights into the interactions between FLC and SVP, which will be valuable for further studies on the molecular regulation mechanisms of the regulation of flowering time in B. juncea and other Brassicaceae.

  20. Spatial organization of heterologous metabolic system in vivo based on TALE.

    Science.gov (United States)

    Zhu, Lv-yun; Qiu, Xin-Yuan; Zhu, Ling-Yun; Wu, Xiao-Min; Zhang, Yuan; Zhu, Qian-Hui; Fan, Dong-Yu; Zhu, Chu-Shu; Zhang, Dong-Yi

    2016-05-17

    For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

  1. Malignant phyllodes tumor of the breast with heterologous high-grade angiosarcoma

    Directory of Open Access Journals (Sweden)

    Ghassan Tranesh

    2017-03-01

    Full Text Available Phyllodes tumors (PTs account for <3% of fibroepithelial breast lesions and for 0.3% to 1.0% of primary breast tumors. They occur predominantly in middle-aged women (mean age range, 40–50 years. PTs can be categorized into benign, borderline, and malignant; the first 2 categories are distinguished only by degree of cellular atypia and mitotic activity. Malignant PTs are more frequent among persons of Hispanic ethnicity, especially those born in Central America or South America. Heterologous sarcomatous elements may be present in malignant PTs, predominantly liposarcoma and rarely fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, osteosarcoma, and chondrosarcoma. Breast angiosarcoma (BA is a rare heterologous, sarcomatous element that may arise secondary to malignant PT. We report a 47-year-old woman with no history of previous surgery or radiation therapy who presented to the emergency department with a painful right breast mass. She admittedly noticed the right breast mass for many years; however, recently it increased in size. Mammography and ultrasonography identified a partially cystic mass. Core needle biopsy showed dense hyalinized fibrous tissue with old blood clots, suggestive of infarcted fibroadenoma. The patient received antibiotics and analgesics; however, she reported intractable pain and a worsening skin rash of her right breast. Chest computed tomography and magnetic resonance imaging showed a doubling in mass size, with pectoralis major muscle involvement. Incisional biopsy showed malignant PT with heterologous high-grade angiosarcoma. The diagnosis of angiosarcoma was confirmed through immunoreactivity for CD31, FLI1, and ERG immunostains.

  2. Heterologous Prime-Boost HIV-1 Vaccination Regimens in Pre-Clinical and Clinical Trials

    Directory of Open Access Journals (Sweden)

    Julia L. Hurwitz

    2010-02-01

    Full Text Available Currently, there are more than 30 million people infected with HIV-1 and thousands more are infected each day. Vaccination is the single most effective mechanism for prevention of viral disease, and after more than 25 years of research, one vaccine has shown somewhat encouraging results in an advanced clinical efficacy trial. A modified intent-to-treat analysis of trial results showed that infection was approximately 30% lower in the vaccine group compared to the placebo group. The vaccine was administered using a heterologous prime-boost regimen in which both target antigens and delivery vehicles were changed during the course of inoculations. Here we examine the complexity of heterologous prime-boost immunizations. We show that the use of different delivery vehicles in prime and boost inoculations can help to avert the inhibitory effects caused by vector-specific immune responses. We also show that the introduction of new antigens into boost inoculations can be advantageous, demonstrating that the effect of ‘original antigenic sin’ is not absolute. Pre-clinical and clinical studies are reviewed, including our own work with a three-vector vaccination regimen using recombinant DNA, virus (Sendai virus or vaccinia virus and protein. Promising preliminary results suggest that the heterologous prime-boost strategy may possibly provide a foundation for the future prevention of HIV-1 infections in humans.

  3. Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

    Directory of Open Access Journals (Sweden)

    Alderete JF

    2005-03-01

    Full Text Available Abstract Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs, a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

  4. Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

    Directory of Open Access Journals (Sweden)

    Selma Majerowicz

    1994-09-01

    Full Text Available Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11. Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

  5. Prior mucosal exposure to heterologous cells alters the pathogenesis of cell-associated mucosal feline immunodeficiency virus challenge

    Directory of Open Access Journals (Sweden)

    Leavell Sarah

    2010-05-01

    Full Text Available Abstract Background Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control and then cats were vaginally challenged with cell-associated or cell-free FIV. Results Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls. Conclusions The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus can be modulated by mucosal exposure to uninfected heterologous cells.

  6. Open-access MIMIC-II database for intensive care research.

    Science.gov (United States)

    Lee, Joon; Scott, Daniel J; Villarroel, Mauricio; Clifford, Gari D; Saeed, Mohammed; Mark, Roger G

    2011-01-01

    The critical state of intensive care unit (ICU) patients demands close monitoring, and as a result a large volume of multi-parameter data is collected continuously. This represents a unique opportunity for researchers interested in clinical data mining. We sought to foster a more transparent and efficient intensive care research community by building a publicly available ICU database, namely Multiparameter Intelligent Monitoring in Intensive Care II (MIMIC-II). The data harnessed in MIMIC-II were collected from the ICUs of Beth Israel Deaconess Medical Center from 2001 to 2008 and represent 26,870 adult hospital admissions (version 2.6). MIMIC-II consists of two major components: clinical data and physiological waveforms. The clinical data, which include patient demographics, intravenous medication drip rates, and laboratory test results, were organized into a relational database. The physiological waveforms, including 125 Hz signals recorded at bedside and corresponding vital signs, were stored in an open-source format. MIMIC-II data were also deidentified in order to remove protected health information. Any interested researcher can gain access to MIMIC-II free of charge after signing a data use agreement and completing human subjects training. MIMIC-II can support a wide variety of research studies, ranging from the development of clinical decision support algorithms to retrospective clinical studies. We anticipate that MIMIC-II will be an invaluable resource for intensive care research by stimulating fair comparisons among different studies.

  7. Synthesis and evaluation of cardiac glycoside mimics as potential anticancer drugs

    DEFF Research Database (Denmark)

    Jensen, Marie; Schmidt, Steffen; Fedosova, Natalya

    2011-01-01

    recent years cardiac glycosides have furthermore been suggested to possess valuable anticancer activity. To mimic the labile trisaccharide of digitoxin with a stabile carbohydrate surrogate, we have used sulfur linked ethylene glycol moieties of varying length (mono-, di-, tri- or tetra-ethylene glycol...... the shortest mimics were found to have highest efficacy, with the best ligand having a monoethylene glycol unit (IC(50) 0.24 μM), which was slightly better than digitoxigenin (IC(50) 0.64 μM), while none of the novel cardiac glycoside mimics display an in vitro effect as high as digitoxin (IC(50) 0.02 μM)....

  8. Potential Therapeutic Uses of p19ARF Mimics in Mammary Tumorigenesis

    National Research Council Canada - National Science Library

    Hann, Stephen R

    2005-01-01

    Since many breast tumors have deregulated c-Myc we hypothesize that an ARF mimic would be a valuable therapeutic agent for breast cancer to inhibit c-Myc-induced transformation/tumorigenesis without...

  9. [Discussion of the implementation of MIMIC database in emergency medical study].

    Science.gov (United States)

    Li, Kaiyuan; Feng, Cong; Jia, Lijing; Chen, Li; Pan, Fei; Li, Tanshi

    2018-05-01

    To introduce Medical Information Mart for Intensive Care (MIMIC) database and elaborate the approach of critically emergent research with big data based on the feature of MIMIC and updated studies both domestic and overseas, we put forward the feasibility and necessity of introducing medical big data to research in emergency. Then we discuss the role of MIMIC database in emergency clinical study, as well as the principles and key notes of experimental design and implementation under the medical big data circumstance. The implementation of MIMIC database in emergency medical research provides a brand new field for the early diagnosis, risk warning and prognosis of critical illness, however there are also limitations. To meet the era of big data, emergency medical database which is in accordance with our national condition is needed, which will provide new energy to the development of emergency medicine.

  10. Pattern Differences of Small Hand Muscle Atrophy in Amyotrophic Lateral Sclerosis and Mimic Disorders.

    Science.gov (United States)

    Fang, Jia; Liu, Ming-Sheng; Guan, Yu-Zhou; Du, Hua; Li, Ben-Hong; Cui, Bo; Ding, Qing-Yun; Cui, Li-Ying

    2016-04-05

    Amyotrophic lateral sclerosis (ALS) and some mimic disorders, such as distal-type cervical spondylotic amyotrophy (CSA), Hirayama disease (HD), and spinobulbar muscular atrophy (SBMA) may present with intrinsic hand muscle atrophy. This study aimed to investigate different patterns of small hand muscle involvement in ALS and some mimic disorders. We compared the abductor digiti minimi/abductor pollicis brevis (ADM/APB) compound muscle action potential (CMAP) ratios between 200 ALS patients, 95 patients with distal-type CSA, 88 HD patients, 43 SBMA patients, and 150 normal controls. The ADM/APB CMAP amplitude ratio was significantly higher in the ALS patients (P mimic disorders presumably reflect distinct pathophysiological mechanisms underlying different disorders, and may aid in distinguishing between ALS and mimic disorders.

  11. MOCVD process technology for affordable, high-yield, high-performance MESFET structures. Phase 3: MIMIC

    Science.gov (United States)

    1993-01-01

    Under the MIMIC Program, Spire has pursued improvements in the manufacturing of low cost, high quality gallium arsenide MOCVD wafers for advanced MIMIC FET applications. As a demonstration of such improvements, Spire was tasked to supply MOCVD wafers for comparison to MBE wafers in the fabrication of millimeter and microwave integrated circuits. In this, the final technical report for Spire's two-year MIMIC contract, we report the results of our work. The main objectives of Spire's MIMIC Phase 3 Program, as outlined in the Statement of Work, were as follows: Optimize the MOCVD growth conditions for the best possible electrical and morphological gallium arsenide. Optimization should include substrate and source qualification as well as determination of the optimum reactor growth conditions; Perform all work on 75 millimeter diameter wafers, using a reactor capable of at least three wafers per run; and Evaluate epitaxial layers using electrical, optical, and morphological tests to obtain thickness, carrier concentration, and mobility data across wafers.

  12. Selective chromo-fluorogenic detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) with a unique probe based on a boron dipyrromethene (BODIPY) dye.

    Science.gov (United States)

    Barba-Bon, Andrea; Costero, Ana M; Gil, Salvador; Martínez-Máñez, Ramón; Sancenón, Félix

    2014-11-21

    A novel colorimetric probe (P4) for the selective differential detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) was prepared. Probe P4 contains three reactive sites; i.e. (i) a nucleophilic phenol group able to undergo phosphorylation with nerve gases, (ii) a carbonyl group as a reactive site for cyanide; and (iii) a triisopropylsilyl (TIPS) protecting group that is known to react with fluoride. The reaction of P4 with DCNP in acetonitrile resulted in both the phosphorylation of the phenoxy group and the release of cyanide, which was able to react with the carbonyl group of P4 to produce a colour modulation from pink to orange. In contrast, phosphorylation of P4 with DFP in acetonitrile released fluoride that hydrolysed the TIPS group in P4 to yield a colour change from pink to blue. Probe P4 was able to discriminate between DFP and DCNP with remarkable sensitivity; limits of detection of 0.36 and 0.40 ppm for DCNP and DFP, respectively, were calculated. Besides, no interference from other organophosphorous derivatives or with presence of acid was observed. The sensing behaviour of P4 was also retained when incorporated into silica gel plates or onto polyethylene oxide membranes, which allowed the development of simple test strips for the colorimetric detection of DCNP and DFP in the vapour phase. P4 is the first probe capable of colorimetrically differentiating between a Tabun mimic (DCNP) and a Sarin and Soman mimic (DFP).

  13. In vitro enzyme-mimic activity and in vivo therapeutic potential of HSJ-0017, a novel Mn porphyrin-based antioxidant enzyme mimic.

    Science.gov (United States)

    Li, Bao-qiu; Dong, Xin; Li, Na; Gao, Ji-you; Yuan, Qiang; Fang, Shi-hong; Gong, Xian-chang; Wang, Shu-juan; Wang, Feng-shan

    2014-10-01

    Manganese (III) 5, 10, 15, 20-tetrakis [3-(2-(2-methoxy)-ethoxy) ethoxy] phenyl porphyrin chloride, designated HSJ-0017, is a novel antioxidant enzyme mimic. The aim of the present study was to investigate the enzyme-mimic activity and the therapeutic potential of HSJ-0017 in free radical-related diseases. Superoxide dismutase (SOD) mimic activity was measured by the nitroblue tetrazolium chloride monohydrate reduction assay. Catalase (CAT) mimic activity was measured based on the decomposition of hydrogen peroxide. The antitumor, radioprotective and chemoprotective effects of HSJ-0017 were evaluated in H22 or S180 tumor-bearing Kunming mice. The anti-inflammatory and hepatoprotective effects were, respectively, evaluated in histamine-induced edema model and CCl4-induced hepatic damage model in Wistar rats. HSJ-0017 over a concentration range of 0.001-10 µmol/L significantly inhibited the generation of superoxide anion. Significant hydrogen peroxide scavenging activity was observed when the concentration of HSJ-0017 was higher than 0.01 µmol/L. HSJ-0017 at a dose of 3.0 mg/kg exhibited significant antitumor effect on S180 tumor xenografts, whereas no significant antitumor effect was observed in H22 tumor xenografts. HSJ-0017 at a dose of 3.0 mg/kg enhanced the antitumor effects of radiotherapy and chemotherapy, and reduced their toxicity. However, HSJ-0017 counteracted the antitumor effects of radiotherapy when administered simultaneously with radiotherapy. HSJ-0017 showed significant anti-inflammatory and hepatoprotective effects. Our results demonstrate that HSJ-0017 exhibits antioxidant, antitumor, anti-inflammatory, radioprotective, chemoprotective, and hepatoprotective effects. It is a potent dual SOD/CAT mimic. © 2014 by the Society for Experimental Biology and Medicine.

  14. Brucella suis strain 2 vaccine is safe and protective against heterologous Brucella spp. infections.

    Science.gov (United States)

    Zhu, Liangquan; Feng, Yu; Zhang, Ge; Jiang, Hui; Zhang, Zhen; Wang, Nan; Ding, Jiabo; Suo, Xun

    2016-01-12

    Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated B. suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2-3 weeks, and a burst production of IFN-γ at one week as well as a two-fold increase in TNF-α production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Sygmund Christoph

    2011-12-01

    Full Text Available Abstract Background FAD dependent glucose dehydrogenase (GDH currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum spp. Results Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L-1 of GDH activity (57 mg L-1. Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. Conclusions The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

  16. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris.

    Science.gov (United States)

    Sygmund, Christoph; Staudigl, Petra; Klausberger, Miriam; Pinotsis, Nikos; Djinović-Carugo, Kristina; Gorton, Lo; Haltrich, Dietmar; Ludwig, Roland

    2011-12-12

    FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp. Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L⁻¹ of GDH activity (57 mg L⁻¹). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

  17. Heterologous Production of a Novel Cyclic Peptide Compound, KK-1, in Aspergillus oryzae

    Directory of Open Access Journals (Sweden)

    Akira Yoshimi

    2018-04-01

    Full Text Available A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb. The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.

  18. Heterologous Production of a Novel Cyclic Peptide Compound, KK-1, in Aspergillus oryzae.

    Science.gov (United States)

    Yoshimi, Akira; Yamaguchi, Sigenari; Fujioka, Tomonori; Kawai, Kiyoshi; Gomi, Katsuya; Machida, Masayuki; Abe, Keietsu

    2018-01-01

    A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata . It consists of 10 amino acid residues, including five N -methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae . The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR , were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae , gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata . Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae , although there may be unknown factors limiting productivity in this species.

  19. Longitudinal study of experimental induction of AA amyloidosis in mice seeded with homologous and heterologous AA fibrils.

    Science.gov (United States)

    Muhammad, Naeem; Murakami, Tomoaki; Inoshima, Yasuo; Ishiguro, Naotaka

    2016-09-01

    To investigate pathogenesis and kinetics of experimentally induced murine AA amyloidosis seeded with homologous (murine) and heterologous (bovine) AA fibrils. Experimental AA amyloidosis was induced by administration of inflammatory stimulus and preformed AA fibrils to a total of 111 female C57/Black mice. In this longitudinal study, heterologous (bovine) as well as homologous (murine) AA fibrils were injected intraperitoneally to mice in various combinations. Re-stimulation was done at 120 or 300 days post first inoculation. To analyze the intensity of amyloid depositions in mice organs, immunohistochemical techniques and image J software were used. Assessment of cytokines level in sera was done using a Mouse Th1/Th2/Th17 Cytokine CBA Kit. Incidence and severity of AA amyloidosis were quite low in mice inoculated with heterologous bovine AA fibrils than homologous murine one. Homologous AA fibrils administration at first and second inoculation caused maximum amount of amyloid depositions and severe systemic form of amyloidosis. Increase in the level of pro-inflammatory cytokine IL-6 was observed after first inoculation, while second inoculation caused a further increase in the level of anti-inflammatory cytokine IL-10. AA amyloidosis can be induced by heterologous as well as homologous AA fibrils. Severity of AA amyloidosis induced with homologous AA fibrils is higher compared to heterologous AA fibrils.

  20. Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae.

    Science.gov (United States)

    Jin, Feng-Jie; Katayama, Takuya; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

    2016-11-01

    Genomic mapping of mutations using next-generation sequencing technologies has facilitated the identification of genes contributing to fundamental biological processes, including human diseases. However, few studies have used this approach to identify mutations contributing to heterologous protein production in industrial strains of filamentous fungi, such as Aspergillus oryzae. In a screening of A. oryzae strains that hyper-produce human lysozyme (HLY), we previously isolated an AUT1 mutant that showed higher production of various heterologous proteins; however, the underlying factors contributing to the increased heterologous protein production remained unclear. Here, using a comparative genomic approach performed with whole-genome sequences, we attempted to identify the genes responsible for the high-level production of heterologous proteins in the AUT1 mutant. The comparative sequence analysis led to the detection of a gene (AO090120000003), designated autA, which was predicted to encode an unknown cytoplasmic protein containing an alpha/beta-hydrolase fold domain. Mutation or deletion of autA was associated with higher production levels of HLY. Specifically, the HLY yields of the autA mutant and deletion strains were twofold higher than that of the control strain during the early stages of cultivation. Taken together, these results indicate that combining classical mutagenesis approaches with comparative genomic analysis facilitates the identification of novel genes involved in heterologous protein production in filamentous fungi.

  1. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes

    DEFF Research Database (Denmark)

    Albertsen, Line; Chen, Yun; Bach, Lars Stougaard

    2011-01-01

    be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes...... increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology...

  2. Heterologous expression of plasmodial proteins for structural studies and functional annotation

    CSIR Research Space (South Africa)

    Birkholtz, LM

    2008-01-01

    Full Text Available Malaria Journal Open AcceReview Heterologous expression of plasmodial proteins for structural studies and functional annotation Lyn-Marie Birkholtz1, Gregory Blatch2, Theresa L Coetzer3, Heinrich C Hoppe1,4, Esmaré Human1, Elizabeth J Morris1,5, Zoleka Ngcete..., Kwadlangezwa, South Africa Email: Lyn-Marie Birkholtz - lbirkholtz@up.ac.za; Gregory Blatch - G.Blatch@ru.ac.za; Theresa L Coetzer - theresa.coetzer@nhls.ac.za; Heinrich C Hoppe - hhoppe@csir.co.za; Esmaré Human - esmare.human@up.ac.za; Elizabeth J Morris...

  3. Heterologous prime-boost vaccinations for poverty-related diseases: advantages and future prospects.

    Science.gov (United States)

    Radosević, Katarina; Rodriguez, Ariane; Lemckert, Angelique; Goudsmit, Jaap

    2009-05-01

    Classical vaccination approaches, based on a single vaccine administered in a homologous prime-boost schedule and optimized to induce primarily neutralizing antibodies, are unlikely to be sufficiently efficacious to prevent TB, malaria or HIV infections. Novel vaccines, capable of inducing a more powerful immune response, in particular T-cell immunity, are desperately needed. Combining different vaccine modalities that are able to complement each other and induce broad and sustainable immunity is a promising approach. This review provides an overview of heterologous prime-boost vaccination modalities currently in development for the 'big three' poverty-related diseases and emphasizes the need for innovative vaccination approaches.

  4. Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts

    International Nuclear Information System (INIS)

    Massou, S.; Puech, V.; Talmont, F.; Demange, P.; Lindley, N.D.; Tropis, M.; Milon, A.

    1999-01-01

    Methylotrophic yeast has previously been shown to be an excellent system for the cost-effective production of perdeuterated biomass and for the heterologous expression of membrane receptors. A protocol for the expression of 85% deuterated, functional human μ-opiate receptor was established. For partially deuterated biomass, deuteration level and distribution were determined for fatty acids, amino acids and carbohydrates. It was shown that prior to biosynthesis of lipids and amino acids (and of carbohydrates, to a lower extent), exchange occurs between water and methanol hydrogen atoms, so that 80%-90% randomly deuterated biomass and over-expressed proteins may be obtained using only deuterated water

  5. Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways

    DEFF Research Database (Denmark)

    Vavitsas, Konstantinos; Rue, Emil Østergaard; Stefánsdóttir, Lára Kristín

    2017-01-01

    BACKGROUND: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity...... different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids...

  6. High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae.

    Science.gov (United States)

    Yamashita, Nobuo; Komori, Yumiko; Okumura, Yoshiyuki; Uchiya, Kei-Ichi; Matsui, Takeshi; Nishimura, Akira; Ogawa, Kenji; Nikai, Toshiaki

    2011-08-01

    AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Long-Lasting Effects of BCG Vaccination on Both Heterologous Th1/Th17 Responses and Innate Trained Immunity

    DEFF Research Database (Denmark)

    Kleinnijenhuis, Johanneke; Quintin, Jessica; Preijers, Frank

    2013-01-01

    '. In the present study we assessed whether BCG was able to induce long-lasting effects on both trained immunity and heterologous T helper 1 (Th1) and Th17 immune responses 1 year after vaccination. The production of TNFα and IL-1β to mycobacteria or unrelated pathogens was higher after 2 weeks and 3 months...... in proinflammatory cytokine production after stimulation with the TLR4 ligand lipopolysaccharide. The heterologous production of Th1 (IFN-γ) and Th17 (IL-17 and IL-22) immune responses to nonmycobacterial stimulation remained strongly elevated even 1 year after BCG vaccination. In conclusion, BCG induces sustained...... changes in the immune system associated with a nonspecific response to infections both at the level of innate trained immunity and at the level of heterologous Th1/Th17 responses. © 2013 S. Karger AG, Basel....

  8. Systematic Assessment of Strategies for Lung-targeted Delivery of MicroRNA Mimics

    Science.gov (United States)

    Schlosser, Kenny; Taha, Mohamad; Stewart, Duncan J.

    2018-01-01

    There is considerable interest in the use of synthetic miRNA mimics (or inhibitors) as potential therapeutic agents in pulmonary vascular disease; however, the optimal delivery method to achieve high efficiency, selective lung targeting has not been determined. Here, we sought to investigate the relative merits of different lung-targeted strategies for delivering miRNA mimics in rats. Methods: Tissue levels of a synthetic miRNA mimic, cel-miR-39-3p (0.5 nmol in 50 µL invivofectamine/PBS vehicle) were compared in male rats (n=3 rats/method) after delivery by commonly used lung-targeting strategies including intratracheal liquid instillation (IT-L), intratracheal aerosolization with (IT-AV) or without ventilator assistance (IT-A), intranasal liquid instillation (IN-L) and intranasal aerosolization (IN-A). Intravenous (IV; via jugular vein), intraperitoneal (IP) and subcutaneous (SC) delivery served as controls. Relative levels of cel-miR-39 were quantified by RT-qPCR. Results: At 2 h post delivery, IT-L showed the highest lung mimic level, which was significantly higher than levels achieved by all other methods (from ~10- to 10,000-fold, pMimic levels remained detectable in the lung 24 h after delivery, but were 10- to 100-fold lower. The intrapulmonary distribution of cel-miR-39 was comparable when delivered as either a liquid or aerosol, with evidence of mimic distribution to both the left and right lung lobes and penetration to distal regions. All lung-targeted strategies showed lung-selective mimic uptake, with mimic levels 10- to 100-fold lower in heart and 100- to 10,000-fold lower in liver, kidney and spleen. In contrast, IV, SC and IP routes showed comparable or higher mimic levels in non-pulmonary tissues. Conclusions: miRNA uptake in the lungs differed markedly by up to 4 orders of magnitude, demonstrating that the choice of delivery strategy could have a significant impact on potential therapeutic outcomes in preclinical investigations of miRNA-based drug

  9. A Host-Produced Autoinducer-2 Mimic Activates Bacterial Quorum Sensing.

    Science.gov (United States)

    Ismail, Anisa S; Valastyan, Julie S; Bassler, Bonnie L

    2016-04-13

    Host-microbial symbioses are vital to health; nonetheless, little is known about the role crosskingdom signaling plays in these relationships. In a process called quorum sensing, bacteria communicate with one another using extracellular signal molecules called autoinducers. One autoinducer, AI-2, is proposed to promote interspecies bacterial communication, including in the mammalian gut. We show that mammalian epithelia produce an AI-2 mimic activity in response to bacteria or tight-junction disruption. This AI-2 mimic is detected by the bacterial AI-2 receptor, LuxP/LsrB, and can activate quorum-sensing-controlled gene expression, including in the enteric pathogen Salmonella typhimurium. AI-2 mimic activity is induced when epithelia are directly or indirectly exposed to bacteria, suggesting that a secreted bacterial component(s) stimulates its production. Mutagenesis revealed genes required for bacteria to both detect and stimulate production of the AI-2 mimic. These findings uncover a potential role for the mammalian AI-2 mimic in fostering crosskingdom signaling and host-bacterial symbioses. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. MIMIC II: a massive temporal ICU patient database to support research in intelligent patient monitoring

    Science.gov (United States)

    Saeed, M.; Lieu, C.; Raber, G.; Mark, R. G.

    2002-01-01

    Development and evaluation of Intensive Care Unit (ICU) decision-support systems would be greatly facilitated by the availability of a large-scale ICU patient database. Following our previous efforts with the MIMIC (Multi-parameter Intelligent Monitoring for Intensive Care) Database, we have leveraged advances in networking and storage technologies to develop a far more massive temporal database, MIMIC II. MIMIC II is an ongoing effort: data is continuously and prospectively archived from all ICU patients in our hospital. MIMIC II now consists of over 800 ICU patient records including over 120 gigabytes of data and is growing. A customized archiving system was used to store continuously up to four waveforms and 30 different parameters from ICU patient monitors. An integrated user-friendly relational database was developed for browsing of patients' clinical information (lab results, fluid balance, medications, nurses' progress notes). Based upon its unprecedented size and scope, MIMIC II will prove to be an important resource for intelligent patient monitoring research, and will support efforts in medical data mining and knowledge-discovery.

  11. Enzyme-Triggered Defined Protein Nanoarrays: Efficient Light-Harvesting Systems to Mimic Chloroplasts.

    Science.gov (United States)

    Zhao, Linlu; Zou, Haoyang; Zhang, Hao; Sun, Hongcheng; Wang, Tingting; Pan, Tiezheng; Li, Xiumei; Bai, Yushi; Qiao, Shanpeng; Luo, Quan; Xu, Jiayun; Hou, Chunxi; Liu, Junqiu

    2017-01-24

    The elegance and efficiency by which chloroplasts harvest solar energy and conduct energy transfer have been a source of inspiration for chemists to mimic such process. However, precise manipulation to obtain orderly arranged antenna chromophores in constructing artificial chloroplast mimics was a great challenge, especially from the structural similarity and bioaffinity standpoints. Here we reported a design strategy that combined covalent and noncovalent interactions to prepare a protein-based light-harvesting system to mimic chloroplasts. Cricoid stable protein one (SP1) was utilized as a building block model. Under enzyme-triggered covalent protein assembly, mutant SP1 with tyrosine (Tyr) residues at the designated sites can couple together to form nanostructures. Through controlling the Tyr sites on the protein surface, we can manipulate the assembly orientation to respectively generate 1D nanotubes and 2D nanosheets. The excellent stability endowed the self-assembled protein architectures with promising applications. We further integrated quantum dots (QDs) possessing optical and electronic properties with the 2D nanosheets to fabricate chloroplast mimics. By attaching different sized QDs as donor and acceptor chromophores to the negatively charged surface of SP1-based protein nanosheets via electrostatic interactions, we successfully developed an artificial light-harvesting system. The assembled protein nanosheets structurally resembled the natural thylakoids, and the QDs can achieve pronounced FRET phenomenon just like the chlorophylls. Therefore, the coassembled system was meaningful to explore the photosynthetic process in vitro, as it was designed to mimic the natural chloroplast.

  12. Cross-amplification of heterologous microsatellite markers in Rhamdia quelen and Leporinus elongatus

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    Nelson Mauricio Lopera-Barrero

    2016-02-01

    Full Text Available Native fish species in Brazil are an asset in fish farming, but their natural stocks have been significantly reduced in recent years. To mitigate this negative impact, studies on fish conservation are being conducted and genetic tools for the discrimination of population parameters are increasingly achieving great importance. Current analysis evaluates a set of microsatellite heterologous primers in the jundiá (Rhamdia quelen and in the piapara (Leporinus elongatus. Samples from the caudal fin of 15 broodstock from each species were analyzed. DNA extraction was performed with NaCl protocol and the integrity of the extracted DNA was checked with agarose gel 1%. Twenty primers developed for Piaractus mesopotamicus, Colossoma macropomum, Prochilodus lineatus, Brycon opalinus and Oreochromis niloticus were evaluated. Cross amplification of four primers of the B. opalinus and P. lineatus species (BoM12, Pli43 and Pli60 in R. quelen and BoM2, Pli43 and Pli60 in L. elongatus was assessed. Primers of P. mesopotamicus, C. macropomum and O. niloticus showed no cross amplification in the two species analyzed. Results revealed the possibility of using the four amplified heterologous primers in genetic studies for R. quelen and L. elongatus.

  13. Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases

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    Rui Miao

    2017-12-01

    Full Text Available Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH for isobutanol production. A strain expressing an introduced kivd without any additional copy of ADH produced 3 mg L−1 OD750−1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L−1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.

  14. Dedifferentiated Leiomyosarcoma of the Uterus with Heterologous Elements: A Potential Diagnostic Pitfall

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    Kojo R. Rawish

    2012-01-01

    Full Text Available Dedifferentiation is a phenomenon that is well characterized in a variety of tumors and is defined by the occurrence of a high-grade or undifferentiated tumor, typically unrecognizable regarding its line of differentiation, from a low-grade/borderline neoplasm. This phenomenon has previously been described in 2 uterine leiomyosarcomas, but both were devoid of heterologous elements. The authors describe herein a case of a dedifferentiated leiomyosarcoma of the uterus with osteoid heterologous elements, believed to be the first such reported case. The original tumor was a high-grade leiomyosarcoma with large low-grade and leiomyoma-like areas and whose constituent cells displayed intense nuclear immunoreactivity for both estrogen receptor (ER and progesterone receptor (PR in approximately 30% of cells. The tumor recurred six months after its resection as an undifferentiated sarcoma that was negative for smooth muscle markers, but which remained positive for ER and PR. Osteoid production was only identified in the recurrent tumor and was significant in extent therein. This case highlights the immunophenotypic changes that may occur in dedifferentiated leiomyosarcomas, and this possibility should be a consideration when an apparently undifferentiated sarcoma is identified in a patient with a history of uterine leiomyosarcoma. In our case, the expression of ER and PR provided significant supportive evidence of the uterine origin of the recurrent tumor.

  15. Heterologous expression of enterocin AS-48 in several strains of lactic acid bacteria.

    Science.gov (United States)

    Fernández, M; Martínez-Bueno, M; Martín, M C; Valdivia, E; Maqueda, M

    2007-05-01

    Enterococcus faecalis produces a cationic and circular enterocin, AS-48, of 7149 Da, the genetic determinants of which are located within the pMB2 plasmid. We have compared enterocin AS-48 production by different enterococci species with that of other 'safe' lactic acid bacteris (LAB) (GRAS status) and looked into the subsequent application of this enterocin in food production. In an effort to exploit this system for the heterologous expression of enterocin AS-48, a number of vectors containing the as-48 cluster were constructed and used to transform several LAB strains (genera Enterococcus, Lactococcus and Lactobacillus) Heterologous production of enterocin AS-48 failed when bacteria other than those belonging to the genus Enterococcus were used as hosts, although expression of a partial level of resistance against AS-48 were always detected, ruling out the possibility of a lack of recognition of the enterococcal promoters. Our results reveal the special capacity of species from the genus Enterococcus to produce AS-48, an enterocin that requires a post-transcriptional modification to generate a circular peptide with a wide range of inhibitory activity against pathogenic and spoilage bacteria. Preliminary experiments in foodstuffs using nonvirulent enterococci with interesting functional properties reveal the possibility of a biotechnological application of these transformants.

  16. Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae

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    Kevin V. Solomon

    2016-12-01

    Full Text Available Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV, and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1 were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae. We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production. Keywords: Pseudomonas, Isobutanol, Dehydrogenase, Mitochondria, Saccharomyces cerevisiae, Metabolic engineering

  17. Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

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    Fabian Machens

    2017-10-01

    Full Text Available Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

  18. Heterologous Synapsis and Crossover Suppression in Heterozygotes for a Pericentric Inversion in the Zebra Finch.

    Science.gov (United States)

    del Priore, Lucía; Pigozzi, María I

    2015-01-01

    In the zebra finch, 2 alternative morphs regarding centromere position were described for chromosome 6. This polymorphism was interpreted to be the result of a pericentric inversion, but other causes of the centromere repositioning were not ruled out. We used immunofluorescence localization to examine the distribution of MLH1 foci on synaptonemal complexes to test the prediction that pericentric inversions cause synaptic irregularities and/or crossover suppression in heterozygotes. We found complete suppression of crossing over in the region involved in the rearrangement in male and female heterozygotes. In contrast, the same region showed high levels of crossing over in homozygotes for the acrocentric form of this chromosome. No inversion loops or synaptic irregularities were detected along bivalent 6 in heterozygotes suggesting that heterologous pairing is achieved during zygotene or early pachytene. Altogether these findings strongly indicate that the polymorphic chromosome 6 originated by a pericentric inversion. Since inversions are common rearrangements in karyotypic evolution in birds, it seems likely that early heterologous pairing could help to fix these rearrangements, preventing crossing overs in heterozygotes and their deleterious effects on fertility. © 2015 S. Karger AG, Basel.

  19. Homologous and Heterologous Protection of Nonhuman Primates by Ebola and Sudan Virus-Like Particles

    Science.gov (United States)

    Warfield, Kelly L.; Dye, John M.; Wells, Jay B.; Unfer, Robert C.; Holtsberg, Frederick W.; Shulenin, Sergey; Vu, Hong; Swenson, Dana L.; Bavari, Sina; Aman, M. Javad

    2015-01-01

    Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components. PMID:25793502

  20. Homologous and heterologous protection of nonhuman primates by Ebola and Sudan virus-like particles.

    Directory of Open Access Journals (Sweden)

    Kelly L Warfield

    Full Text Available Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV and Marburg virus (MARV following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system and then demonstrate protection against Sudan virus (SUDV and Taï Forest virus (TAFV. Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.

  1. Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride.

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    Edgar Balcázar-López

    Full Text Available Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc. To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.

  2. Heterologous Expression and Characterization of a Thermostable Exo-β-D-Glucosaminidase from Aspergillus oryzae.

    Science.gov (United States)

    Wu, Dingxin; Wang, Linchun; Li, Yuwei; Zhao, Shumiao; Peng, Nan; Liang, Yunxiang

    2016-02-01

    An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of Vmax and kcat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

  3. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

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    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  4. Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host.

    Science.gov (United States)

    Kim, Mina; Jin, Yerin; An, Hyun-Joo; Kim, Jaehan

    2017-07-28

    The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

  5. Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis▿

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-01-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs. PMID:19251885

  6. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

  7. Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker

    Science.gov (United States)

    Yoshizaki, G.; Patino, R.; Thomas, P.; Bolamba, D.; Chang, Xiaotian

    2001-01-01

    A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown /maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker. ?? 2001 Elsevier Science.

  8. Estimating the size of non-observed economy in Croatia using the MIMIC approach

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    Vjekoslav Klarić

    2011-03-01

    Full Text Available This paper gives a quick overview of the approaches that have been used in the research of shadow economy, starting with the definitions of the terms “shadow economy” and “non-observed economy”, with the accent on the ISTAT/Eurostat framework. Several methods for estimating the size of the shadow economy and the non-observed economy are then presented. The emphasis is placed on the MIMIC approach, one of the methods used to estimate the size of the nonobserved economy. After a glance at the theory behind it, the MIMIC model is then applied to the Croatian economy. Considering the described characteristics of different methods, a previous estimate of the size of the non-observed economy in Croatia is chosen to provide benchmark values for the MIMIC model. Using those, the estimates of the size of non-observed economy in Croatia during the period 1998-2009 are obtained.

  9. Reproducing a Prospective Clinical Study as a Computational Retrospective Study in MIMIC-II.

    Science.gov (United States)

    Kury, Fabrício S P; Huser, Vojtech; Cimino, James J

    2015-01-01

    In this paper we sought to reproduce, as a computational retrospective study in an EHR database (MIMIC-II), a recent large prospective clinical study: the 2013 publication, by the Japanese Association for Acute Medicine (JAAM), about disseminated intravascular coagulation, in the journal Critical Care (PMID: 23787004). We designed in SQL and Java a set of electronic phenotypes that reproduced the study's data sampling, and used R to perform the same statistical inference procedures. All produced source code is available online at https://github.com/fabkury/paamia2015. Our program identified 2,257 eligible patients in MIMIC-II, and the results remarkably agreed with the prospective study. A minority of the needed data elements was not found in MIMIC-II, and statistically significant inferences were possible in the majority of the cases.

  10. Bicelles and Other Membrane Mimics: Comparison of Structure, Properties, and Dynamics from MD Simulations

    DEFF Research Database (Denmark)

    Vestergaard, Mikkel; Kraft, Johan Frederik; Vosegaard, Thomas

    2015-01-01

    present molecular dynamics simulations to elucidate structural and dynamic properties of small bicelles and compare them to a large alignable bicelle, a small nanodisc, and a lipid bilayer. Properties such as lipid packing and properties related to embedding both an α-helical peptide and a transmembrane...... protein are investigated. The small bicelles are found to be very dynamic and mainly assume a prolate shape substantiating that small bicelles cannot be regarded as well-defined disclike structures. However, addition of a peptide results in an increased tendency to form disc-shaped bicelles. The small......The increased interest in studying membrane proteins has led to the development of new membrane mimics such as bicelles and nanodiscs. However, only limited knowledge is available of how these membrane mimics are affected by embedded proteins and how well they mimic a lipid bilayer. Herein, we...

  11. MicroRNA-30c Mimic Mitigates Hypercholesterolemia and Atherosclerosis in Mice*

    Science.gov (United States)

    Irani, Sara; Pan, Xiaoyue; Peck, Bailey C. E.; Iqbal, Jahangir; Sethupathy, Praveen; Hussain, M. Mahmood

    2016-01-01

    High plasma cholesterol levels are a major risk factor for atherosclerosis. Plasma cholesterol can be reduced by inhibiting lipoprotein production; however, this is associated with steatosis. Previously we showed that lentivirally mediated hepatic expression of microRNA-30c (miR-30c) reduced hyperlipidemia and atherosclerosis in mice without causing hepatosteatosis. Because viral therapy would be formidable, we examined whether a miR-30c mimic can be used to mitigate hyperlipidemia and atherosclerosis without inducing steatosis. Delivery of a miR-30c mimic to the liver diminished diet-induced hypercholesterolemia in C57BL/6J mice. Reductions in plasma cholesterol levels were significantly correlated with increases in hepatic miR-30c levels. Long term dose escalation studies showed that miR-30c mimic caused sustained reductions in plasma cholesterol with no obvious side effects. Furthermore, miR-30c mimic significantly reduced hypercholesterolemia and atherosclerosis in Apoe−/− mice. Mechanistic studies showed that miR-30c mimic had no effect on LDL clearance but reduced lipoprotein production by down-regulating microsomal triglyceride transfer protein expression. MiR-30c had no effect on fatty acid oxidation but reduced lipid synthesis. Additionally, whole transcriptome analysis revealed that miR-30c mimic significantly down-regulated hepatic lipid synthesis pathways. Therefore, miR-30c lowers plasma cholesterol and mitigates atherosclerosis by reducing microsomal triglyceride transfer protein expression and lipoprotein production and avoids steatosis by diminishing lipid syntheses. It mitigates atherosclerosis most likely by reducing lipoprotein production and plasma cholesterol. These findings establish that increasing hepatic miR-30c levels is a viable treatment option for reducing hypercholesterolemia and atherosclerosis. PMID:27365390

  12. Antimicrobial Effects of Novel Triple Antibiotic Paste-Mimic Scaffolds on Actinomyces naeslundii Biofilm.

    Science.gov (United States)

    Albuquerque, Maria T P; Ryan, Stuart J; Münchow, Eliseu A; Kamocka, Maria M; Gregory, Richard L; Valera, Marcia C; Bottino, Marco C

    2015-08-01

    Actinomyces naeslundii has been recovered from traumatized permanent teeth diagnosed with necrotic pulps. In this work, a triple antibiotic paste (TAP)-mimic scaffold is proposed as a drug-delivery strategy to eliminate A. naeslundii dentin biofilm. Metronidazole, ciprofloxacin, and minocycline were added to a polydioxanone (PDS) polymer solution and spun into fibrous scaffolds. Fiber morphology, mechanical properties, and drug release were investigated by using scanning electron microscopy, microtensile testing, and high-performance liquid chromatography, respectively. Human dentin specimens (4 × 4 × 1 mm(3), n = 4/group) were inoculated with A. naeslundii (ATCC 43146) for 7 days for biofilm formation. The infected dentin specimens were exposed to TAP-mimic scaffolds, TAP solution (positive control), and pure PDS (drug-free scaffold). Dentin infected (7-day biofilm) specimens were used for comparison (negative control). Confocal laser scanning microscopy was done to determine bacterial viability. Scaffolds displayed a submicron mean fiber diameter (PDS = 689 ± 312 nm and TAP-mimic = 718 ± 125 nm). Overall, TAP-mimic scaffolds showed significantly (P ≤ .040) lower mechanical properties than PDS. Within the first 24 hours, a burst release for all drugs was seen. A sustained maintenance of metronidazole and ciprofloxacin was observed over 4 weeks, but not for minocycline. Confocal laser scanning microscopy demonstrated complete elimination of all viable bacteria exposed to the TAP solution. Meanwhile, TAP-mimic scaffolds led to a significant (P mimic scaffolds hold significant potential in the eradication/elimination of bacterial biofilm, a critical step in regenerative endodontics. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  13. MicroRNA-30c Mimic Mitigates Hypercholesterolemia and Atherosclerosis in Mice.

    Science.gov (United States)

    Irani, Sara; Pan, Xiaoyue; Peck, Bailey C E; Iqbal, Jahangir; Sethupathy, Praveen; Hussain, M Mahmood

    2016-08-26

    High plasma cholesterol levels are a major risk factor for atherosclerosis. Plasma cholesterol can be reduced by inhibiting lipoprotein production; however, this is associated with steatosis. Previously we showed that lentivirally mediated hepatic expression of microRNA-30c (miR-30c) reduced hyperlipidemia and atherosclerosis in mice without causing hepatosteatosis. Because viral therapy would be formidable, we examined whether a miR-30c mimic can be used to mitigate hyperlipidemia and atherosclerosis without inducing steatosis. Delivery of a miR-30c mimic to the liver diminished diet-induced hypercholesterolemia in C57BL/6J mice. Reductions in plasma cholesterol levels were significantly correlated with increases in hepatic miR-30c levels. Long term dose escalation studies showed that miR-30c mimic caused sustained reductions in plasma cholesterol with no obvious side effects. Furthermore, miR-30c mimic significantly reduced hypercholesterolemia and atherosclerosis in Apoe(-/-) mice. Mechanistic studies showed that miR-30c mimic had no effect on LDL clearance but reduced lipoprotein production by down-regulating microsomal triglyceride transfer protein expression. MiR-30c had no effect on fatty acid oxidation but reduced lipid synthesis. Additionally, whole transcriptome analysis revealed that miR-30c mimic significantly down-regulated hepatic lipid synthesis pathways. Therefore, miR-30c lowers plasma cholesterol and mitigates atherosclerosis by reducing microsomal triglyceride transfer protein expression and lipoprotein production and avoids steatosis by diminishing lipid syntheses. It mitigates atherosclerosis most likely by reducing lipoprotein production and plasma cholesterol. These findings establish that increasing hepatic miR-30c levels is a viable treatment option for reducing hypercholesterolemia and atherosclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Design and synthesis of an antigenic mimic of the Ebola glycoprotein

    OpenAIRE

    Rutledge, Ryan D.; Huffman, Brian J.; Cliffel, David E.; Wright, David W.

    2008-01-01

    An antigenic mimic of the Ebola glycoprotein was synthesized and tested for its ability to be recognized by an anti-Ebola glycoprotein antibody. Epitope-mapping procedures yielded a suitable epitope that, when presented on the surface of a nanoparticle, forms a structure that is recognized by an antibody specific for the native protein. This mimic-antibody interaction has been quantitated through ELISA and QCM-based methods and yielded an affinity (Kd = 12 × 10−6 M) within two orders of magni...

  15. Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

    Science.gov (United States)

    Hoang, Huy-Dung; Maruyama, Jun-ichi

    2014-01-01

    Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

  16. [High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae].

    Science.gov (United States)

    Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi

    2013-11-04

    High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering

  17. Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

    Science.gov (United States)

    Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

    2014-12-16

    Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory

  18. Using heterologous expression systems to characterize potassium and sodium transport activities.

    Science.gov (United States)

    Rodríguez, Alonso; Benito, Begoña; Cagnac, Olivier

    2012-01-01

    The expression of plant transporters in simple well-characterized cell systems is an irreplaceable technique for gaining insights into the kinetic and energetic features of plant transporters. Among all the available expression systems, yeast cells offer the highest simplicity and have the capacity to mimic the in vivo properties of plant transporters. Here, we describe the use of yeast mutants to express K(+) and Na(+) plant transporters and discuss some experimental problems that can produce misleading results.

  19. Heterologous Immunological Effects of Early BCG Vaccination in Low-Birth-Weight Infants in Guinea-Bissau

    DEFF Research Database (Denmark)

    Jensen, Kristoffer Jarlov; Larsen, Nanna; Biering-Sørensen, Sofie

    2015-01-01

    or -7/8, or purified protein derivative (PPD). RESULTS:  Among 467 infants, BCG significantly increased the in vitro cytokine responses to purified protein derivative of Mycobacterium tuberculosis (PPD), as expected. BCG was also associated with increased responses to heterologous innate stimulation...

  20. LCR/MEL: A versatile system for high-level expression of heterologous proteins in erythroid cells.

    NARCIS (Netherlands)

    M. Needham; C. Gooding; K. Hudson; M. Antoniou (Michael); F.G. Grosveld (Frank); M. Hollis

    1992-01-01

    textabstractWe have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin

  1. Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

    Science.gov (United States)

    Katoch, Meenu; Mazmouz, Rabia; Chau, Rocky; Pearson, Leanne A.; Pickford, Russell

    2016-01-01

    ABSTRACT Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens. PMID:27520810

  2. Multi-omic profiling of EPO producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    Heterologous protein production in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to characterize the physiological impact of erythropoietin production, and discover production bottlenecks, ...

  3. Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied...

  4. Suppression of adaptive immunity to heterologous antigens during Plasmodium infection through hemozoin-induced failure of dendritic cell function

    Directory of Open Access Journals (Sweden)

    Phillips R

    2006-04-01

    Full Text Available Abstract Background Dendritic cells (DCs are central to the initiation and regulation of the adaptive immune response during infection. Modulation of DC function may therefore allow evasion of the immune system by pathogens. Significant depression of the host's systemic immune response to both concurrent infections and heterologous vaccines has been observed during malaria infection, but the mechanisms underlying this immune hyporesponsiveness are controversial. Results Here, we demonstrate that the blood stages of malaria infection induce a failure of DC function in vitro and in vivo, causing suboptimal activation of T cells involved in heterologous immune responses. This effect on T-cell activation can be transferred to uninfected recipients by DCs isolated from infected mice. Significantly, T cells activated by these DCs subsequently lack effector function, as demonstrated by a failure to migrate to lymphoid-organ follicles, resulting in an absence of B-cell responses to heterologous antigens. Fractionation studies show that hemozoin, rather than infected erythrocyte (red blood cell membranes, reproduces the effect of intact infected red blood cells on DCs. Furthermore, hemozoin-containing DCs could be identified in T-cell areas of the spleen in vivo. Conclusion Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function, providing a potential explanation for epidemiological studies linking endemic malaria with secondary infections and reduced vaccine efficacy.

  5. Mucosal vaccination with heterologous viral vectored vaccine targeting subdominant SIV accessory antigens strongly inhibits early viral replication

    DEFF Research Database (Denmark)

    Xu, Huanbin; Andersson, Anne-Marie Carola; Ragonnaud, Emeline

    2017-01-01

    Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat...

  6. Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha

    NARCIS (Netherlands)

    Gietl, Christine; Faber, Klaas Nico; Klei, Ida J. van der; Veenhuis, Marten

    1994-01-01

    We have studied the significance of the N-terminal presequence of watermelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase; EC 1.1.1.37] in microbody targeting. The yeast Hansenula polymorpha was used as heterologous host for the in vivo expression of

  7. Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production

    Directory of Open Access Journals (Sweden)

    Paul N. Schwarz

    2018-03-01

    Full Text Available The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

  8. Development of novel miR-129 mimics with enhanced efficacy to eliminate chemoresistant colon cancer stem cells

    Science.gov (United States)

    Ju, Jingfang

    2018-01-01

    Background Resistance to 5-Fluorouracil (5-FU) based chemotherapy is the major reason for failure of treating patients with advanced colorectal cancer. Materials and methods In this study, we developed a novel miR-129 mimic with potent efficacy in eliminating resistant colon cancer stem cells both in vitro and in vivo. We integrated 5-FU into miR-129 by replacing Uracil (U) to generate 5-FU-miR-129 mimics (Mimic-1). Results Mimic-1 is a strong therapeutic candidate with a number of unique features. Mimic-1 can be delivered to cancer cells without any transfection reagents (e.g. lipids, viral vector, nanoparticles). Mimic-1 is more potent at inhibiting cell proliferation and inducing cell cycle arrest at G1 phase than native miR-129 and the other mimics tested, while retaining target specificity. Mimic-1 prevents colon cancer metastasis in vivo without toxicity. Conclusion This represents a significant advancement in the development of a nontoxic and highly potent miRNA based cancer therapeutics and establishes a foundation for further developing Mimic-1 as a novel anti-cancer therapeutic for treating colorectal cancer. PMID:29507661

  9. Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair.

    Science.gov (United States)

    Mozafari, Roghayeh; Kyrylenko, Sergiy; Castro, Mateus Vidigal; Ferreira, Rui Seabra; Barraviera, Benedito; Oliveira, Alexandre Leite Rodrigues

    2018-01-01

    Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F + T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. The experiments indicated that

  10. Nucleolytic degradation of homologous and heterologous deoxyribonucleic acid molecules at the surface of competent pneumococci

    International Nuclear Information System (INIS)

    Seto, H.; Lopez, R.; Garrigan, O.; Tomasz, A.

    1975-01-01

    Competent pneumococci can catalyze the rapid and quantitative degradation of extracellular deoxyribonucleic acid (DNA) molecules through the activity of surface-located nucleases (endo- and, possibly, exonucleases as well). Both homologous and heterologous DNAs are degraded by a mechanism that seems to involve a cyclic process: (i) attachment of DNA to the cell surface followed by (ii) nucleolytic attack, and (iii) release to the medium. Processes (ii) and (iii) are both inhibited by ethylenediaminetetraacetate. Whereas surface nuclease activity is specific for competent cells, the bulk of this activity is not coupled to irreversible DNA uptake (deoxyribonuclease-resistant binding). Pneumococcal DNA treated with ultraviolet irradiation or nitrous acid (cross-linking) is selectively impaired in the ability to irreversibly bind to competent cells, whereas reversible binding is normal. (U.S.)

  11. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

  12. Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina

    Directory of Open Access Journals (Sweden)

    Nam Kyu Kang

    2015-12-01

    Full Text Available Oleaginous microalgae of the Nannochloropsis genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in Nannochloropsis salina in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Shble under the control of TUB and UEP promoters, cloned from N. salina, was used to confer resistance to Zeocin antibiotics, resulting in 5.9 and 4.7 transformants per 108 cells, respectively. Stable integration of the markers into the genome was confirmed using a restriction enzyme site-directed amplification (RESDA PCR. The expression of sfCherry fluorescent protein was confirmed by Western blot analysis and confocal microscopy. These results suggest new possibilities of efficient genetic engineering of Nannochloropsis for the production of biofuels and other biochemicals.

  13. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S. cerevisiae

    DEFF Research Database (Denmark)

    Rudashevskaya, Elena; Ye, Juanying; Young, Clifford

     It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...

  14. Recombinant lactic acid bacteria as delivery vectors of heterologous antigens: the future of vaccination?

    Science.gov (United States)

    Trombert, A

    2015-01-01

    Lactic acid bacteria (LABs) are good candidates for the development of new oral vaccines and are attractive alternatives to attenuated pathogens. This review focuses on the use of wild-type and recombinant lactococci and lactobacilli with emphasis on their molecular design, immunomodulation and treatment of bacterial infections. The majority of studies related to recombinant LABs have focused on Lactococcus lactis, however, molecular tools have been successfully used for Lactobacillus spp. Recombinant lactobacilli and lactococci have several health benefits, such as immunomodulation, restoration of the microbiota, synthesis of antimicrobial substances and inhibition of virulence factors. In addition, protective immune responses that are well tolerated are induced by the expression of heterologous antigens from recombinant probiotics.

  15. Transmission of a heterologous clade C Symbiodinium in a model anemone infection system via asexual reproduction

    Directory of Open Access Journals (Sweden)

    Wan-Nan U. Chen

    2016-08-01

    Full Text Available Anemones of genus Exaiptasia are used as model organisms for the study of cnidarian-dinoflagellate (genus Symbiodinium endosymbiosis. However, while most reef-building corals harbor Symbiodinium of clade C, Exaiptasia spp. anemones mainly harbor clade B Symbiodinium (ITS2 type B1 populations. In this study, we reveal for the first time that bleached Exaiptasia pallida anemones can establish a symbiotic relationship with a clade C Symbiodinium (ITS2 type C1. We further found that anemones can transmit the exogenously supplied clade C Symbiodinium cells to their offspring by asexual reproduction (pedal laceration. In order to corroborate the establishment of stable symbiosis, we used microscopic techniques and genetic analyses to examine several generations of anemones, and the results of these endeavors confirmed the sustainability of the system. These findings provide a framework for understanding the differences in infection dynamics between homologous and heterologous dinoflagellate types using a model anemone infection system.

  16. Application of image cytometry to characterize heterologous lipid flippases in yeast

    DEFF Research Database (Denmark)

    Jensen, Maria Stumph; Costa, Sara; Theorin, Lisa

    2016-01-01

    Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number...... is typically monitored by flow cytometry, a costly and maintenance-intensive method. Here, we have optimized a protocol to use an automated image-based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical...... for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry....

  17. Streptomyces rimosus GDS(L Lipase: Production, Heterologous Overexpression and Structure-Stability Relationship

    Directory of Open Access Journals (Sweden)

    Marija Abramić

    2003-01-01

    Full Text Available Streptomyces rimosus lipase gene has been overexpressed in a heterologous host, S. lividans TK23. The maximal lipase activity was determined in the culture filtrates of the late stationary phase. Time course of lipase production was monitored by a modified plate assay. S. rimosus lipase gene has been located on the AseI B fragment approximately 2 Mb far from the left end of the S. rimosus linear chromosome. Out of eight examined streptomycetes, the presence of this rare type of bacterial lipase gene was detected in two belonging to the S. rimosus taxonomic cluster, and in one non-related species. Comparison of protein sequences of the Streptomyces lipolytic enzymes was performed. The result indicated the best structural stability of the putative S. coelicolor lipase-2.

  18. Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.

    Science.gov (United States)

    Yamada, Osamu; Sakamoto, Kazutoshi; Tominaga, Mihoko; Nakayama, Tasuku; Koseki, Takuya; Fujita, Akiko; Akita, Osamu

    2005-03-01

    We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

  19. Process strategies to improve heterologous protein production in Escherichia coli under lactose or IPTG induction

    DEFF Research Database (Denmark)

    Kilikian, B. V.; Surarez, I. D.; Liria, C. W.

    2000-01-01

    Cells of Escherichia coli BL21 bearing the chicken muscle Troponin C (TnC) gene under the control of the lacUV5 promoter were induced under different cultivation conditions and the consequences on growth and cell protein content were investigated. The type of inducer molecule (lactose or IPTG...... per gram dry cell weight (DCW), was achieved when isopropyl-beta-D-thiogalactoside (IPTG) was the inducer. Under lactose induction, a value of 96 mg per gram DCW was attained. However, the high metabolic load imposed by IPTG, when compared with lactose induction, as assessed by the cell protein...... content and stability, indicates that lactose is probably the most appropriate inducer for the synthesis of this heterologous protein. (C) 2000 Elsevier Science Ltd. All rights reserved....

  20. Effects of introducing heterologous pathways on microbial metabolism with respect to metabolic optimality

    DEFF Research Database (Denmark)

    Kim, Hyun Uk; Kim, Byoungjin; Seung, Do Young

    2014-01-01

    reactions are more frequently introduced into various microbial hosts. The genome-scale metabolic simulations of Escherichia coli strains engineered to produce 1,4-butanediol, 1,3-propanediol, and amorphadiene suggest that microbial metabolism shows much different responses to the introduced heterologous...... reactions in a strain-specific manner than typical gene knockouts in terms of the energetic status (e.g., ATP and biomass generation) and chemical production capacity. The 1,4-butanediol and 1,3-propanediol producers showed greater metabolic optimality than the wild-type strains and gene knockout mutants...... for the energetic status, while the amorphadiene producer was metabolically less optimal. For the optimal chemical production capacity, additional gene knockouts were most effective for the strain producing 1,3-propanediol, but not for the one producing 1,4-butanediol. These observations suggest that strains having...

  1. Synergistic Effect of MiR-146a Mimic and Cetuximab on Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Suning Huang

    2014-01-01

    Full Text Available Previously, we found that the expression of microRNA-146a (miR-146a was downregulated in hepatocellular carcinoma (HCC formalin-fixed paraffin-embedded (FFPE tissues compared to the adjacent noncancerous hepatic tissues. In the current study, we have explored the in vitro effect of miR-146a on the malignant phenotypes of HCC cells. MiR-146a mimic could suppress cell growth and increase cellular apoptosis in HCC cell lines HepG2, HepB3, and SNU449, as assessed by spectrophotometry, fluorimetry, and fluorescence microscopy, respectively. Furthermore, western blot showed that miR-146a mimic downregulated EGFR, ERK1/2, and stat5 signalings. These effects were less potent compared to that of a siRNA targeting EGFR, a known target gene of miR-146a. Moreover, miR-146a mimic could enhance the cell growth inhibition and apoptosis induction impact of various EGFR targeting agents. The most potent combination was miR-146a mimic with cetuximab, presenting a synergistic effect. In conclusion, miR-146a plays a vital role in the cell growth and apoptosis of HCC cells and inducing miR-146a level might be a critical targeted molecular therapy strategy for HCC.

  2. Unmasking Hyperthyroidism in the Elderly: How to distinguish hyperthyroidism from conditions that mimic the symptoms

    OpenAIRE

    Gordon, Michael; Isenberg, Yoel; Bain, Jerald

    1992-01-01

    Hyperthyroidism in the elderly can mimic symptoms suggestive of aging or concomitant illnesses. The disease can cause serious problems and disabilities in older individuals. A strong sense of suspicion, the appropriate use of screening tests, and, when indicated, more definitive investigations usually results in the accurate diagnosis of a condition for which there is effective treatment.

  3. Vesicles to concentrate iron in low-Iron media: An attempt to mimic marine siderophores

    Czech Academy of Sciences Publication Activity Database

    Bednárová, Lucie; Brandel, J.; d'Hardemare, A. d. M.; Bednár, J.; Serratrice, G.; Pierre, J. L.

    2008-01-01

    Roč. 14, č. 12 (2008), s. 3680-3686 ISSN 0947-6539 Institutional research plan: CEZ:AV0Z40550506 Keywords : iron * marine siderophore mimics * micelles * self-assembly * vesicles Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 5.454, year: 2008

  4. Families, schools, and student achievement inequality: a multilevel MIMIC model approach

    Czech Academy of Sciences Publication Activity Database

    Tsai, S. L.; Smith, Michael; Hauser, R. M.

    2017-01-01

    Roč. 90, č. 1 (2017), s. 64-88 ISSN 0038-0407 R&D Projects: GA ČR GCP404/12/J006; GA ČR GB14-36154G Institutional support: RVO:67985998 Keywords : MIMIC model * educational inequality * academic performance Subject RIV: AO - Sociology, Demography OBOR OECD: Sociology Impact factor: 2.697, year: 2016

  5. Fluorescent discrimination between traces of chemical warfare agents and their mimics.

    Science.gov (United States)

    Díaz de Greñu, Borja; Moreno, Daniel; Torroba, Tomás; Berg, Alexander; Gunnars, Johan; Nilsson, Tobias; Nyman, Rasmus; Persson, Milton; Pettersson, Johannes; Eklind, Ida; Wästerby, Pär

    2014-03-19

    An array of fluorogenic probes is able to discriminate between nerve agents, sarin, soman, tabun, VX and their mimics, in water or organic solvent, by qualitative fluorescence patterns and quantitative multivariate analysis, thus making the system suitable for the in-the-field detection of traces of chemical warfare agents as well as to differentiate between the real nerve agents and other related compounds.

  6. Chemical UV Filters Mimic the Effect of Progesterone on Ca(2+) Signaling in Human Sperm Cells

    DEFF Research Database (Denmark)

    Rehfeld, A; Dissing, S; Skakkebæk, N E

    2016-01-01

    Progesterone released by cumulus cells surrounding the egg induces a Ca(2+) influx into human sperm cells via the cationic channel of sperm (CatSper) Ca(2+) channel and controls multiple Ca(2+)-dependent responses essential for fertilization. We hypothesized that chemical UV filters may mimic...

  7. The immune impact of mimic endoscopic retrograde appendicitis therapy and appendectomy on rabbits of acute appendicitis.

    Science.gov (United States)

    Liu, Suqin; Pei, Fenghua; Wang, Xinhong; Li, Deliang; Zhao, Lixia; Song, Yanyan; Chen, Zhendong; Liu, Bingrong

    2017-09-12

    This study was conducted to evaluate the immune impact of mimic endoscopic retrograde appendicitis therapy and appendectomy on rabbits of acute suppurative appendicitis and to determine whether TLR4/MYD88/NF-κB signaling pathway was activated in this process. 48 rabbits were assigned into 4 groups: group I, the mimic endoscopic retrograde appendicitis therapy group; group II, the appendectomy group; group III, the model group; and group IV, the blank group. White blood cells decreased, while levels of C-reactive protein, tumor necrosis factor-α, interleukin-6, interleukin-4, and interleukin-10 increased on the 2 nd day in group I and II. IgA in feces decreased at 2 weeks, while fecal microbiota changed at 2 and 4 weeks after appendectomy. CD8 + cells in appendix of group I increased within 8 weeks. Upregulated expression of TLR4, MYD88, and nuclear NF-κB were detected on the 2 nd day in group I and II. Mimic endoscopic retrograde appendicitis therapy and appendectomy are effective ways for acute suppurative appendicitis. Mimic endoscopic retrograde appendicitis therapy was more preferable due to its advantage in maintaining intestinal immune function. TLR4/MYD88/NF-κB signaling pathway was activated in acute phase of appendicitis.

  8. Self-Assembly of Multi-nanozymes to Mimic an Intracellular Antioxidant Defense System.

    Science.gov (United States)

    Huang, Yanyan; Liu, Zhen; Liu, Chaoqun; Ju, Enguo; Zhang, Yan; Ren, Jinsong; Qu, Xiaogang

    2016-06-01

    In this work, for the first time, we constructed a novel multi-nanozymes cooperative platform to mimic intracellular antioxidant enzyme-based defense system. V2 O5 nanowire served as a glutathione peroxidase (GPx) mimic while MnO2 nanoparticle was used to mimic superoxide dismutase (SOD) and catalase (CAT). Dopamine was used as a linker to achieve the assembling of the nanomaterials. The obtained V2 O5 @pDA@MnO2 nanocomposite could serve as one multi-nanozyme model to mimic intracellular antioxidant enzyme-based defense procedure in which, for example SOD, CAT, and GPx co-participate. In addition, through assembling with dopamine, the hybrid nanocomposites provided synergistic antioxidative effect. Importantly, both in vitro and in vivo experiments demonstrated that our biocompatible system exhibited excellent intracellular reactive oxygen species (ROS) removal ability to protect cell components against oxidative stress, showing its potential application in inflammation therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Recent advances in compartmentalized synthetic architectures as drug carriers, cell mimics and artificial organelles

    DEFF Research Database (Denmark)

    York-Durán, María José; Gallardo, Maria Godoy; Labay, Cédric Pierre

    2017-01-01

    significant research attention and these assemblies are proposed as candidate materials for a range of biomedical applications. In this Review article, the recent successes of multicompartment architectures as carriers for the delivery of therapeutic cargo or the creation of micro- and nanoreactors that mimic...

  10. A novel nitrogen-dependent gene associates with the lesion mimic trait in wheat

    Science.gov (United States)

    Lesion mimic (LM) refers to hypersensitive reaction-like (HRL) symptoms that appears on leaf tissue in the absence of plant pathogens. In a wheat line P7001, LM showed up on the leaves under the 0 g nitrogen (N) treatment, but disappeared when sufficient N was supplied, suggesting that LM is N-respo...

  11. Antibacterial TAP-mimic electrospun polymer scaffold: effects on P. gingivalis-infected dentin biofilm.

    Science.gov (United States)

    Albuquerque, Maria Tereza P; Evans, Joshua D; Gregory, Richard L; Valera, Marcia C; Bottino, Marco C

    2016-03-01

    This study sought to investigate, in vitro, the effects of a recently developed triple antibiotic paste (TAP)-mimic polymer nanofibrous scaffold against Porphyromonas gingivalis-infected dentin biofilm. Dentin specimens (4 × 4 × 1 mm(3)) were prepared from human canines. The specimens were sterilized, inoculated with P. gingivalis (ATCC 33277), and incubated for 1 week to allow for biofilm formation. Infected dentin specimens were exposed for 3 days to the following treatments: antibiotic-free polydioxanone scaffold (PDS, control), PDS + 25 wt% TAP [25 mg of each antibiotic (metronidazole, ciprofloxacin, and minocycline) per mL of the PDS polymer solution], or a saturated TAP-based solution (50 mg of each antibiotic per mL of saline solution). In order to serve as the negative control, infected dentin specimens were left untreated (bacteria only). To determine the antimicrobial efficacy of the TAP-mimic scaffold, a colony-forming unit (CFU) per milliliter (n = 10/group) measurement was performed. Furthermore, additional specimens (n = 2/group) were prepared to qualitatively study biofilm inhibition via scanning electron microscopy (SEM). Statistics were performed, and significance was set at the 5% level. Both the TAP-mimic scaffold and the positive control (TAP solution) led to complete bacterial elimination, differing statistically (p mimic scaffold against an established P. gingivalis-infected dentin biofilm. Collectively, the data suggest that the proposed nanofibrous scaffold might be used as an alternative to the advocated clinical gold standard (i.e., TAP) for intracanal disinfection prior to regenerative endodontics.

  12. Evaluation of MIMIC-Model Methods for DIF Testing with Comparison to Two-Group Analysis

    Science.gov (United States)

    Woods, Carol M.

    2009-01-01

    Differential item functioning (DIF) occurs when an item on a test or questionnaire has different measurement properties for 1 group of people versus another, irrespective of mean differences on the construct. This study focuses on the use of multiple-indicator multiple-cause (MIMIC) structural equation models for DIF testing, parameterized as item…

  13. Synergistic effect of MiR-146a mimic and cetuximab on hepatocellular carcinoma cells.

    Science.gov (United States)

    Huang, Suning; He, Rongquan; Rong, Minhua; Dang, Yiwu; Chen, Gang

    2014-01-01

    Previously, we found that the expression of microRNA-146a (miR-146a) was downregulated in hepatocellular carcinoma (HCC) formalin-fixed paraffin-embedded (FFPE) tissues compared to the adjacent noncancerous hepatic tissues. In the current study, we have explored the in vitro effect of miR-146a on the malignant phenotypes of HCC cells. MiR-146a mimic could suppress cell growth and increase cellular apoptosis in HCC cell lines HepG2, HepB3, and SNU449, as assessed by spectrophotometry, fluorimetry, and fluorescence microscopy, respectively. Furthermore, western blot showed that miR-146a mimic downregulated EGFR, ERK1/2, and stat5 signalings. These effects were less potent compared to that of a siRNA targeting EGFR, a known target gene of miR-146a. Moreover, miR-146a mimic could enhance the cell growth inhibition and apoptosis induction impact of various EGFR targeting agents. The most potent combination was miR-146a mimic with cetuximab, presenting a synergistic effect. In conclusion, miR-146a plays a vital role in the cell growth and apoptosis of HCC cells and inducing miR-146a level might be a critical targeted molecular therapy strategy for HCC.

  14. MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

    Science.gov (United States)

    Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A.; Pan, Hongling; He, Yuchun; Evans-Holm, Martha; Carlson, Joseph W.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2011-01-01

    We demonstrate the versatility of a collection of insertions of the transposon Minos mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp system. Insertions within coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the Drosophila melanogaster toolkit. PMID:21985007

  15. Validation of Triphasic Mixture Theory for a Mimic of Intervertebral Disk Tissue

    NARCIS (Netherlands)

    Oomens, C.W.J.; Heus, de H.J.; Huyghe, J.M.R.J.; Nelissen, J.G.L.; Janssen, J.D.

    1995-01-01

    This paper describes experimental studies an synthetic madel materials that mimic the mechanical behavior af intervertebral disk tissue. The results are used ta validate the triphasic mixture theory to describe soft charged hydrated materiais. Permeability and swelling pressure experiments were used

  16. Bourgeois Males of the Peacock Blenny, Salaria pavo, Discriminate Female Mimics from Females?

    DEFF Research Database (Denmark)

    Gonçalves, David; Matos, Ricardo Jorge Santa Clara; Fagundes, Teresa

    2005-01-01

    In a Portuguese population of Salaria pavo, two types of reproductively active males occur: large bourgeois males that defend nests and have fully developed secondary sex characters (SSC) and small sneaker males that mimic the females' morphology and behaviour to approach nests and parasitize...

  17. α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

    Science.gov (United States)

    Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  18. Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

    Science.gov (United States)

    Law, Wenjing; Wuescher, Leah M; Ortega, Amanda; Hapiak, Vera M; Komuniecki, Patricia R; Komuniecki, Richard

    2015-04-01

    Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification

  19. Lactobacillus priming of the respiratory tract: Heterologous immunity and protection against lethal pneumovirus infection.

    Science.gov (United States)

    Garcia-Crespo, Katia E; Chan, Calvin C; Gabryszewski, Stanislaw J; Percopo, Caroline M; Rigaux, Peter; Dyer, Kimberly D; Domachowske, Joseph B; Rosenberg, Helene F

    2013-03-01

    We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We show here that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for 5months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity. We undertook the present study in order to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (CXCL1, CCL3, CCL2, CXCL10, TNF-alpha and IL-17A) but not Th1 cytokines. IFNγ does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities. Published by Elsevier B.V.

  20. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    Science.gov (United States)

    Yurong, Chai; Yumin, Lu; Tianyun, Wang; Weihong, Hou; Lexun, Xue

    2006-12-01

    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  1. Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

    Directory of Open Access Journals (Sweden)

    Tyo Keith EJ

    2012-03-01

    Full Text Available Abstract Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor or a larger protein (α-amylase. Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a degradation of protein/recycling amino acids, (b overall transcription/translation repression, and (c oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

  2. The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

    Science.gov (United States)

    Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

    2010-01-01

    Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

  3. The role of heterologous chloroplast sequence elements in transgene integration and expression.

    Science.gov (United States)

    Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

    2010-04-01

    Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

  4. Production of antibacterial peptide from bee venom via a new strategy for heterologous expression.

    Science.gov (United States)

    Hou, Chunsheng; Guo, Liqiong; Lin, Junfang; You, Linfeng; Wu, Wuhua

    2014-12-01

    Honey bee is important economic insect that not only pollinates fruits and crops but also provides products with various physiological activities. Bee venom is a functional agent that is widely applied in clinical treatment and pharmacy. Secapin is one of these agents that have a significant role in therapy. The functions of secapin from the bee venom have been documented, but little information is known about its heterologous expression under natural condition. Moreover, few scholars verified experimentally the functions of secapin from bee venom in vitro. In this study, we successfully constructed a heterologous expression vector, which is different from conventional expression system. A transgenic approach was established for transformation of secapin gene from the venom of Apis mellifera carnica (Ac-sec) into the edible fungi, Coprinus cinereus. Ac-sec was encoded by a 234 bp nucleotide that contained a signal peptide domain and two potential phosphorylation sites. The sequence exhibited highly homology with various secapins characterized from honey bee and related species. Southern blot data indicated that Ac-sec was present as single or multiple copy loci in the C. cinereus genome. By co-transformation and double-layer active assay, Ac-sec was expressed successfully in C. cinereus and the antibacterial activity of the recombinants was identified, showing notable antibacterial activities on different bacteria. Although Ac-sec is from the venom of Apidae, phylogenetic analysis demonstrated that Ac-sec was more closely related to that of Vespid than to bee species from Apidae. The molecular characteristics of Ac-sec and the potential roles of small peptides in biology were discussed.

  5. Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses.

    Directory of Open Access Journals (Sweden)

    Songsong Wu

    2017-03-01

    Full Text Available Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4, could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility loci. Reactive oxygen species (ROS plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

  6. Trivalent combination vaccine induces broad heterologous immune responses to norovirus and rotavirus in mice.

    Directory of Open Access Journals (Sweden)

    Kirsi Tamminen

    Full Text Available Rotavirus (RV and norovirus (NoV are the two major causes of viral gastroenteritis (GE in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1 derived virus-like particles (VLPs of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6, the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined in vitro. The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50% as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization (GII-4 NO, GII-12 and GI-1 VLPs and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes.

  7. Heterologous production of Pseudomonas aeruginosa rhamnolipid under anaerobic conditions for microbial enhanced oil recovery.

    Science.gov (United States)

    Zhao, F; Shi, R; Zhao, J; Li, G; Bai, X; Han, S; Zhang, Y

    2015-02-01

    The ex situ application of rhamnolipid to enhance oil recovery is costly and complex in terms of rhamnolipid production and transportation, while in situ production of rhamnolipid is restricted by the oxygen-deficient environments of oil reservoirs. To overcome the oxygen-limiting conditions and to circumvent the complex regulation of rhamnolipid biosynthesis in Pseudomonas aeruginosa, an engineered strain Pseudomonas stutzeri Rhl was constructed for heterologous production of rhamnolipid under anaerobic conditions. The rhlABRI genes for rhamnolipid biosynthesis were cloned into a facultative anaerobic strain Ps. stutzeri DQ1 to construct the engineered strain Rhl. Anaerobic production of rhamnolipid was confirmed by thin layer chromatography and Fourier transform infrared analysis. Rhamnolipid product reduced the air-water surface tension to 30.3 mN m(-1) and the oil-water interfacial tension to 0.169 mN m(-1). Rhl produced rhamnolipid of 1.61 g l(-1) using glycerol as the carbon source. Rhl anaerobic culture emulsified crude oil up to EI24 ≈ 74. An extra 9.8% of original crude oil was displaced by Rhl in the core flooding test. Strain Rhl achieved anaerobic production of rhamnolipid and worked well for enhanced oil recovery in the core flooding model. The rhamnolipid produced by Rhl was similar to that of the donor strain SQ6. This is the first study to achieve anaerobic and heterologous production of rhamnolipid. Results demonstrated the potential feasibility of Rhl as a promising strain to enhance oil recovery through anaerobic production of rhamnolipid. © 2014 The Society for Applied Microbiology.

  8. Partial protection of SIV-infected rhesus monkeys against superinfection with a heterologous SIV isolate

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette [Los Alamos National Laboratory

    2009-01-01

    Although there is increasing evidence that individuals already infected with human immunodeficiency virus type 1 (HIV-1) can be infected with a heterologous strain of the virus, the extent of protection against superinfection conferred by the first infection and the biologic consequences of superinfection are not well understood. We explored these questions in the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV-1/AIDS. We infected cohorts of rhesus monkeys with either SIVmac251 or SIVsmE660 and then exposed animals to the reciprocal virus through intrarectal inoculations. Employing a quantitative real-time PCR assay, we determined the replication kinetics of the two strains of virus for 20 weeks. We found that primary infection with a replication-competent virus did not protect against acquisition of infection by a heterologous virus but did confer relative control of the superinfecting virus. In animals that became superinfected, there was a reduction in peak replication and rapid control of the second virus. The relative susceptibility to superinfection was not correlated with CD4(+) T-cell count, CD4(+) memory T-cell subsets, cytokine production by virus-specific CD8(+) or CD4(+) cells, or neutralizing antibodies at the time of exposure to the second virus. Although there were transient increases in viral loads of the primary virus and a modest decline in CD4(+) T-cell counts after superinfection, there was no evidence of disease acceleration. These findings indicate that an immunodeficiency virus infection confers partial protection against a second immunodeficiency virus infection, but this protection may be mediated by mechanisms other than classical adaptive immune responses.

  9. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    Science.gov (United States)

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Heterologous expression of mlrA in a photoautotrophic host - Engineering cyanobacteria to degrade microcystins.

    Science.gov (United States)

    Dexter, Jason; Dziga, Dariusz; Lv, Jing; Zhu, Junqi; Strzalka, Wojciech; Maksylewicz, Anna; Maroszek, Magdalena; Marek, Sylwia; Fu, Pengcheng

    2018-06-01

    In this report, we establish proof-of-principle demonstrating for the first time genetic engineering of a photoautotrophic microorganism for bioremediation of naturally occurring cyanotoxins. In model cyanobacterium Synechocystis sp. PCC 6803 we have heterologously expressed Sphingopyxis sp. USTB-05 microcystinase (MlrA) bearing a 23 amino acid N-terminus secretion peptide from native Synechocystis sp. PCC 6803 PilA (sll1694). The resultant whole cell biocatalyst displayed about 3 times higher activity against microcystin-LR compared to a native MlrA host (Sphingomonas sp. ACM 3962), normalized for optical density. In addition, MlrA activity was found to be almost entirely located in the cyanobacterial cytosolic fraction, despite the presence of the secretion tag, with crude cellular extracts showing MlrA activity comparable to extracts from MlrA expressing E. coli. Furthermore, despite approximately 9.4-fold higher initial MlrA activity of a whole cell E. coli biocatalyst, utilization of a photoautotrophic chassis resulted in prolonged stability of MlrA activity when cultured under semi-natural conditions (using lake water), with the heterologous MlrA biocatalytic activity of the E. coli culture disappearing after 4 days, while the cyanobacterial host displayed activity (3% of initial activity) after 9 days. In addition, the cyanobacterial cell density was maintained over the duration of this experiment while the cell density of the E. coli culture rapidly declined. Lastly, failure to establish a stable cyanobacterial isolate expressing native MlrA (without the N-terminus tag) via the strong cpcB560 promoter draws attention to the use of peptide tags to positively modulate expression of potentially toxic proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

    Directory of Open Access Journals (Sweden)

    Wenjing Law

    2015-04-01

    Full Text Available Monoamines, such as 5-HT and tyramine (TA, paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for

  12. Extensive preclinical investigation of polymersomal formulation of doxorubicin versus Doxil-mimic formulation.

    Science.gov (United States)

    Alibolandi, Mona; Abnous, Khalil; Mohammadi, Marzieh; Hadizadeh, Farzin; Sadeghi, Fatemeh; Taghavi, Sahar; Jaafari, Mahmoud Reza; Ramezani, Mohammad

    2017-10-28

    Due to the severe cardiotoxicity of doxorubicin, its usage is limited. This shortcoming could be overcome by modifying pharmacokinetics of the drugs via preparation of various nanoplatforms. Doxil, a well-known FDA-approved nanoplatform of doxorubicin as antineoplastic agent, is frequently used in clinics in order to reduce cardiotoxicity of doxorubicin. Since Doxil shows some shortcomings in clinics including hand and food syndrome and very slow release pattern thus, there is a demand for the development and preparation of new doxorubicin nanoformulation with fewer side effects. The new formulation of the doxorubicin, synthesized previously by our group was extensively examined in the current study. This new formulation is doxorubicin encapsulated in PEG-PLGA polymersomes (PolyDOX). The main aim of the study was to compare the distribution and treatment efficacy of a new doxorubicin-polymersomal formulation (PolyDOX) with regular liposomal formulation (Doxil-mimic) in murine colon adenocarcinoma model. Additionally, the pathological, hematological changes, pharmacodynamics, biodistribution, tolerated dose and survival rate in vivo were evaluated and compared. Murine colon cancer model was induced by subcutaneous inoculation of BALB/c mice with C26 cells. Afterwards, either Doxil-mimic or PolyDOX was administered intravenously. The obtained results from biodistribution study showed a remarkable difference in the distribution of drugs in murine organs. In this regard, Doxil-mimic exhibited prolonged (48h) presence within liver tissues while PolyDOX preferentially accumulate in tumor and the presence in liver 48h post-treatment was significantly lower than that of Doxil-mimic. Obtained results demonstrated comparable final length of life for mice receiving either Doxil-mimic or PolyDOX formulations whereas tolerated dose of mice receiving Doxil-mimic was remarkably higher than those receiving PolyDOX. Therapeutic efficacy of formulation in term of tumor growth rate

  13. Triazacyclophane (TAC)-scaffolded histidine and aspartic acid residues as mimics of non-heme metalloenzyme active sites

    NARCIS (Netherlands)

    Albada, H.B.; Soulimani, F.; Jacobs, H.J.F.; Versluis, C.; Weckhuysen, B.M.; Liskamp, R.M.J.

    2012-01-01

    We describe the synthesis and coordination behaviour to copper(II) of two close structural triazacyclophane-based mimics of two often encountered aspartic acid and histidine containing metalloenzyme active sites. Coordination of these mimics to copper(I) and their reaction with molecular oxygen

  14. How well does cholesteryl hemisuccinate mimic cholesterol in saturated phospholipid bilayers?

    DEFF Research Database (Denmark)

    Kulig, W.; Tynkkynen, J.; Javanainen, M.

    2014-01-01

    Cholesteryl hemisuccinate is a detergent that is often used to replace cholesterol in crystallization of membrane proteins. Here we employ atomistic molecular dynamics simulations to characterize how well the properties of cholesteryl hemisuccinate actually match those of cholesterol in saturated...... protein-free lipid membranes. We show that the protonated form of cholesteryl hemisuccinate mimics many of the membrane properties of cholesterol quite well, while the deprotonated form of cholesteryl hemisuccinate is less convincing in this respect. Based on the results, we suggest that cholesteryl...... hemisuccinate in its protonated form is a quite faithful mimic of cholesterol for membrane protein crystallization, if specific cholesterol-protein interactions (not investigated here) are not playing a crucial role....

  15. Biomaterial delivery of morphogens to mimic the natural healing cascade in bone.

    Science.gov (United States)

    Mehta, Manav; Schmidt-Bleek, Katharina; Duda, Georg N; Mooney, David J

    2012-09-01

    Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Sensitive fluorescence on-off probes for the fast detection of a chemical warfare agent mimic.

    Science.gov (United States)

    Khan, Muhammad Shar Jhahan; Wang, Ya-Wen; Senge, Mathias O; Peng, Yu

    2018-01-15

    Two highly sensitive probes bearing a nucleophilic imine moiety have been utilized for the selective detection of chemical warfare agent (CWA) mimics. Diethyl chlorophosphate (DCP) was used as mimic CWAs. Both iminocoumarin-benzothiazole-based probes not only demonstrated a remarkable fluorescence ON-OFF response and good recognition, but also exhibited fast response times (10s) along with color changes upon addition of DCP. Limits of detection for the two sensors 1 and 2 were calculated as 0.065μM and 0.21μM, respectively, which are much lower than most other reported probes. These two probes not only show high sensitivity and selectivity in solution, but can also be applied for the recognition of DCP in the gas state, with significant color changes easily observed by the naked eye. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Knockdown of Rice microRNA166 by Short Tandem Target Mimic (STTM).

    Science.gov (United States)

    Teotia, Sachin; Zhang, Dabing; Tang, Guiliang

    2017-01-01

    Small RNAs, including microRNAs (miRNAs), are abundant in plants and play key roles in controlling plant development and physiology. miRNAs regulate the expression of the target genes involved in key plant processes. Due to functional redundancy among miRNA family members in plants, an ideal approach to silence the expression of all members simultaneously, for their functional characterization, is desirable. Target mimic (TM) was the first approach to achieve this goal. Short tandem target mimic (STTM) is a potent approach complementing TM for silencing miRNAs in plants. STTMs have been successfully used in dicots to block miRNA functions. Here, we describe in detail the protocol for designing STTM construct to block miRNA functions in rice. Such approach can be applied to silence miRNAs in other monocots as well.

  18. Structural Mimics of the [Fe]-Hydrogenase: A Complete Set for Group VIII Metals.

    Science.gov (United States)

    Barik, Chandan Kr; Ganguly, Rakesh; Li, Yongxin; Leong, Weng Kee

    2018-06-18

    A set of structural mimics of the [Fe]-hydrogenase active site comprising all the group VIII metals, viz., [M(2-NHC(O)C 5 H 4 N)(CO) 2 (2-S-C 5 H 4 N)], has been synthesized. They exist as a mixture of isomers in solution, and the relative stability of the isomers depends on the nature of the metal and the substituent at the 6-position of the pyridine ligand.

  19. Artificial cell mimics as simplified models for the study of cell biology.

    Science.gov (United States)

    Salehi-Reyhani, Ali; Ces, Oscar; Elani, Yuval

    2017-07-01

    Living cells are hugely complex chemical systems composed of a milieu of distinct chemical species (including DNA, proteins, lipids, and metabolites) interconnected with one another through a vast web of interactions: this complexity renders the study of cell biology in a quantitative and systematic manner a difficult task. There has been an increasing drive towards the utilization of artificial cells as cell mimics to alleviate this, a development that has been aided by recent advances in artificial cell construction. Cell mimics are simplified cell-like structures, composed from the bottom-up with precisely defined and tunable compositions. They allow specific facets of cell biology to be studied in isolation, in a simplified environment where control of variables can be achieved without interference from a living and responsive cell. This mini-review outlines the core principles of this approach and surveys recent key investigations that use cell mimics to address a wide range of biological questions. It will also place the field in the context of emerging trends, discuss the associated limitations, and outline future directions of the field. Impact statement Recent years have seen an increasing drive to construct cell mimics and use them as simplified experimental models to replicate and understand biological phenomena in a well-defined and controlled system. By summarizing the advances in this burgeoning field, and using case studies as a basis for discussion on the limitations and future directions of this approach, it is hoped that this minireview will spur others in the experimental biology community to use artificial cells as simplified models with which to probe biological systems.

  20. A concise flow synthesis of indole-3-carboxylic ester and its derivatisation to an auxin mimic

    Directory of Open Access Journals (Sweden)

    Marcus Baumann

    2017-11-01

    Full Text Available An assembled suite of flow-based transformations have been used to rapidly scale-up the production of a novel auxin mimic-based herbicide which was required for preliminary field trials. The overall synthetic approach and optimisation studies are described along with a full description of the final reactor configurations employed for the synthesis as well as the downstream processing of the reaction streams.

  1. Acute aortic dissection mimics acute inferoposterior wall myocardial infarction in a Marfan syndrome patient

    OpenAIRE

    Phowthongkum, Prasit

    2010-01-01

    A 30-year old man with acute chest pain was diagnosed with acute inferoposterior wall myocardial infarction following electrocardiography. After a failed coronary angiography, an echocardiogram revealed an aortic intimal flap after which acute aortic dissection was diagnosed. The patient received a successful Bentall operation without immediate complication. Retrospective examination then confirmed the diagnosis of Marfan syndrome. This case demonstrates acute aortic dissection may mimic acut...

  2. Acute aortic dissection mimics acute inferoposterior wall myocardial infarction in a Marfan syndrome patient.

    Science.gov (United States)

    Phowthongkum, Prasit

    2010-01-01

    A 30-year old man with acute chest pain was diagnosed with acute inferoposterior wall myocardial infarction following electrocardiography. After a failed coronary angiography, an echocardiogram revealed an aortic intimal flap after which acute aortic dissection was diagnosed. The patient received a successful Bentall operation without immediate complication. Retrospective examination then confirmed the diagnosis of Marfan syndrome. This case demonstrates acute aortic dissection may mimic acute myocardial infarction.

  3. Noise analysis and mimic experiments for loose part accident in the primary coolant loop of PWR

    International Nuclear Information System (INIS)

    Yang Xiuzhou; Cheng Tingxiang; Zhang Bin

    1994-01-01

    The basic principle of loose part monitoring is to detect and measure the structure transfer sound generated by impacting of metal loose part with accelerators and to identify and diagnose by the micro-processor. This paper introduces the theoretical base of loose part monitoring, the location and mass estimation of loose part, and three mimic experiment applying noise analysis techniques. It provides some useful preparations for the development of loose part monitoring system

  4. A sensitive colorimetric aptasensor based on trivalent peroxidase-mimic DNAzyme and magnetic nanoparticles.

    Science.gov (United States)

    Liu, Shuwen; Xu, Naihan; Tan, Chunyan; Fang, Wei; Tan, Ying; Jiang, Yuyang

    2018-08-14

    In this study, a novel colorimetric aptasensor was prepared by coupling trivalent peroxidase-mimic DNAzyme and magnetic nanoparticles for highly sensitive and selective detection of target proteins. A three G-quadruplex (G4) DNA-hemin complex was employed as the trivalent peroxidase-mimic DNAzyme, in which hemin assisted the G4-DNA to fold into a catalytic conformation and act as an enzyme. The design of the aptasensor includes magnetic nanoparticles (MNPs), complementary DNA (cDNA) modified with biotin, and a label-free single strand DNA (ssDNA) including the aptamer and trivalent peroxidase-mimic DNAzyme. The trivalent DNAzyme, which has the highest catalytic activity among multivalent DNAzymes, catalyzed the H 2 O 2 -mediated oxidation of ABTS. The colorless ABTS was oxidized to produce a blue-green product that can be clearly distinguished by the naked eye. The aptamer and trivalent peroxidase-mimic DNAzyme promote the specificity and sensitivity of this detection method, which can be generalized for other targets by simply replacing the corresponding aptamers. To demonstrate the feasible use of the aptasensor for target detection, a well-known tumor biomarker MUC1 was evaluated as the model target. The limits of detection were determined to be 5.08 and 5.60 nM in a linear range of 50-1000 nM in a buffer solution and 10% serum system, respectively. This colorimetric and label-free aptasensor with excellent sensitivity and strong anti-interference ability has potential application in disease diagnoses, prognosis tracking, and therapeutic evaluation. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Characterization and fine mapping of a light-dependent leaf lesion mimic mutant 1 in rice.

    Science.gov (United States)

    Wang, Jing; Ye, Bangquan; Yin, Junjie; Yuan, Can; Zhou, Xiaogang; Li, Weitao; He, Min; Wang, Jichun; Chen, Weilan; Qin, Peng; Ma, Bintian; Wang, Yuping; Li, Shigui; Chen, Xuewei

    2015-12-01

    Plants that spontaneously produce lesion mimics or spots, without any signs of obvious adversity, such as pesticide and mechanical damage, or pathogen infection, are so-called lesion mimic mutants (lmms). In rice, many lmms exhibit enhanced resistance to pathogens, which provides a unique opportunity to uncover the molecular mechanism underlying lmms. We isolated a rice light-dependent leaf lesion mimic mutant 1 (llm1). Lesion spots appeared in the leaves of the llm1 mutant at the tillering stage. Furthermore, the mutant llm1 had similar agronomic traits to wild type rice. Trypan blue and diamiobenzidine staining analyses revealed that the lesion spot formation on the llm1 mutant was due to programmed cell death and reactive oxygen species. The chloroplasts were severely damaged in the llm1 mutant, suggesting that chloroplast damage was associated with the formation of lesion spots in llm1. More importantly, llm1 exhibited enhanced resistance to bacterial blight pathogens within increased expression of pathogenesis related genes (PRs). Using a map-based cloning approach, we delimited the LLM1 locus to a 121-kb interval between two simple sequence repeat markers, RM17470 and RM17473, on chromosome 4. We sequenced the candidate genes on the interval and found that a base mutation had substituted adenine phosphate for thymine in the last exon of LOC_Os04g52130, which led to an amino acid change (Asp(388) to Val) in the llm1 mutant. Our investigation showed that the putative coproporphyrinogen III oxidase (CPOX) encoded by LOC_Os04g52130 was produced by LLM1 and that amino acid Asp(388) was essential for CPOX function. Our study provides the basis for further investigations into the mechanism underlying lesion mimic initiation associated with LLM1. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  6. Independent monitor unit calculation for intensity modulated radiotherapy using the MIMiC multileaf collimator

    International Nuclear Information System (INIS)

    Chen Zhe; Xing Lei; Nath, Ravinder

    2002-01-01

    A self-consistent monitor unit (MU) and isocenter point-dose calculation method has been developed that provides an independent verification of the MU for intensity modulated radiotherapy (IMRT) using the MIMiC (Nomos Corporation) multileaf collimator. The method takes into account two unique features of IMRT using the MIMiC: namely the gantry-dynamic arc delivery of intensity modulated photon beams and the slice-by-slice dose delivery for large tumor volumes. The method converts the nonuniform beam intensity planned at discrete gantry angles of 5 deg. or 10 deg. into conventional nonmodulated beam intensity apertures of elemental arc segments of 1 deg. This approach more closely simulates the actual gantry-dynamic arc delivery by MIMiC. Because each elemental arc segment is of uniform intensity, the MU calculation for an IMRT arc is made equivalent to a conventional arc with gantry-angle dependent beam apertures. The dose to the isocenter from each 1 deg. elemental arc segment is calculated by using the Clarkson scatter summation technique based on measured tissue-maximum-ratio and output factors, independent of the dose calculation model used in the IMRT planning system. For treatments requiring multiple treatment slices, the MU for the arc at each treatment slice takes into account the MU, leakage and scatter doses from other slices. This is achieved by solving a set of coupled linear equations for the MUs of all involved treatment slices. All input dosimetry data for the independent MU/isocenter point-dose calculation are measured directly. Comparison of the MU and isocenter point dose calculated by the independent program to those calculated by the Corvus planning system and to direct measurements has shown good agreement with relative difference less than ±3%. The program can be used as an independent initial MU verification for IMRT plans using the MIMiC multileaf collimators

  7. Glutathione peroxidase mimic ebselen improves glucose-stimulated insulin secretion in murine islets.

    Science.gov (United States)

    Wang, Xinhui; Yun, Jun-Won; Lei, Xin Gen

    2014-01-10

    Glutathione peroxidase (GPX) mimic ebselen and superoxide dismutase (SOD) mimic copper diisopropylsalicylate (CuDIPs) were used to rescue impaired glucose-stimulated insulin secretion (GSIS) in islets of GPX1 and(or) SOD1-knockout mice. Ebselen improved GSIS in islets of all four tested genotypes. The rescue in the GPX1 knockout resulted from a coordinated transcriptional regulation of four key GSIS regulators and was mediated by the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α)-mediated signaling pathways. In contrast, CuDIPs improved GSIS only in the SOD1 knockout and suppressed gene expression of the PGC-1α pathway. Islets from the GPX1 and(or) SOD1 knockout mice provided metabolically controlled intracellular hydrogen peroxide (H2O2) and superoxide conditions for the present study to avoid confounding effects. Bioinformatics analyses of gene promoters and expression profiles guided the search for upstream signaling pathways to link the ebselen-initiated H2O2 scavenging to downstream key events of GSIS. The RNA interference was applied to prove PGC-1α as the main mediator for that link. Our study revealed a novel metabolic use and clinical potential of ebselen in rescuing GSIS in the GPX1-deficient islets and mice, along with distinct differences between the GPX and SOD mimics in this regard. These findings highlight the necessities and opportunities of discretional applications of various antioxidant enzyme mimics in treating insulin secretion disorders. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Regina Brigelius-Flohe, Vadim Gladyshev, Dexing Hou, and Holger Steinbrenner.

  8. Heterologous microarray experiments allow the identification of the early events associated with potato tuber cold sweetening

    Directory of Open Access Journals (Sweden)

    Vitulli Federico

    2008-04-01

    Full Text Available Abstract Background Since its discovery more than 100 years ago, potato (Solanum tuberosum tuber cold-induced sweetening (CIS has been extensively investigated. Several carbohydrate-associated genes would seem to be involved in the process. However, many uncertainties still exist, as the relative contribution of each gene to the process is often unclear, possibly as the consequence of the heterogeneity of experimental systems. Some enzymes associated with CIS, such as β-amylases and invertases, have still to be identified at a sequence level. In addition, little is known about the early events that trigger CIS and on the involvement/association with CIS of genes different from carbohydrate-associated genes. Many of these uncertainties could be resolved by profiling experiments, but no GeneChip is available for the potato, and the production of the potato cDNA spotted array (TIGR has recently been discontinued. In order to obtain an overall picture of early transcriptional events associated with CIS, we investigated whether the commercially-available tomato Affymetrix GeneChip could be used to identify which potato cold-responsive gene family members should be further studied in detail by Real-Time (RT-PCR (qPCR. Results A tomato-potato Global Match File was generated for the interpretation of various aspects of the heterologous dataset, including the retrieval of best matching potato counterparts and annotation, and the establishment of a core set of highly homologous genes. Several cold-responsive genes were identified, and their expression pattern was studied in detail by qPCR over 26 days. We detected biphasic behaviour of mRNA accumulation for carbohydrate-associated genes and our combined GeneChip-qPCR data identified, at a sequence level, enzymatic activities such as β-amylases and invertases previously reported as being involved in CIS. The GeneChip data also unveiled important processes accompanying CIS, such as the induction of redox

  9. Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

    Science.gov (United States)

    Pascual, Elena; Mata, Carlos P.; Carrascosa, José L.; Castón, José R.

    2017-12-01

    Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases

  10. Gibberellic and kaurenoic hybrid strigolactone mimics for seed germination of parasitic weeds.

    Science.gov (United States)

    Pereira, Rondinelle G; Cala, Antonio; Fernández-Aparicio, Mónica; Molinillo, José Mg; Boaventura, Maria Ad; Macías, Francisco A

    2017-12-01

    Parasitic weeds are widespread and cause significant losses in important crops. Their germination requires the detection of crop-derived molecules such as strigolactones. Strigolactone mimics are germination-inducing molecules with the potential to apply a suicidal germination strategy for seed bank control of parasitic weeds. The D-ring, which is instrumental in the germination process of seeds of parasitic weeds, was attached to gibberellin (GA 3 ) and kaurenoic acid as the scaffold. It was shown that indeed strigolactone mimics prepared from GA 3 and kaurenoic acid are active as stimulants when a D-ring is present; some of the mimics are as active as GR24. The starting molecules were plant hormones that had previous growth-regulating activity in other organisms and the products showed enhanced activity towards parasitic weeds. The information generated may contribute to a better understanding of the germination biochemistry of the weed species used. Further research is required in this area but it is clear that the results are promising. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. MIMIC: An Innovative Methodology for Determining Mobile Laser Scanning System Point Density

    Directory of Open Access Journals (Sweden)

    Conor Cahalane

    2014-08-01

    Full Text Available Understanding how various Mobile Mapping System (MMS laser hardware configurations and operating parameters exercise different influence on point density is important for assessing system performance, which in turn facilitates system design and MMS benchmarking. Point density also influences data processing, as objects that can be recognised using automated algorithms generally require a minimum point density. Although obtaining the necessary point density impacts on hardware costs, survey time and data storage requirements, a method for accurately and rapidly assessing MMS performance is lacking for generic MMSs. We have developed a method for quantifying point clouds collected by an MMS with respect to known objects at specified distances using 3D surface normals, 2D geometric formulae and line drawing algorithms. These algorithms were combined in a system called the Mobile Mapping Point Density Calculator (MIMIC and were validated using point clouds captured by both a single scanner and a dual scanner MMS. Results from MIMIC were promising: when considering the number of scan profiles striking the target, the average error equated to less than 1 point per scan profile. These tests highlight that MIMIC is capable of accurately calculating point density for both single and dual scanner MMSs.

  12. A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Marco Todesco

    2010-07-01

    Full Text Available Many targets of plant microRNAs (miRNAs are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

  13. Instructions to mimic improve facial emotion recognition in people with sub-clinical autism traits.

    Science.gov (United States)

    Lewis, Michael B; Dunn, Emily

    2017-11-01

    People tend to mimic the facial expression of others. It has been suggested that this helps provide social glue between affiliated people but it could also aid recognition of emotions through embodied cognition. The degree of facial mimicry, however, varies between individuals and is limited in people with autism spectrum conditions (ASC). The present study sought to investigate the effect of promoting facial mimicry during a facial-emotion-recognition test. In two experiments, participants without an ASC diagnosis had their autism quotient (AQ) measured. Following a baseline test, they did an emotion-recognition test again but half of the participants were asked to mimic the target face they saw prior to making their responses. Mimicry improved emotion recognition, and further analysis revealed that the largest improvement was for participants who had higher scores on the autism traits. In fact, recognition performance was best overall for people who had high AQ scores but also received the instruction to mimic. Implications for people with ASC are explored.

  14. Early diagnosis of amyotrophic lateral sclerosis mimic syndromes: pros and cons of current clinical diagnostic criteria.

    Science.gov (United States)

    Cortés-Vicente, Elena; Pradas, Jesús; Marín-Lahoz, Juan; De Luna, Noemi; Clarimón, Jordi; Turon-Sans, Janina; Gelpí, Ellen; Díaz-Manera, Jordi; Illa, Isabel; Rojas-Garcia, Ricard

    2017-08-01

    To describe the frequency and clinical characteristics of patients referred to a tertiary neuromuscular clinic as having amyotrophic lateral sclerosis (ALS) but who were re-diagnosed as having an ALS mimic syndrome, and to identify the reasons that led to the revision of the diagnosis. We reviewed the final diagnosis of all patients prospectively registered in the Sant Pau-MND register from 1 January 2004 to 31 December 2015. A detailed clinical evaluation and a clinically-guided electrophysiological study were performed at first evaluation. Twenty of 314 (6.4%) patients included were re-diagnosed as having a condition other than ALS, in 18 cases already at first evaluation. An alternative specific diagnosis was identified in 17 of those 20, consisting of a wide range of conditions. The main finding leading to an alternative diagnosis was the result of the electrophysiological study. Fifty per cent did not fulfil the El Escorial revised criteria (EECr) for ALS. The most common clinical phenotype at onset in patients with ALS mimic syndromes was progressive muscular atrophy (PMA). Misdiagnosing ALS is still a common problem. Early identification of ALS mimic syndromes is possible based on atypical clinical features and a clinically-guided electrophysiological study. Patients should be attended in specialised centres. The application of EECr helps to identify ALS misdiagnoses.

  15. MicroRNA-122 mimic transfection contributes to apoptosis in HepG2 cells.

    Science.gov (United States)

    Huang, Hongyan; Zhu, Yueyong; Li, Shaoyang

    2015-11-01

    There is currently a requirement for effective treatment strategies for human hepatocellular carcinoma (HCC), a leading cause of cancer‑associated mortality. MicroRNA-122 (miR-122), a repressor of the endogenous apoptosis regulator Bcl‑w, is frequently downregulated in HCC. Thus, it is hypothesized that the activation of miR‑122 may induce selective hepatocellular apoptosis via caspase activation in a model of HCC. In the present study, an miR‑122 mimic transfection was performed in HepG2 cells, and used to investigate the role and therapeutic potential of miR‑122 in the regulation of HCC‑derived cell lines. The apoptotic rates of HepG2 cells were significantly increased following miR‑122 mimic transfection. Reverse transcription‑polymerase chain reaction analysis revealed that Bcl‑w mRNA was significantly reduced, while the mRNA levels of caspase‑9 and caspase‑3 were markedly increased. The immunocytochemistry results supported the mRNA trends. Collectively, the present results suggest that endogenous miR‑122 contributes to HepG2 apoptosis and that transfection of mimic miR‑122 normalizes apoptotic levels in a model of HCC.

  16. Fast and efficient Drosophila melanogaster gene knock-ins using MiMIC transposons.

    Science.gov (United States)

    Vilain, Sven; Vanhauwaert, Roeland; Maes, Ine; Schoovaerts, Nils; Zhou, Lujia; Soukup, Sandra; da Cunha, Raquel; Lauwers, Elsa; Fiers, Mark; Verstreken, Patrik

    2014-10-08

    Modern molecular genetics studies necessitate the manipulation of genes in their endogenous locus, but most of the current methodologies require an inefficient donor-dependent homologous recombination step to locally modify the genome. Here we describe a methodology to efficiently generate Drosophila knock-in alleles by capitalizing on the availability of numerous genomic MiMIC transposon insertions carrying recombinogenic attP sites. Our methodology entails the efficient PhiC31-mediated integration of a recombination cassette flanked by unique I-SceI and/or I-CreI restriction enzyme sites into an attP-site. These restriction enzyme sites allow for double-strand break-mediated removal of unwanted flanking transposon sequences, while leaving the desired genomic modifications or recombination cassettes. As a proof-of-principle, we mutated LRRK, tau, and sky by using different MiMIC elements. We replaced 6 kb of genomic DNA encompassing the tau locus and 35 kb encompassing the sky locus with a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional LRRK(HA) knock in allele. Given that ~92% of the Drosophila genes are located within the vicinity (MiMIC element, our methodology enables the efficient manipulation of nearly every locus in the fruit fly genome without the need for inefficient donor-dependent homologous recombination events. Copyright © 2014 Vilain et al.

  17. Mimic Carbonic Anhydrase Using Metal-Organic Frameworks for CO2 Capture and Conversion.

    Science.gov (United States)

    Jin, Chaonan; Zhang, Sainan; Zhang, Zhenjie; Chen, Yao

    2018-02-19

    Carbonic anhydrase (CA) is a zinc-containing metalloprotein, in which the Zn active center plays the key role to transform CO 2 into carbonate. Inspired by nature, herein we used metal-organic frameworks (MOFs) to mimic CA for CO 2 conversion, on the basis of the structural similarity between the Zn coordination in MOFs and CA active center. The biomimetic activity of MOFs was investigated by detecting the hydrolysis of para-nitrophenyl acetate, which is a model reaction used to evaluate CA activity. The biomimetic materials (e.g., CFA-1) showed good catalytic activity, and excellent reusability, and solvent and thermal stability, which is very important for practical applications. In addition, ZIF-100 and CFA-1 were used to mimic CA to convert CO 2 gas, and exhibited good efficiency on CO 2 conversion compared with those of other porous materials (e.g., MCM-41, active carbon). This biomimetic study revealed a novel CO 2 treatment method. Instead of simply using MOFs to absorb CO 2 , ZIF-100 and CFA-1 were used to mimic CA for in situ CO 2 conversion, which provides a new prospect in the biological and industrial applications of MOFs.

  18. Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy.

    Science.gov (United States)

    Zhang, Linqun; Liu, Yuanjian; Li, Ying; Zhao, Yuewu; Wei, Wei; Liu, Songqin

    2016-08-24

    A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6](3+), RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL(-1) to 10 U mL(-1), with a detection limit down to 0.03 U mL(-1). Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens

    Science.gov (United States)

    McGrann, Graham R. D.; Steed, , Andrew; Burt, Christopher; Nicholson, Paul; Brown, James K. M.

    2015-01-01

    Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. PMID:25873675

  20. Settlement pattern of Posidonia oceanica epibionts along a gradient of ocean acidification: an approach with mimics

    Directory of Open Access Journals (Sweden)

    L. DONNARUMMA

    2014-02-01

    Full Text Available Effects of ocean acidification (OA on the colonization/settlement pattern of the epibiont community of the leaves and rhizomesof the Mediterranean seagrass,Posidoniaoceanica, have been studied at volcanic CO2vents off Ischia (Italy, using “mimics”as artificial substrates. The experiments were conducted in shallowPosidoniastands (2-3 m depth, in three stations on the northand three on the south sides of the study area, distributed along a pH gradient. At each station, 4 rhizome mimics and 6 artificialleaves were collected every three months (Sept 2009-Sept 2010. The epibionts on both leaf and rhizome mimics showed clearchanges along the pH gradient; coralline algae and calcareous invertebrates (bryozoans, serpulid polychaetes and barnacles weredominant at control stations but progressively disappeared at the most acidified stations. In these extremely low pH sites theassemblage was dominated by filamentous algae and non calcareous taxa such as hydroids and tunicates. Settlement pattern onthe artificial leaves and rhizome mimics over time showed a consistent distribution pattern along the pH gradient and highlightedthe peak of recruitment of the various organisms in different periods according to their life history.Posidoniamimics at theacidified station showed a poor and very simplified assemblage where calcifying epibionts seemed less competitive for space. Thisprofound difference in epiphyte communities in low pH conditions suggests cascading effects on the food web of the meadow and,consequently, on the functioning of the system

  1. Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

    Science.gov (United States)

    Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-08-03

    Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum

    Directory of Open Access Journals (Sweden)

    D Tagu

    2009-12-01

    Full Text Available Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

  3. Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Steffensen, L.; Pedersen, P. A.

    2006-01-01

    -ATPase also induced GCN4 translation. Derepression of GCN4 translation required phosphorylation of eIF-2 , the tRNA binding domain of Gcn2p, and the ribosome-associated proteins Gcn1p and Gcn20p. The increase in Gcn4p density in response to heterologous expression did not induce transcription from the HIS4...... promoter, a traditional Gcn4p target.......This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2 (eIF-2 ) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4...

  4. Malignant pleural mesothelioma with heterologous osteoblastic differentiation: case report of the characteristic CT and bone scan findings

    International Nuclear Information System (INIS)

    Cho, Young Jun; Kim, Joung Sook; Kim, Ji Young; Choi, Soo Jeon; Choi, Sang Bong

    2008-01-01

    Malignant pleural mesothelioma is an uncommon neoplasm which is accompanied extremely rarely by osteoblastic heterologous elements. The CT manifestations of this tumor have been reported in several references. And, to our knowledge, only one case report provides a description of the bone scan findings. Here, we report the case of a rapidly progressing malignant pleural mesothelioma with heterologous osteoblastic elements. A CT scan reveals diffuse irregular pleural thickening and very coarse nodular calcifications along the right pleura and major fissure. A bone scan revealed an area of extensive increased radioactivity consistent with the pleural calcifications on the CT scan in the right hemithorax. A follow-up CT scan performed 40 days later suggests the presence of rapidly progressing nodular coarse calcifications

  5. Malignant pleural mesothelioma with heterologous osteoblastic differentiation: case report of the characteristic CT and bone scan findings

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Young Jun; Kim, Joung Sook; Kim, Ji Young; Choi, Soo Jeon; Choi, Sang Bong [Sanggye Paik Hospital, Inje University College of Medicine, Seoul (Korea, Republic of)

    2008-06-15

    Malignant pleural mesothelioma is an uncommon neoplasm which is accompanied extremely rarely by osteoblastic heterologous elements. The CT manifestations of this tumor have been reported in several references. And, to our knowledge, only one case report provides a description of the bone scan findings. Here, we report the case of a rapidly progressing malignant pleural mesothelioma with heterologous osteoblastic elements. A CT scan reveals diffuse irregular pleural thickening and very coarse nodular calcifications along the right pleura and major fissure. A bone scan revealed an area of extensive increased radioactivity consistent with the pleural calcifications on the CT scan in the right hemithorax. A follow-up CT scan performed 40 days later suggests the presence of rapidly progressing nodular coarse calcifications.

  6. P143 proteins from heterologous nucleopolyhedroviruses induce apoptosis in BM-N cells derived from the silkworm Bombyx mori.

    Science.gov (United States)

    Hamajima, Rina; Kobayashi, Michihiro; Ikeda, Motoko

    2017-04-02

    We previously demonstrated that ribosomal RNA (rRNA) of Bombyx mori BM-N cells is rapidly degraded upon infection with heterologous nucleopolyhedroviruses (NPVs), including Autographa californica multiple NPV (AcMNPV), Hyphantria cunea MNPV, Spodoptera exigua MNPV and S. litura MNPV, and that this response is triggered by viral P143 proteins. The transient expression of P143 proteins from heterologous NPVs was also shown to induce apoptosis and caspase-3-like protease activation in BM-N cells. In the present study, we conducted a transient expression assay using BM-N cells expressing mutant AcMNPV P143 (Ac-P143) proteins and demonstrated that five amino acid residues cooperatively participate in Ac-P143 protein-triggered apoptosis of BM-N cells. Notably, these five residues were previously shown to be required for triggering rRNA degradation in BM-N cells. As rRNA degradation in BM-N cells does not result from apoptosis, the present results suggest that Ac-P143-triggered rRNA degradation is the upstream signal for apoptosis induction in BM-N cells. We further showed that P143 protein-triggered apoptosis does not occur in S. frugiperda Sf9 or Lymantria dispar Ld652Y cells, indicating that apoptosis induction by heterologous P143 proteins is a BM-N cell-specific response. In addition, the observed induction of apoptosis in BM-N cells was found to be mediated by activation of the initiator caspase Bm-Dronc. Taken together, these results suggest that BM-N cells evolved a unique antiviral system that recognizes heterologous NPV P143 proteins to induce rRNA degradation and caspase-dependent apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. miR-370 mimic inhibits replication of Japanese encephalitis virus in glioblastoma cells.

    Science.gov (United States)

    Li, Wenjuan; Cheng, Peng; Nie, Shangdan; Cui, Wen

    2016-01-01

    Japanese encephalitis (JE) is one of the most severe viral infections of the central nervous system. No effective treatment for JE currently exists, because its pathogenesis remains largely unknown. The present study was designed to screen the potential microRNAs (miRNAs) involved in JE. Glioblastoma cells were collected, after being infected with the Japanese encephalitis virus (JEV). Total miRNAs were extracted and analyzed using an miRNA chip. One of the most severely affected miRNAs was selected, and the role of miR-370 in JEV infection was investigated. Cell viability and apoptosis of the host cells were evaluated. JEV replication was detected via analysis of gene E expression. Real-time polymerase chain reaction was used to determine the levels of endogenous miR-370 and expression of innate immunity-related genes. Following JEV infection, 114 miRNAs were affected, as evidenced by the miRNA chip. Among them, 30 miRNAs were upregulated and 84 were downregulated. The changes observed in five miRNAs were confirmed by real-time polymerase chain reaction. One of the significantly downregulated miRNAs was miR-370. Therefore, miR-370 mimic was transfected into the cells, following which the levels of endogenous miR-370 were significantly elevated. Concurrently, JEV replication was significantly reduced 24 hours after transfection of miR-370 mimic. Functionally, miR-370 mimic mitigated both JEV-induced apoptosis and the inhibition of host cell proliferation. Following JEV infection, interferon-β and nuclear factor-kappa B were upregulated, whereas miR-370 mimic prevented the upregulation of the genes induced by JEV infection. The present study demonstrated that miR-370 expression in host cells is downregulated following JEV infection, which further mediates innate immunity-related gene expression. Taken together, miR-370 mimic might be useful to prevent viral replication and infection-induced host cell injury.

  8. The Effect of Small Sample Size on Measurement Equivalence of Psychometric Questionnaires in MIMIC Model: A Simulation Study

    Directory of Open Access Journals (Sweden)

    Jamshid Jamali

    2017-01-01

    Full Text Available Evaluating measurement equivalence (also known as differential item functioning (DIF is an important part of the process of validating psychometric questionnaires. This study aimed at evaluating the multiple indicators multiple causes (MIMIC model for DIF detection when latent construct distribution is nonnormal and the focal group sample size is small. In this simulation-based study, Type I error rates and power of MIMIC model for detecting uniform-DIF were investigated under different combinations of reference to focal group sample size ratio, magnitude of the uniform-DIF effect, scale length, the number of response categories, and latent trait distribution. Moderate and high skewness in the latent trait distribution led to a decrease of 0.33% and 0.47% power of MIMIC model for detecting uniform-DIF, respectively. The findings indicated that, by increasing the scale length, the number of response categories and magnitude DIF improved the power of MIMIC model, by 3.47%, 4.83%, and 20.35%, respectively; it also decreased Type I error of MIMIC approach by 2.81%, 5.66%, and 0.04%, respectively. This study revealed that power of MIMIC model was at an acceptable level when latent trait distributions were skewed. However, empirical Type I error rate was slightly greater than nominal significance level. Consequently, the MIMIC was recommended for detection of uniform-DIF when latent construct distribution is nonnormal and the focal group sample size is small.

  9. Characterization and mapping of complementary lesion-mimic genes lm1 and lm2 in common wheat.

    Science.gov (United States)

    Yao, Qin; Zhou, Ronghua; Fu, Tihua; Wu, Weiren; Zhu, Zhendong; Li, Aili; Jia, Jizeng

    2009-10-01

    A lesion-mimic phenotype appeared in a segregating population of common wheat cross Yanzhan 1/Zaosui 30. The parents had non-lesion normal phenotypes. Shading treatment and histochemical analyses showed that the lesions were caused by light-dependent cell death and were not associated with pathogens. Studies over two cropping seasons showed that some lines with more highly expressed lesion-mimic phenotypes exhibited significantly lower grain yields than those with the normal phenotype, but there were no significant effects in the lines with weakly expressed lesion-mimic phenotypes. Among yield traits, one-thousand grain weight was the most affected by lesion-mimic phenotypes. Genetic analysis indicated that this was a novel type of lesion mimic, which was caused by interaction of recessive genes derived from each parent. The lm1 (lesion mimic 1) locus from Zaosui 30 was flanked by microsatellite markers Xwmc674 and Xbarc133/Xbarc147 on chromosome 3BS, at genetic distances of 1.2 and 3.8 cM, respectively, whereas lm2 from Yanzhan 1 was mapped between microsatellite markers Xgwm513 and Xksum154 on chromosome 4BL, at genetic distances of 1.5 and 3 cM, respectively. The linked microsatellite makers identified in this study might be useful for evaluating whether potential parents with normal phenotype are carriers of lesion-mimic alleles.

  10. The Effect of Small Sample Size on Measurement Equivalence of Psychometric Questionnaires in MIMIC Model: A Simulation Study.

    Science.gov (United States)

    Jamali, Jamshid; Ayatollahi, Seyyed Mohammad Taghi; Jafari, Peyman

    2017-01-01

    Evaluating measurement equivalence (also known as differential item functioning (DIF)) is an important part of the process of validating psychometric questionnaires. This study aimed at evaluating the multiple indicators multiple causes (MIMIC) model for DIF detection when latent construct distribution is nonnormal and the focal group sample size is small. In this simulation-based study, Type I error rates and power of MIMIC model for detecting uniform-DIF were investigated under different combinations of reference to focal group sample size ratio, magnitude of the uniform-DIF effect, scale length, the number of response categories, and latent trait distribution. Moderate and high skewness in the latent trait distribution led to a decrease of 0.33% and 0.47% power of MIMIC model for detecting uniform-DIF, respectively. The findings indicated that, by increasing the scale length, the number of response categories and magnitude DIF improved the power of MIMIC model, by 3.47%, 4.83%, and 20.35%, respectively; it also decreased Type I error of MIMIC approach by 2.81%, 5.66%, and 0.04%, respectively. This study revealed that power of MIMIC model was at an acceptable level when latent trait distributions were skewed. However, empirical Type I error rate was slightly greater than nominal significance level. Consequently, the MIMIC was recommended for detection of uniform-DIF when latent construct distribution is nonnormal and the focal group sample size is small.

  11. Heterologous Synthesis and Recovery of Advanced Biofuels from Bacterial Cell Factories.

    Science.gov (United States)

    Malik, Sana; Afzal, Ifrah; Mehmood, Muhammad Aamer; Al Doghaither, Huda; Rahimuddin, Sawsan Abdulaziz; Gull, Munazza; Nahid, Nazia

    2018-01-01

    Microbial engineering to produce advanced biofuels is currently the most encouraging approach in renewable energy. Heterologous synthesis of biofuels and other useful industrial chemicals using bacterial cell factories has radically diverted the attentions from the native synthesis of these compounds. However, recovery of biofuels from the media and cellular toxicity are the main hindrances to successful commercialization of advanced biofuels. Therefore, membrane transporter engineering is gaining increasing attentions from all over the world. The main objective of this review is to explore the ways to increase the microbial production of biofuels by counteracting the cellular toxicity and facilitating their easier recovery from media. Microbial synthesis of industrially viable compounds such as biofuels has been increased due to genomic revolution. Moreover, advancements in protein engineering, gene regulation, pathway portability, metabolic engineering and synthetic biology led the focus towards the development of robust and cost-effective systems for biofuel production. The most convenient way to combat cellular toxicity and to secrete biofuels is the use of membrane transport system. The use of membrane transporters is currently a serious oversight as do not involve chemical changes and contribute greatly to efflux biofuels in extracellular milieu. However, overexpression of transport systems can also be detrimental to cell, so, in future, structure-based engineering of transporters can be employed to evaluate optimum expression range, to increase biofuel specificity and transport rate through structural studies of biofuel molecules. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans▿

    Science.gov (United States)

    Simeonova, Diliana Dancheva; Wilson, Marlena Marie; Metcalf, William W.; Schink, Bernhard

    2010-01-01

    Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays. PMID:20622064

  13. Identification and heterologous expression of genes involved in anaerobic dissimilatory phosphite oxidation by Desulfotignum phosphitoxidans.

    Science.gov (United States)

    Simeonova, Diliana Dancheva; Wilson, Marlena Marie; Metcalf, William W; Schink, Bernhard

    2010-10-01

    Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.

  14. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

    Science.gov (United States)

    Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk

    2012-01-01

    A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272

  15. Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

    Directory of Open Access Journals (Sweden)

    Langella P.

    1999-01-01

    Full Text Available Lactic acid bacteria (LAB are Gram-positive bacteria and are generally regarded as safe (GRAS organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV epitope-protein fusion (BCV-Nuc. BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

  16. The pea SAD short-chain dehydrogenase/reductase: quinone reduction, tissue distribution, and heterologous expression.

    Science.gov (United States)

    Scherbak, Nikolai; Ala-Häivälä, Anneli; Brosché, Mikael; Böwer, Nathalie; Strid, Hilja; Gittins, John R; Grahn, Elin; Eriksson, Leif A; Strid, Åke

    2011-04-01

    The pea (Pisum sativum) tetrameric short-chain alcohol dehydrogenase-like protein (SAD) family consists of at least three highly similar members (SAD-A, -B, and -C). According to mRNA data, environmental stimuli induce SAD expression. The aim of this study was to characterize the SAD proteins by examining their catalytic function, distribution in pea, and induction in different tissues. In enzyme activity assays using a range of potential substrates, the SAD-C enzyme was shown to reduce one- or two-ring-membered quinones lacking long hydrophobic hydrocarbon tails. Immunological assays using a specific antiserum against the protein demonstrated that different tissues and cell types contain small amounts of SAD protein that was predominantly located within epidermal or subepidermal cells and around vascular tissue. Particularly high local concentrations were observed in the protoderm of the seed cotyledonary axis. Two bow-shaped rows of cells in the ovary and the placental surface facing the ovule also exhibited considerable SAD staining. Ultraviolet-B irradiation led to increased staining in epidermal and subepidermal cells of leaves and stems. The different localization patterns of SAD suggest functions both in development and in responses to environmental stimuli. Finally, the pea SAD-C promoter was shown to confer heterologous wound-induced expression in Arabidopsis (Arabidopsis thaliana), which confirmed that the inducibility of its expression is regulated at the transcriptional level.

  17. [Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

    Science.gov (United States)

    Wang, Jian; Qi, Meng-Die; Guo, Juan; Shen, Ye; Lin, Hui-Xin; Huang, Lu-Qi

    2017-03-01

    Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis. Copyright© by the Chinese Pharmaceutical Association.

  18. Heterología. La ciencia (imposible de los residuos violentos

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    Sergio Tonkonoft

    2015-01-01

    Full Text Available EI presente trabajo busca mostrar que en la obra de Bataille existe una teoría social general, articulada en torno a la noción de lo sagrado. Nuestra hipótesis es que Bataille profundizó y generalizó d movimiento iniciado por Durkheim, quien postula la sintaxis de lo sagrado arcaico como la parte fundamental de la gramática social, pero interpretando este legado a la luz del concepto de multitud, entendido como el otro nombre de la economía general de las pasiones colectivas. Para dlo, se sirvió de dementos deI psicoanálisis y la fenomenología alemana, produciendo la teoría original dd lazo social, su estructuración y sus dinâmicas, con las que reformuló la idea durkheimniana de lo sagrado y procedió a analizar los malestares de su época. Dada la antropología trágica que enmarcó esta empresa intelectual, resultó de ello la teoría social transgredida o imposible que lleva eI nombre de heterología.

  19. Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

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    Bey Mathieu

    2011-12-01

    Full Text Available Abstract Background Cellobiose dehydrogenase (CDH is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i the production of a large amount of gluconic acid, (ii increased hemicellulose degradation, and (iii increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM. Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

  20. Heterologous expression of the Halothiobacillus neapolitanus carboxysomal gene cluster in Corynebacterium glutamicum.

    Science.gov (United States)

    Baumgart, Meike; Huber, Isabel; Abdollahzadeh, Iman; Gensch, Thomas; Frunzke, Julia

    2017-09-20

    Compartmentalization represents a ubiquitous principle used by living organisms to optimize metabolic flux and to avoid detrimental interactions within the cytoplasm. Proteinaceous bacterial microcompartments (BMCs) have therefore created strong interest for the encapsulation of heterologous pathways in microbial model organisms. However, attempts were so far mostly restricted to Escherichia coli. Here, we introduced the carboxysomal gene cluster of Halothiobacillus neapolitanus into the biotechnological platform species Corynebacterium gluta-micum. Transmission electron microscopy, fluorescence microscopy and single molecule localization microscopy suggested the formation of BMC-like structures in cells expressing the complete carboxysome operon or only the shell proteins. Purified carboxysomes consisted of the expected protein components as verified by mass spectrometry. Enzymatic assays revealed the functional production of RuBisCO in C. glutamicum both in the presence and absence of carboxysomal shell proteins. Furthermore, we could show that eYFP is targeted to the carboxysomes by fusion to the large RuBisCO subunit. Overall, this study represents the first transfer of an α-carboxysomal gene cluster into a Gram-positive model species supporting the modularity and orthogonality of these microcompartments, but also identified important challenges which need to be addressed on the way towards biotechnological application. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Metabolism of homologous and heterologous serum proteins in garter snakes (Thamnophis ordinoides)

    International Nuclear Information System (INIS)

    Leong, D.; Coe, J.E.

    1978-01-01

    The half-life (Tsub(1/2) of serum immunoglobulin (Ig) and albumin from snakes and mammals were determined in both garter snakes (Thamnophis ordinoides) and mice (Mus musculus). Metabolism of serum proteins in snakes was similar to mammalian protein metabolism in that homologous serum albumin had shorter Tsub(1/2) (16 days) than IgG (38 days). Also, reptilian and mammalian serum proteins had a relatively longer Tsub(1/2) when injected into closely related species. Thus mammalian serum Ig (rabbit gamma globulin (RGG)) had a shorter Tsub(1/2) (6.3 days) in snake than did homologous snake IgG (38 days), whereas in mice, RGG had a longer Tsub(1/2) (3.8 days) than snake Ig (0.9 days). Differences between metabolism of homologous and heterologous albumins were apparent only in snakes in which the Tsub(1/2) of homologous albumin was approximately 8-fold greater than mammalian albumin. These results indicate that metabolism of both Ig and albumin in snakes is regulated by specific receptors whereas albumin receptors have been difficult to demonstrate in mammals. The results of this study suggest that one of the factors determining the metabolism of a protein is its foreignness to the host perhaps because of receptor cross reactions. (author)

  2. Heterologous production of the stain solving peptidase PPP1 from Pleurotus pulmonarius.

    Science.gov (United States)

    Leonhardt, Robin-Hagen; Krings, Ulrich; Berger, Ralf G; Linke, Diana

    2016-05-01

    A novel stain solving subtilisin-like peptidase (PPP1) was identified from the culture supernatant of the agaricomycete Pleurotus pulmonarius. It was purified to homogeneity using a sequence of preparative isoelectric focusing, anion exchange and size exclusion chromatography. Peptides were identified by ab initio sequencing (nLC-ESI-QTOF-MS/MS), characterizing the enzyme as a member of the subtilase family (EC 3.4.21.X). An expression system was established featuring the pPIC9K vector, an alternative Kozak sequence, the codon optimized gene ppp1 gene without the native signal sequence with C-terminal hexa-histidine tag, and Pichia pastoris GS115 as expression host. Intracellular active enzyme was obtained from cultivations in shake flasks and in a five liter bioreactor. With reaction optima of 40 °C and a pH > 8.5, considerable bleaching of pre-stained fabrics (blood, milk and India ink), and the possibility of larger-scale production, the heterologous enzyme is well suitable for detergent applications, especially at lower temperatures as part of a more energy- and cost-efficient washing process. Showing little sequence similarity to other subtilases, this unique peptidase is the first subtilisin-like peptidase from Basidiomycota, which has been functionally produced in Pichia pastoris.

  3. Benchmarking two commonly used Saccharomyces cerevisiae strains for heterologous vanillin-β-glucoside production

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    Tomas Strucko

    2015-12-01

    Full Text Available The yeast Saccharomyces cerevisiae is a widely used eukaryotic model organism and a key cell factory for production of biofuels and wide range of chemicals. From the broad palette of available yeast strains, the most popular are those derived from laboratory strain S288c and the industrially relevant CEN.PK strain series. Importantly, in recent years these two strains have been subjected to comparative “-omics” analyzes pointing out significant genotypic and phenotypic differences. It is therefore possible that the two strains differ significantly with respect to their potential as cell factories for production of specific compounds. To examine this possibility, we have reconstructed a de novo vanillin-β-glucoside pathway in an identical manner in S288c and CEN.PK strains. Characterization of the two resulting strains in two standard conditions revealed that the S288c background strain produced up to 10-fold higher amounts of vanillin-β-glucoside compared to CEN.PK. This study demonstrates that yeast strain background may play a major role in the outcome of newly developed cell factories for production of a given product. Keywords: Yeast, Cell factory, Strain choice, Heterologous production, Vanillin-glucoside, Shikimate pathway

  4. Improved cellulolytic efficacy in Penicilium decumbens via heterologous expression of Hypocrea jecorina endoglucanase II

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    Qin Yuqi

    2013-01-01

    Full Text Available Hypocrea jecorina endoglucanase II (Hjegl2 was heterologously expressed in Penicillium decumbens (yielding strain Pd::Hjegl2. After induction in cellulose containing media, strain Pd::Hjeg2 displayed increased carboxymethylcellulase activity (CMCase, 5.77 IU/ml, representing a 21% increase and cellulose degradation determined with a filter paper assay (FPA, 0.40 IU/ml, 67% increase, as compared to the parent strain. In media supplemented with glucose (2%, Pd::Hjegl2, displayed 51.2-fold and 3-fold higher CMCase and FPA activities, respectively, as compared to the parent strain. No changes in the expression levels of the four main native cellulase genes of P. decumbens (Pdegl1, Pdegl2, Pdcbh1, and Pdcbh2 were noted between the transformant and wild-type strains. These data support the idea that Hjegl2 cleaves both internal and terminal glycosidic residues, in a relatively random and processive manner. In situ polyacrylamide gelactivity staining of extracts derived from wild-type and Pd::Hjegl2 revealed two additional active fractions in the latter strain; one with a molecular mass ~50-65 KDa and another ~80-116 kDa.

  5. Babesia divergens: protection of intact calves against heterologous challenge by the injection of irradiated piroplasms

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, D; Purnell, R E; Brocklesby, D W [Agricultural Research Council, Compton (UK). Inst. for Research on Animal Diseases

    1980-03-01

    Blood from a splenectomized calf infected with Babesia divergens was divided into 20 ml aliquots which were ..gamma..-irradiated at doses of 0, 24, 28, 32, 36 and 40 krad and then inoculated into groups of three intact calves. Animals receiving non-irradiated blood had typical mild B. divergens reactions, but those receiving irradiated blood had either very mild reactions or no overt reaction. When the calves were challenged with a similar number (7.5 X 10/sup 9/) of heterologous parasites of a recently-isolated field strain, those which had received blood irradiated at 0, 24, 28 and 32 krad were all immune whereas four of the five surviving animals which had received blood irradiated at 36 or 40 krad were susceptible. The immune status of individual cattle was reflected accurately in the results of the micro-ELISA test, which detected a significant rise in serum antibody titre of the four susceptible animals by day 21 after challenge.

  6. Malignant Solitary Fibrous Tumor with Heterologous Rhabdomyosarcomatous Differentiation: A Case Report

    Directory of Open Access Journals (Sweden)

    Jeong-Hwa Kwon

    2017-03-01

    Full Text Available Malignant solitary fibrous tumor (MSFT is a well-described entity, from which heterologous differentiation is extremely rare. We encountered a case of MSFT with rhabdomyosarcomatous differentiation in a 56-year-old man. This patient presented with a large mass in his posterior thigh. He had been treated with chemoradiation for sarcoma involving the cervical spine, right femoral head, and both lungs 6 months earlier. A wide excision was performed. The mass measured 10.6 cm and showed a fish-flesh cut surface with necrotic foci. Microscopically, the tumor showed heterogeneous cellularity with a hemangiopericytic vascular pattern. A hypercellular area showed spindle cells or epithelioid cells with high mitotic activity (63/10 high-power fields and immunoreactivity for CD34 and CD99. A hypocellular area and a cystic area showed pleomorphic rhabdoid cells with immunoreactivity for desmin and myogenin. This is a report of a rare case of MSFT with rhabdomyosarcomatous differentiation and presents new histologic features of MSFT.

  7. Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

    Directory of Open Access Journals (Sweden)

    Zi Yang

    Full Text Available Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q/asparagine (N-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller "seeds." We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI(+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI(+] or [URE3] prions. We explore in detail the events leading to the loss (curing of [PSI(+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI(+].

  8. Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2016-08-01

    Full Text Available Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1–8 from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases.

  9. Heterologous expression and characterization of a new heme-catalase in Bacillus subtilis 168.

    Science.gov (United States)

    Philibert, Tuyishime; Rao, Zhiming; Yang, Taowei; Zhou, Junping; Huang, Genshu; Irene, Komera; Samuel, Niyomukiza

    2016-06-01

    Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784 U/mg expressed in Bacillus subtilis 168 and 98.097 µmol/min/mg peroxidatic activity, the apparent K m of catalytic activity was 59.6 ± 13 mM with higher turnover rate (K cat = 322.651 × 10(3) s(-1)). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40 °C and the recombinant heme-catalase exhibited a strong Fe(2+) preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.

  10. Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris).

    Science.gov (United States)

    Chessa, Rossella; Landolfo, Sara; Ciani, Maurizio; Budroni, Marilena; Zara, Severino; Ustun, Murat; Cakar, Zeynep Petek; Mannazzu, Ilaria

    2017-04-01

    The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with β-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has β-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.

  11. Engineering multifunctional protein nanoparticles by in vitro disassembling and reassembling of heterologous building blocks

    Science.gov (United States)

    Unzueta, Ugutz; Serna, Naroa; Sánchez-García, Laura; Roldán, Mónica; Sánchez-Chardi, Alejandro; Mangues, Ramón; Villaverde, Antonio; Vázquez, Esther

    2017-12-01

    The engineering of protein self-assembling at the nanoscale allows the generation of functional and biocompatible materials, which can be produced by easy biological fabrication. The combination of cationic and histidine-rich stretches in fusion proteins promotes oligomerization as stable protein-only regular nanoparticles that are composed by a moderate number of building blocks. Among other applications, these materials are highly appealing as tools in targeted drug delivery once empowered with peptidic ligands of cell surface receptors. In this context, we have dissected here this simple technological platform regarding the controlled disassembling and reassembling of the composing building blocks. By applying high salt and imidazole in combination, nanoparticles are disassembled in a process that is fully reversible upon removal of the disrupting agents. By taking this approach, we accomplish here the in vitro generation of hybrid nanoparticles formed by heterologous building blocks. This fact demonstrates the capability to generate multifunctional and/or multiparatopic or multispecific materials usable in nanomedical applications.

  12. Induction of divalent cation permeability by heterologous expression of a voltage sensor domain.

    Science.gov (United States)

    Arima, Hiroki; Tsutsui, Hidekazu; Sakamoto, Ayako; Yoshida, Manabu; Okamura, Yasushi

    2018-01-06

    The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K + channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Heterologous production of human papillomavirus type-16 L1 protein by a lactic acid bacterium

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    Bermúdez-Humarán Luis G

    2009-08-01

    Full Text Available Abstract Background The expression of vaccine antigens in lactic acid bacteria (LAB is a safe and cost-effective alternative to traditional expression systems. In this study, we investigated i the expression of Human papillomavirus type 16 (HPV-16 L1 major capsid protein in the model LAB Lactococcus lactis and ii the ability of the resulting recombinant strain to produce either capsomer-or virus-like particles (VLPs. Results and conclusion HPV-16 L1 gene was cloned into two vectors, pCYT and pSEC, designed for controlled intra- or extracellular heterologous expression in L. lactis, respectively. The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays. Electron microscopy analysis suggests that, L1 protein produced by recombinant lactococci can self-assemble into structures morphologically similar to VLPs intracellularly. The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody. Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.

  14. Babesia divergens: protection of intact calves against heterologous challenge by the injection of irradiated piroplasms

    International Nuclear Information System (INIS)

    Lewis, D.; Purnell, R.E.; Brocklesby, D.W.

    1980-01-01

    Blood from a splenectomized calf infected with Babesia divergens was divided into 20 ml aliquots which were γ-irradiated at doses of 0, 24, 28, 32, 36 and 40 krad and then inoculated into groups of three intact calves. Animals receiving non-irradiated blood had typical mild B. divergens reactions, but those receiving irradiated blood had either very mild reactions or no overt reaction. When the calves were challenged with a similar number (7.5 X 10 9 ) of heterologous parasites of a recently-isolated field strain, those which had received blood irradiated at 0, 24, 28 and 32 krad were all immune whereas four of the five surviving animals which had received blood irradiated at 36 or 40 krad were susceptible. The immune status of individual cattle was reflected accurately in the results of the micro-ELISA test, which detected a significant rise in serum antibody titre of the four susceptible animals by day 21 after challenge. (Auth.)

  15. A heterologous radioimmunoassay for avian prolactin: Application to the measurement of prolactin in the turkey

    International Nuclear Information System (INIS)

    McNeilly, A.S.; Etches, R.J.; Friesen, H.G.

    1978-01-01

    A specific heterologous double-antibody radioimmunoassay has been developed to measure turkey prolactin (PRL) using a guinea pig anti-hPRL antiserum and 125 I-labelled ovine PRL [ 125 I]oPRL. Turkey pituitary prolactin and serum give parallel dose-response curves and no cross-rection is seen with turkey growth hormone, LH or FSH, or mammalian LH, FSH, TSH, GH or placental lactogens. The RIA is accurate and precise and is sufficiently sensitive to measure PRL in all physiological situations investigated in the turkey. The RIA will measure PRL in several avian species including the chicken, duck, goose, pheasant, pheasant x chicken F 1 hybrid, pigeon, quail, and rook. Plasma PRL concentrations in laying and broody turkey hens were not significantly different (46.5 +- 2.5 vs. 39.7 +- 3.8 ng/ml) but both were significantly higher (P < 0.001) than in non-laying turkey hens (4.6 +- 0.7 ng/ml). Oestradiol injection into laying hens did not alter PRL levels while the same injection in non-laying hens caused a significant three-fold increse in plasma PRL levels. (author)

  16. Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.

    Science.gov (United States)

    Cheng, L; Chen, G; Ding, G; Zhao, Z; Dong, T; Hu, Z

    2015-04-27

    The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.

  17. Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays

    Directory of Open Access Journals (Sweden)

    Jouventin Pierre

    2010-05-01

    Full Text Available Abstract Background Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. Results We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. Conclusions MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.

  18. Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    João Vitor Dutra Molino

    Full Text Available Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.

  19. Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins.

    Science.gov (United States)

    Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2011-02-01

    Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

  20. Improved heterologous protein production by a tripeptidyl peptidase gene (AosedD) disruptant of the filamentous fungus Aspergillus oryzae.

    Science.gov (United States)

    Zhu, Lin; Nemoto, Takeshi; Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2012-01-01

    Proteolytic degradation is one of the serious bottlenecks limiting the yields of heterologous protein production by Aspergillus oryzae. In this study, we selected a tripeptidyl peptidase gene AosedD (AO090166000084) as a candidate potentially degrading the heterologous protein, and performed localization analysis of the fusion protein AoSedD-EGFP in A. oryzae. As a result, the AoSedD-EGFP was observed in the septa and cell walls as well as in the culture medium, suggesting that AoSedD is a secretory enzyme. An AosedD disruptant was constructed to investigate an effect of AoSedD on the production level of heterologous proteins and protease activity. Both of the total protease and tripeptidyl peptidase activities in the culture medium of the AosedD disruptant were decreased as compared to those of the control strain. The maximum yields of recombinant bovine chymosin (CHY) and human lysozyme (HLY) produced by the AosedD disruptants showed approximately 2.9- and 1.7-fold increases, respectively, as compared to their control strains. These results suggest that AoSedD is one of the major proteases involved in the proteolytic degradation of recombinant proteins in A. oryzae.

  1. In vivo preclinical evaluation of the accuracy of toroidal-shaped HIFU treatments using a tumor-mimic model

    International Nuclear Information System (INIS)

    N'Djin, W A; Melodelima, D; Parmentier, H; Chapelon, J Y; Rivoire, M

    2010-01-01

    The pig is an ideal animal model for preclinical evaluation of HIFU treatments, especially in the liver. However, there is no liver tumor model available for pigs. In this work, we propose to study an in vivo tumor-mimic model as a tool for evaluating if a sonographycally guided HIFU treatment, delivered by a toroidal-shaped device dedicated for the treatment of liver metastases, is correctly located in the liver. One centimeter tumor-mimics were created in liver tissues. These tumor-mimics were detectable on ultrasound imaging and on gross pathology. Two studies were carried out. First, an in vivo study of tolerance at mid-term (30 days, 10 pigs) revealed that tumor-mimics are suitable for studying HIFU treatments at a preclinical stage, since local and biological tolerances were excellent. The dimensions of the tumor-mimics were reproducible (diameter at day 0: 9.7 ± 2.0 mm) and were the same as a function of time (p = 0.64). A second in vivo study was carried out in ten pigs. Tumor mimics were used as targets in liver tissues in order to determine if the HIFU treatment is correctly located in the liver. A procedure of extensive HIFU ablation using multiple HIFU lesions juxtaposed manually was then tested on eight tumor-mimics. In 88% of the cases (seven out of eight), tumor-mimics were treated with negative margins (≥1 mm) in all directions. On average, negative margins measured 10.0 ± 6.7 mm. These tumor-mimics constitute an excellent reference for studying in vivo the accuracy of HIFU therapy in the liver.

  2. Identification of Chemical Agents (Mimics) Residues after Destructive Adsorption Using TPD and UV-vis-IR and Raman

    National Research Council Canada - National Science Library

    Klabunde, Kenneth J

    2001-01-01

    Instrumentation was purchased that helps characterize the chemical structures and chemical reactions that occur when nanocrystalline metal oxides carry out destructive adsorption of chemical agents mimics. The residues (adducts...

  3. Effectiveness of a large mimic panel in an existing nuclear power plant central control board

    International Nuclear Information System (INIS)

    Kubota, Ryuji; Satoh, Hiroyuki; Sasajima, Katsuhiro; Kawano, Ryutaro; Shibuya Shinya

    1999-01-01

    We conducted the analysis of the nuclear power plant (NPP) operators' behaviors under emergency conditions by using training simulators as a joint research project by Japanese BWR groups for twelve years. In the phase-IV of this project we executed two kinds of experiments to evaluate the effectiveness of the interfaces. One was for evaluations of the interfaces such as CRTs with touch screen, a large mimic panel, and a hierarchical annunciator system introduced in the newly developed ABWR type central control board. The other was that we analyzed the operators' behaviors in emergency conditions by using the first generation BWR type central control board which was added new interfaces such as a large display screen and demarcation on the board to help operators to understand the plant. The demarcation is one of the visual interface improvements and its technique is that a line enclosing several components causes them to be perceived as a group.The result showed that both the large display panel Introduced in ABWR central control board and the large display screen in the existing BWR type central control board improved the performance of the NPP operators in the experiments. It was expected that introduction of the large mimic panel into the existing BWR type central control boards would improve operators' performance. However, in the case of actual installation of the large display board into the existing central control boards, there are spatial and hardware constraints. Therefore the size of lamps, lines connecting from symbols of the pumps or valves to the others' will have to be modified under these constraints. It is important to evaluate the displayed information on the large display board before actual installation. We made experiments to solve these problems by using TEPCO's research simulator which is added a large mimic panel. (author)

  4. Synthetic miR-145 Mimic Enhances the Cytotoxic Effect of the Antiangiogenic Drug Sunitinib in Glioblastoma.

    Science.gov (United States)

    Liu, Hongwei; Liu, Zhixiong; Jiang, Bing; Huo, Lei; Liu, Jinfang; Lu, Jingchen

    2015-06-01

    Although aggressive therapeutic regimen has been applied in the treatment of Glioblastoma (GBM), the prognosis of patients with GBM remains poor. Preclinical studies have demonstrated the efficacy of Suntinib in GBM both in vitro and in vivo. In this study, we showed that the cytotoxicity was enhanced by transfection with miR-145 mimic. In addition, we suggested that the enhanced cytotoxicity of Sunitinib by miR-145 mimic was mediated by inhibition of both P-gp and Bcrp.

  5. Creation of a Tumor-Mimic Model Using a Muscle Paste for Radiofrequency Ablation of the Lung

    International Nuclear Information System (INIS)

    Kawai, T.; Kaminou, T.; Sugiura, K.; Hashimoto, M.; Ohuchi, Y.; Adachi, A.; Fujioka, S.; Ito, H.; Nakamura, K.; Ogawa, T.

    2009-01-01

    The purpose of this study was to develop an easily created tumor-mimic model and evaluate its efficacy for radiofrequency ablation (RFA) of the lung. The bilateral lungs of eight living adult swine were used. A tumor-mimic model was made by percutaneous injection of 1.0 ml muscle paste through the bone biopsy needle into the lung. An RFA probe was then inserted into the tumor mimics immediately after tumor creation. Ablation time, tissue impedance, and temperature were recorded. The tumor mimics and their coagulated regions were evaluated microscopically and macroscopically. The muscle paste was easily injected into the lung parenchyma through the bone biopsy needle and well visualized under fluoroscopy. In 10 of 12 sites the tumor mimics were oval shaped, localized, and homogeneous on gross specimens. Ten tumor mimics were successfully ablated, and four locations were ablated in the normal lung parenchyma as controls. In the tumor and normal lung parenchyma, ablation times were 8.9 ± 3.5 and 4.4 ± 1.6 min, respectively; tissue impedances at the start of ablation were 100.6 ± 16.6 and 145.8 ± 26.8 Ω, respectively; and temperatures at the end of ablation were 66.0 ± 7.9 and 57.5 ± 7.6 o C, respectively. The mean size of tumor mimics was 13.9 x 8.2 mm, and their coagulated area was 18.8 x 13.1 mm. In the lung parenchyma, the coagulated area was 15.3 x 12.0 mm. In conclusion, our tumor-mimic model using muscle paste can be easily and safely created and can be ablated using the ablation algorithm in the clinical setting.

  6. Chondromyxoid fibroma of the rib mimics a chondrosarcoma on 18F-FDG PET/CT

    Energy Technology Data Exchange (ETDEWEB)

    Makis, William (Dept. of Nuclear Medicine, Brandon Regional Health Centre, Brandon (Canada)), email: makisw79@yahoo.com; Ciarallo, Anthony; Lisbona, Robert (Dept. of Nuclear Medicine, Royal Victoria Hospital, McGill Univ. Health Centre, Montreal (Canada))

    2011-06-15

    Chondromyxoid fibroma (CMF) is a rare benign bone tumor of chondroid origin that occurs mostly in the metaphyses of long bones. CMF can occasionally mimic a chondrosarcoma on CT, and the literature on the 18F-FDG PET/CT imaging characteristics of CMF tumors is limited. In the presented case, a large histologically proven CMF chest wall mass was initially misinterpreted as a chondrosarcoma. This case highlights a potential pitfall in the PET/CT evaluation of these rare benign bone tumors

  7. Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency.

    Science.gov (United States)

    Blanco, Beatriz; Sedes, Antía; Peón, Antonio; Lamb, Heather; Hawkins, Alastair R; Castedo, Luis; González-Bello, Concepción

    2012-05-14

    Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.

  8. Mucous retention cyst of temporal bone: a mimic of cholesteatoma on DW-MRI.

    Science.gov (United States)

    Karandikar, Amit; Goh, Julian; Loke, Siu Cheng; Yeo, Seng Beng; Tan, Tiong Yong

    2013-01-01

    Non-EPI DW imaging is increasingly being used as a sensitive sequence in detecting cholesteatomas especially if CT findings are not confirmatory. Cholesteatoma appears as a hyperintense focus on DWI. We present two cases of mucous retention cysts in the mastoid temporal bone/middle ear cavity, which present as hyperintense on non-EPI DWI and potentially may mimic cholesteatomas. Differentiating between the two conditions is important, as surgery can be avoided in mucous retention cysts. We have also discussed ways to differentiate between these two conditions on MRI. To our knowledge, this entity is not reported previously. © 2013 Elsevier Inc. All rights reserved.

  9. Estimating the size of non-observed economy in Croatia using the MIMIC approach

    OpenAIRE

    Vjekoslav Klaric

    2011-01-01

    This paper gives a quick overview of the approaches that have been used in the research of shadow economy, starting with the defi nitions of the terms “shadow economy” and “non-observed economy”, with the accent on the ISTAT/Eurostat framework. Several methods for estimating the size of the shadow economy and the non-observed economy are then presented. The emphasis is placed on the MIMIC approach, one of the methods used to estimate the size of the nonobserved economy. After a glance at the ...

  10. Cooperative interplay of let-7 mimic and HuR with MYC RNA

    OpenAIRE

    Gunzburg, Menachem J; Sivakumaran, Andrew; Pendini, Nicole R; Yoon, Je-Hyun; Gorospe, Myriam; Wilce, Matthew Cj; Wilce, Jacqueline A

    2015-01-01

    Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of...

  11. Effector Mimics and Integrated Decoys, the Never-Ending Arms Race between Rice and Xanthomonas oryzae.

    Science.gov (United States)

    Zuluaga, Paola; Szurek, Boris; Koebnik, Ralf; Kroj, Thomas; Morel, Jean-Benoit

    2017-01-01

    Plants are constantly challenged by a wide range of pathogens and have therefore evolved an array of mechanisms to defend against them. In response to these defense systems, pathogens have evolved strategies to avoid recognition and suppress plant defenses (Brown and Tellier, 2011). Three recent reports dealing with the resistance of rice to Xanthomonas oryzae have added a new twist to our understanding of this fascinating co-evolutionary arms race (Ji et al., 2016; Read et al., 2016; Triplett et al., 2016). They show that pathogens also develop sophisticated effector mimics to trick recognition.

  12. Exaggerated Arthropod Bite: A Case Report and Review of the Mimics

    Directory of Open Access Journals (Sweden)

    Sagah Ahmed

    2018-01-01

    Full Text Available Exaggerated arthropod bite reactions causing hemorrhagic or necrotic bullous lesions can mimic other serious conditions such as cutaneous anthrax, brown recluse spider bite, and tularemia. A 55- year-old, healthy woman presented to the emergency department with a 3.5-centimeter painless, collapsed hemorrhagic bulla at the left costal margin. She was afebrile and had no systemic symptoms. Laboratory evaluation was unremarkable. She was prescribed silver sulfadiazine cream and mupirocin ointment. The area denuded two days later and the lesion completely healed. This case illustrates the broad differential to be considered when evaluating patients with hemorrhagic bullous lesions.

  13. Arterial tortuosity syndrome: An extremely rare disease presenting as a mimic of pulmonary sling

    Directory of Open Access Journals (Sweden)

    Amy Farkas, MD

    2018-02-01

    Full Text Available Pulmonary sling is the anatomic variant defined by the aberrant origin of the left pulmonary artery from the right pulmonary artery. This patient presented with a mimic of pulmonary sling as a result of an extremely rare condition, arterial tortuosity syndrome (ATS. The patient was first diagnosed with pulmonary sling on prenatal echocardiogram performed by cardiology. Computed tomography angiography of the chest obtained at birth to evaluate respiratory depression demonstrated ATS. The early detection of ATS has been demonstrated to improve patient outcome. This case provides an overview of the typical imaging features of ATS to aid radiologists in making this uncommon diagnosis.

  14. TAPIR, a web server for the prediction of plant microRNA targets, including target mimics.

    Science.gov (United States)

    Bonnet, Eric; He, Ying; Billiau, Kenny; Van de Peer, Yves

    2010-06-15

    We present a new web server called TAPIR, designed for the prediction of plant microRNA targets. The server offers the possibility to search for plant miRNA targets using a fast and a precise algorithm. The precise option is much slower but guarantees to find less perfectly paired miRNA-target duplexes. Furthermore, the precise option allows the prediction of target mimics, which are characterized by a miRNA-target duplex having a large loop, making them undetectable by traditional tools. The TAPIR web server can be accessed at: http://bioinformatics.psb.ugent.be/webtools/tapir. Supplementary data are available at Bioinformatics online.

  15. Asthma mimic: Case report and literature review of vocal cord nodule associated with wheezing.

    Science.gov (United States)

    Kashif, Muhammad; Singh, Tushi; Aslam, Ahsan; Khaja, Misbahuddin

    2017-01-01

    Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. Various clinical conditions can mimic asthma, such as foreign body aspiration, subglottic stenosis, congestive heart failure, diffuse panbronchiolitis, aortic arch anomalies, reactive airway dysfunction syndrome, chronic obstructive pulmonary disease, retrosternal goiter, vocal cord tumors, other airway tumors, and vocal cord dysfunction. Upper airway obstruction can be a life-threatening emergency. Here, we present the case of a 58-year-old female with recurrent hospital visits for wheezing and exacerbations of asthma, who was later found to have a vocal cord nodule confirmed to be squamous cell carcinoma, which was mimicking like asthma.

  16. Yeast as a Heterologous Model System to Uncover Type III Effector Function.

    Directory of Open Access Journals (Sweden)

    Crina Popa

    2016-02-01

    Full Text Available Type III effectors (T3E are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. "Favourite" targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure-function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations

  17. Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

    Science.gov (United States)

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2016-02-01

    Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  18. Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon, Cucumis melo var. makuwa Makino.

    Directory of Open Access Journals (Sweden)

    Songxiao Cao

    Full Text Available Lipoxygenases (LOXs are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino cultivar "Yumeiren", encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.

  19. Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Saulo Martins Vieira

    2017-07-01

    Full Text Available Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2 structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64 in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold, increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2 from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

  20. Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

    Science.gov (United States)

    Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M

    2015-07-01

    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Heterologous expression and characterization of Bacillus coagulans L-arabinose isomerase.

    Science.gov (United States)

    Zhou, Xingding; Wu, Jin Chuan

    2012-05-01

    Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure L: -lactic acid from both hexose and pentose sugars including L: -arabinose with high yield, titer and productivity under thermophilic conditions. The L: -arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn(2+) was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K (m), V (max) and k (cat)/K (m) for the conversion of L: -arabinose were 106 mM, 84 U/mg and 34.5 mM(-1)min(-1), respectively. The equilibrium ratio of L: -arabinose to L: -ribulose was 78:22 under optimal conditions. L: -ribulose (97 g/L) was obtained from 500 g/l of L: -arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L(-1) h(-1).

  2. Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E

    2016-10-01

    Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

  3. Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene

    Directory of Open Access Journals (Sweden)

    Sônia C. Melo

    2015-06-01

    Full Text Available Heterologous expression of a putative manganese superoxide dismutase gene (SOD2 of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs located the protein of M. perniciosa (MpSod2p in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

  4. Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications.

    Science.gov (United States)

    Juturu, Veeresh; Wu, Jin Chuan

    2018-01-04

    Pichia pastoris is a well-known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent-free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site-specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α-factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co-expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high-quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol-II, and hyaluronic acid. The cell-surface display of enzymes has obviously increased enzyme stability, and high-level intracellular expression of acyl-CoA and ethanol O-acyltransferase, lipase and d-amino acid oxidase has opened up applications in whole-cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High-level expression of various food-grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Heterologous, Fresh, Human Donor Sclera as Patch Graft Material in Glaucoma Drainage Device Surgery.

    Science.gov (United States)

    Tsoukanas, Dimitrios; Xanthopoulou, Paraskevi; Charonis, Alexandros C; Theodossiadis, Panagiotis; Kopsinis, Gerasimos; Filippopoulos, Theodoros

    2016-07-01

    To determine the safety and efficacy of fresh, human sclera allografts as a patch graft material in glaucoma drainage device (GDD) surgery. Retrospective, noncomparative, interventional, consecutive case series. All GDD cases operated between 2008 and 2013 in which fresh human corneoscleral rims were used immediately after the central corneal button was used for penetrating or endothelial keratoplasty. Surgery was performed by 2 surgeons at 2 facilities. The Ahmed Glaucoma Valve (FP-7) was used exclusively in this cohort. Sixty-four eyes of 60 patients were identified; demographic data were recorded along with intraocular pressure (IOP), medication requirements, visual acuity, complications, and subsequent interventions. Incidence of complications. IOP and medication requirements at the last follow-up. Quilified success utilizing Tube Versus Trabeculectomy study criteria. The mean age of the cohort was 66.2±19.1 years; the average preoperative IOP was 33.2±11.1 mm Hg on 4.2±1.3 IOP-lowering agents before GDD surgery. IOP decreased significantly to 14.1±4.7 mm Hg (Pendophthalmitis, and there was 1 case of conjunctival erosion and tube/plate exposure (1.6%) occurring 30 days after surgery. Qualified success was estimated as 90.5% and 81% at 1 and 2 years, respectively, using Tube Versus Trabeculectomy study criteria. Heterologous, fresh, human donor sclera appears to be a safe material for GDD tube coverage. It provides a cost-efficient alternative compared with traditional patch graft materials associated with a low risk of pathogen transmission.

  6. The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains.

    Science.gov (United States)

    Shattuck, Jenifer E; Waechter, Aubrey C; Ross, Eric D

    2017-07-04

    Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN + ], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.

  7. Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

    Science.gov (United States)

    Silva, D A O; Vitaliano, S N; Mineo, T W P; Ferreira, R A; Bevilacqua, E; Mineo, J R

    2005-10-01

    Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

  8. Dual-energy CT for the evaluation of urinary calculi: Image interpretation, pitfalls and stone mimics

    International Nuclear Information System (INIS)

    Jepperson, M.A.; Cernigliaro, J.G.; Sella, D.; Ibrahim, E.; Thiel, D.D.; Leng, S.; Haley, W.E.

    2013-01-01

    Urolithiasis is a common disease with a reported prevalence between 4% and 20% in developed countries. Determination of urinary calculi composition is a key factor in preoperative evaluation, treatment, and stone recurrence prevention. Prior to the introduction of dual-energy computed tomography (DECT), available methods for determining urinary stone composition were only available after stone extraction, and thereby unable to aid in optimized stone management prior to intervention. DECT utilizes the attenuation difference produced by two different x-ray energy spectra to quantify urinary calculi composition as uric acid or non-uric acid (with likely further classification in the future) while still providing the information attained with a conventional CT. Knowledge of DECT imaging pitfalls and stone mimics is important, as the added benefit of dual-energy analysis is the determination of stone composition, which in turn affects all aspects of stone management. This review briefly describes DECT principles, scanner types and acquisition protocols for the evaluation of urinary calculi as they relate to imaging pitfalls (inconsistent characterization of small stones, small dual-energy field of view, and mischaracterization from surrounding material) and stone mimics (drainage devices) that may adversely impact clinical decisions. We utilize our clinical experience from scanning over 1200 patients with this new imaging technique to present clinically relevant examples of imaging pitfalls and possible mechanisms for resolution

  9. Intact Rapid Facial Mimicry as well as Generally Reduced Mimic Responses in Stable Schizophrenia Patients

    Science.gov (United States)

    Chechko, Natalya; Pagel, Alena; Otte, Ellen; Koch, Iring; Habel, Ute

    2016-01-01

    Spontaneous emotional expressions (rapid facial mimicry) perform both emotional and social functions. In the current study, we sought to test whether there were deficits in automatic mimic responses to emotional facial expressions in patients (15 of them) with stable schizophrenia compared to 15 controls. In a perception-action interference paradigm (the Simon task; first experiment), and in the context of a dual-task paradigm (second experiment), the task-relevant stimulus feature was the gender of a face, which, however, displayed a smiling or frowning expression (task-irrelevant stimulus feature). We measured the electromyographical activity in the corrugator supercilii and zygomaticus major muscle regions in response to either compatible or incompatible stimuli (i.e., when the required response did or did not correspond to the depicted facial expression). The compatibility effect based on interactions between the implicit processing of a task-irrelevant emotional facial expression and the conscious production of an emotional facial expression did not differ between the groups. In stable patients (in spite of a reduced mimic reaction), we observed an intact capacity to respond spontaneously to facial emotional stimuli. PMID:27303335

  10. A microbially derived tyrosine-sulfated peptide mimics a plant peptide hormone.

    Science.gov (United States)

    Pruitt, Rory N; Joe, Anna; Zhang, Weiguo; Feng, Wei; Stewart, Valley; Schwessinger, Benjamin; Dinneny, José R; Ronald, Pamela C

    2017-07-01

    The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX mimics the growth-stimulating activity of PSY peptides. Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analyses and reactive oxygen species burst assays to evaluate the activity of RaxX and PSY peptides. Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence. These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide. © 2017 UT-Battelle LLC. New Phytologist © 2017 New Phytologist Trust.

  11. Synthesis and biological investigation of PIM mimics carrying biotin or a fluorescent label for cellular imaging.

    Science.gov (United States)

    Front, Sophie; Bourigault, Marie-Laure; Rose, Stéphanie; Noria, Ségueni; Quesniaux, Valérie F J; Martin, Olivier R

    2013-01-16

    Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-inflammatory agents. Recently, heterocyclic analogues of PIMs in which the inositol is replaced by a piperidine (aza-PIM mimics) or a tetrahydropyran moiety (oxa-PIM mimics) have been prepared by short synthetic sequences and shown to retain the biological activity of the parent PIM structures. In this investigation, the aza-PIM analogue was used as a convenient scaffold to link biotin or a fluorescent label (tetramethyl-rhodamine) by way of an aminocaproyl spacer, with the goal of using these conjugates for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM-biotin conjugate binds to macrophage membranes with rapid kinetics.

  12. Differentiation of meningiomas from histologic mimics via the use of claudin-1

    International Nuclear Information System (INIS)

    Shalaby, A.M.R.; Naquib, S.M.

    2006-01-01

    Meningiomas may be occasionally difficult to distinguish pathologically from other tumors of the central nervous system. Claudin-1 is a tight junction-associated protein recently shown to be expressed in anaplastic meningiomas. This study aimed at assessment whether immunohistochemical staining for claudin-1 could help distinguish meningiomas from histologic mimics, compared with commonly used markers. Tissue sections from 20 meningothelial meningiomas, 40 fibrous meningiomas, 20 atypical meningiomas, 14 solitary fibrous tumors of the meninges, 10 meningeal hemangiopericytomas, and 14 vestibular schwannomas were stained immunohistochemically for claudin-1, epithelial membrane antigen, S-100 protein, CD34, and glial fibrillary acidic protein. In total, 42(53%) of 80 meningiomas were immunoreactive for claudin-1, whereas none of the other tumors were positive. In contrast, there was considerable overlap in the distribution of the other antibodies evaluated. Claudin-1 seems to be a specific marker for meningiomas in this context. Although its sensitivity is relatively low, claudin-1 may be helpful in a panel of immunostains to distinguish meningiomas from histologic mimics. (author)

  13. Intravital Confocal and Two-photon Imaging of Dual-color Cells and Extracellular Matrix Mimics

    Science.gov (United States)

    Bal, Ufuk; Andresen, Volker; Baggett, Brenda; Utzinger, Urs

    2013-01-01

    To optimize imaging of cells in three dimensional culture we studied confocal backscattering, Second Harmonic Generation (SHG) and autofluorescence as source of contrast in extracellular matrix (ECM) mimics and evaluated the attenuation as well as bleaching of endogenous cellular fluorescence signals. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence while still providing good reflectance to detect voids in the embedding medium. We labeled breast cancer cells’ outline with DsRed2 and nucleus with eGFP. DsRed2 can be excited with confocal imaging at 568nm, and with two photon excitation (TPE) in the red and longer NIR. eGFP was excited at 488nm for confocal and in the NIR for TPE. While there is small difference in the bleaching rate for eGFP between confocal and TPE we observed significant difference for DsRed2 where bleaching is strongest during TPE in the red wavelengths and smallest during confocal imaging. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence becomes twice as strong compared to confocal imaging. PMID:23380006

  14. Heterologous Expression of the Cotton NBS-LRR Gene GbaNA1 Enhances Verticillium Wilt Resistance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Nan-Yang Li

    2018-02-01

    Full Text Available Verticillium wilt caused by Verticillium dahliae results in severe losses in cotton, and is economically the most destructive disease of this crop. Improving genetic resistance is the cleanest and least expensive option to manage Verticillium wilt. Previously, we identified the island cotton NBS-LRR-encoding gene GbaNA1 that confers resistance to the highly virulent V. dahliae isolate Vd991. In this study, we expressed cotton GbaNA1 in the heterologous system of Arabidopsis thaliana and investigated the defense response mediated by GbaNA1 following inoculations with V. dahliae. Heterologous expression of GbaNA1 conferred Verticillium wilt resistance in A. thaliana. Moreover, overexpression of GbaNA1 enabled recovery of the resistance phenotype of A. thaliana mutants that had lost the function of GbaNA1 ortholog gene. Investigations of the defense response in A. thaliana showed that the reactive oxygen species (ROS production and the expression of genes associated with the ethylene signaling pathway were enhanced significantly following overexpression of GbaNA1. Intriguingly, overexpression of the GbaNA1 ortholog from Gossypium hirsutum (GhNA1 in A. thaliana did not induce the defense response of ROS production due to the premature termination of GhNA1, which lacks the encoded NB-ARC and LRR motifs. GbaNA1 therefore confers Verticillium wilt resistance in A. thaliana by the activation of ROS production and ethylene signaling. These results demonstrate the functional conservation of the NBS-LRR-encoding GbaNA1 in a heterologous system, and the mechanism of this resistance, both of which may prove valuable in incorporating GbaNA1-mediated resistance into other plant species.

  15. Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Sybirna Kateryna

    2008-09-01

    Full Text Available Abstract Background The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii. Results Based on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase. Conclusion In the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1 of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.

  16. Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal α-Amylase Gene in Aspergillus oryzae.

    Science.gov (United States)

    Yin, Yanchen; Mao, Youzhi; Yin, Xiaolie; Gao, Bei; Wei, Dongzhi

    2015-07-01

    The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30°C. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

  17. Cell surface expression system for the display of heterologous gene products using chimeric flagellin fusions of bacillus halodurans isolate

    CSIR Research Space (South Africa)

    Du Plessis, A

    2006-10-01

    Full Text Available system for the display of heterologous gene products using chimeric flagellin fusions of a Bacillus halodurans isolate Slide 2 © CSIR 2006 www.csir.co.za Bacillus halodurans Alk 36 xrhombus Ability to over-produce cell... for functionality of the His-tag for metal binding. Slide 13 © CSIR 2006 www.csir.co.za PAGE gel showing over-production of chimeric poly-His flagellin proteins 66.2 kDa 45.0 kDa 31.0 kDa 1. LMW ladder 2. NC3 3. NHisC3 4. NC6 5...

  18. Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences

    DEFF Research Database (Denmark)

    Pallesen, L; Poulsen, LK; Christiansen, Gunna

    1995-01-01

    of heterologous DNA segments encoding two reporter sequences. In the selected positions such insertions did not significantly alter the function of the FimH protein with regard to surface location and adhesive ability. The system seemed to be quite flexible, since chimeric versions of the FimH adhesin containing...... as many as 56 foreign amino acids were transported to the bacterial surface as components of the fimbrial organelles. Furthermore, the foreign protein segments were recognized by insert-specific antibodies when expressed within chimeric proteins on the surface of the bacteria. The results from...

  19. ABO hemolytic disease of the fetus and newborn: an iatrogenic complication of heterologous assisted reproductive technology-induced pregnancy.

    Science.gov (United States)

    Zuppa, Antonio Alberto; Cardiello, Valentina; Lai, Marco; Cataldi, Luigi; D'Andrea, Vito; Romagnoli, Costantino

    2010-10-01

    ABO hemolytic disease of the fetus and newborn (ABO HDFN) may manifest itself in cases of mothers belonging to blood group O and newborns of groups A or B and more frequently in group A and less so in group B. The case subjects are twin-birth newborns with ABO HDFN, of group AB born to a mother of group O. These cases of ABO HDFN prove inconsistent with Mendel's law of segregation. This case study finds its explanation in new methods of assisted reproduction, particularly heterologous in vitro fertilization with ovodonation. © 2010 American Association of Blood Banks.

  20. Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

    DEFF Research Database (Denmark)

    Choi, Kyeong Rok; Cho, Jae Sung; Cho, In Jin

    2018-01-01

    Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable...... plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida, a bacterial host strain of increasing academic and industrial interest....

  1. Heterologous amyloid seeding: revisiting the role of acetylcholinesterase in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Létitia Jean

    2007-07-01

    for observations that implicate hAChE in the extent of Abeta deposition in the brain. Furthermore, this process of heterologous amyloid seeding by a proteolytic fragment from another protein may represent a previously underestimated pathological trigger, implying that the abundance of the major amyloidogenic species (Abeta in AD, for example may not be the only important factor in neurodegeneration.

  2. ANTIGENIC RELATEDNESS OF SELECTED FLAVIVIRUSES: STUDY WITH HOMOLOGOUS AND HETEROLOGOUS IMMUNE MOUSE ASCITIC FLUIDS

    Directory of Open Access Journals (Sweden)

    S.S. BABA

    1998-11-01

    Full Text Available The antigenic relationship of 9 flaviviruses, Yellow fever (YF , Wesselsbron (WSL , Uganda S (UGS , Potiskum (POT, West Nile (WN , Banzi (BAN , Zika (ZK , Dengue type 1 (DEN-1 and Dengue type 2 (DEN-2, was assessed by cross-haemagglutination-inhibition (Cross-HI and cross-complement fixation (Cross-CF reactions between each of the viruses and their homologous immune mouse ascitic fluids. Titre ratios were calculated using the heterologous and homologous titres. Cross-CF reactions revealed wider antigenic variations among viruses than Cross-HI reactions. There was no significant antigenic variation between WSL, POT and YF viruses using either of those methods. However, definite differences in antigenicity were observed between them and UGS, BAN and ZK viruses. There were no significant differences between UGS, BAN and ZK or between DEN-1 and DEN-2. The serological relationship among flaviviruses is important in establishing diagnosis and epidemiology of these infections in Africa.A relação antigênica de 9 Flavivirus, Febre amarela (YF, Wesselsbron (WSL, Uganda S (UGS, Potiskum (POT, West Nile (WN, Banzi (BAN, Zika (ZK, Dengue tipo 1 (DEN-1 e Dengue tipo2 (DEN-2, foi avaliada por reação de inibição da hemaglutinação cruzada (cross-HI e reação de fixação do complemento cruzada (Cross-CF entre cada um dos virus e seu fluido ascítico homólogo em camundongos. Médias de títulos foram calculadas usando os títulos heterólogos e homólogos. Reações cruzadas CF revelaram maiores variações antigênicas entre virus do que reações cruzadas HI. Não houve variação antigênica significativa entre virus WSL, POT e YF usando cada um dos métodos. Todavia, diferenças definidas da antigenicidade foram observadas entre eles e os vírus UGS, BAN e ZK. Não existiram diferenças significativas entre UGS, BAN e ZK ou entre DEN-1 e DEN-2. A relação sorológica entre Flavivirus é importante para se estabelecer o diagnóstico e a

  3. Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Kim Dae-Hyuk

    2010-02-01

    Full Text Available Abstract Background A tannic acid-inducible and mycoviral-regulated laccase3 (lac3 from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae. Results Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants. Conclusions The results suggest that expression of the lac3 gene has an inhibitory effect on the growth of

  4. Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

    Science.gov (United States)

    Laugwitz, K L; Weig, H J; Moretti, A; Hoffmann, E; Ueblacker, P; Pragst, I; Rosport, K; Schömig, A; Ungerer, M

    2001-04-13

    In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of c

  5. Heterologous radioimmunoassays for monkey gonadotrophins. I. Assessment of the reagents proposed for the assay of FSH

    Energy Technology Data Exchange (ETDEWEB)

    Khan, S A; Diczfalusy, E [Reproductive Endocrinology Research Unit, Karolinska sjukhuset, Stockholm

    1983-01-01

    The reliability of the reagents used in heterologous immunoassay procedures for FSH in monkey sera was assessed. Re-investigation of the iodination and purification of the human FSH tracer confirmed the presence of different iodinated molecular species, such as a high molecular weight aggregated material, 'intact' human FSH and subunits. Tracers obtained with the chloramine T procedure were superior to those obtained by the lactoperoxidase method. Purification by cellulose adsorption chromatography resulted in partial dissociation of the intact hormone into free subunits, while Sephadex G-25 chromatography did not yield any subunit-like material. Purification by Ultrogel AcA 54 gave the best separation of the 'intact' iodinated FSH. Binding studies indicated that an anti-ovine FSH (oFSH) serum showed a high degree of binding to 'intact' hFSH, although its binding to subunits and to aggregated material was also significant. The highest degree of binding to this antiserum was shown by the 'intact' tracer iodinated with chloramine T and purified sequentially by Sephadex G-25 and Ultrogel (AcA 54) chromatography. Using a highly purified hFSH tracer in combination with three antisera (anti-oFSH, H 31; anti-hFSH, WHO, M-93-2; anti-hFSH, NIH, batch No. 5) displacement curves were investigated with three purified monkey FSH preparations. No displacement was observed with a widely available anti-hFSH (WHO, M 93-2) serum. However, linear dose dependent displacement was found with another anti-hFSH (NIH, batch No. 5) serum and with an anti-oFSH (H 31) serum. Comparison of immunoassay with the human and ovine antiserum revealed significant differences in slope, parallelism, precision and effective range. Significant discrepancies in the FSH potency estimates in monkey sera were also observed with the two assay systems. It is suggested that - until a homologous system is developed - the immunoassay employing an hFSH tracer and an anti-oFSH serum should be used for macaque FSH.

  6. Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge.

    Science.gov (United States)

    Mooney, Alaina J; Gabbard, Jon D; Li, Zhuo; Dlugolenski, Daniel A; Johnson, Scott K; Tripp, Ralph A; He, Biao; Tompkins, S Mark

    2017-12-01

    Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats. IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing

  7. Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.

    Science.gov (United States)

    Zhu, Duolong; Fu, Yuxin; Liu, Fulu; Xu, Haijin; Saris, Per Erik Joakim; Qiao, Mingqiang

    2017-01-03

    The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation. Four large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly. The genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design

  8. Structure of a SUMO-binding-motif mimic bound to Smt3p–Ubc9p: conservation of a noncovalent Ubiquitin-like protein–E2 complex as a platform for selective interactions within a SUMO pathway

    Science.gov (United States)

    Duda, David M.; van Waardenburg, Robert C. A. M.; Borg, Laura A.; McGarity, Sierra; Nourse, Amanda; Waddell, M. Brett; Bjornsti, Mary-Ann; Schulman, Brenda A.

    2007-01-01

    Summary The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast S. cerevisiae, Smt3p and Ubc9p, are encoded by essential genes. Here we describe the 1.9 Å resolution crystal structure of a noncovalent Smt3p–Ubc9p complex. Unexpectedly, a heterologous portion of the crystallized complex derived from the expression construct mimics an SBM, and binds Smt3p in a manner resembling SBM binding to human SUMO family members. In the complex, Smt3p binds a surface distal from Ubc9's catalytic cysteine. The structure implies that a single molecule of Smt3p cannot bind concurrently to both the noncovalent binding site and the catalytic cysteine of a single Ubc9p molecule. However, formation of higher-order complexes can occur, where a single Smt3p covalently linked to one Ubc9p's catalytic cysteine also binds noncovalently to another molecule of Ubc9p. Comparison with other structures from the SUMO pathway suggests that formation of the noncovalent Smt3p–Ubc9p complex occurs mutually exclusively with many other Smt3p and Ubc9p interactions in the conjugation cascade. By contrast, high-resolution insights into how Smt3p–Ubc9p can also interact with downstream recognition machineries come from contacts with the SBM mimic. Interestingly, the overall architecture of the Smt3p–Ubc9p complex is strikingly similar to recent structures from the ubiquitin pathway. The results imply that noncovalent ubiquitin-like protein–E2 complexes are conserved platforms, which function as parts of larger assemblies involved many protein post-translational regulatory pathways. PMID:17475278

  9. IFN-γ Induces Mimic Extracellular Trap Cell Death in Lung Epithelial Cells Through Autophagy-Regulated DNA Damage.

    Science.gov (United States)

    Lin, Chiou-Feng; Chien, Shun-Yi; Chen, Chia-Ling; Hsieh, Chia-Yuan; Tseng, Po-Chun; Wang, Yu-Chih

    2016-02-01

    Treatment of interferon-γ (IFN-γ) causes cell growth inhibition and cytotoxicity in lung epithelial malignancies. Regarding the induction of autophagy related to IFN-γ signaling, this study investigated the link between autophagy and IFN-γ cytotoxicity. In A549 human lung cancer cells, IFN-γ treatment induced concurrent apoptotic and nonapoptotic events. Unexpectedly, the nonapoptotic cells present mimic extracellular trap cell death (ETosis), which was regulated by caspase-3 and by autophagy induction through immunity-related GTPase family M protein 1 and activating transcription factor 6. Furthermore, IFN-γ signaling controlled mimic ETosis through a mechanism involving an autophagy- and Fas-associated protein with death domain-controlled caspase-8/-3 activation. Following caspase-mediated lamin degradation, IFN-γ caused DNA damage-associated ataxia telangiectasia and Rad3-related protein (ATR)/ataxia telangiectasia mutated (ATM)-regulated mimic ETosis. Upon ATR/ATM signaling, peptidyl arginine deiminase 4 (PAD4)-mediated histone 3 citrullination promoted mimic ETosis. Such IFN-γ-induced effects were defective in PC14PE6/AS2 human lung cancer cells, which were unsusceptible to IFN-γ-induced autophagy. Due to autophagy-based caspase cascade activation, IFN-γ triggers unconventional caspase-mediated DNA damage, followed by ATR/ATM-regulated PAD4-mediated histone citrullination during mimic ETosis in lung epithelial malignancy.

  10. Targeted delivery of microRNA 146b mimic to hepatocytes by lactosylated PDMAEMA nanoparticles for the treatment of NAFLD.

    Science.gov (United States)

    He, Shuying; Guo, Weihong; Deng, Feihong; Chen, Kequan; Jiang, Yonghong; Dong, Minyu; Peng, Liang; Chen, Xueqing

    2018-03-21

    Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide, and precision therapeutic will be a benefit for the NAFLD regression. In this study, we observed low microRNA 146 b (miR-146 b) expression in NAFLD mice model induced by methionine-choline-deficient diet (MCD) compared with control group. Furthermore, miR-146b -/- mice induced MCD exhibited severe liver steatosis and hepatitis. A bio-distribution study showed that novel Lactosylated PDMAEMA nanoparticles effectively targeted hepatocytes Lac-PDMAEMA. We coupled miR-146b mimic with Lac-PDMAEMA and then were administrated to NAFLD mice model, which could obviously alleviate the hepatic steatosis. Lac-PDMAEMA effectively delivered miR-146b mimic to hepatocytes with a ∼8-fold upregulation of miR-146b mimic targeting MyD88 and IRAK1, and in turn suppressed the expression of PPARγ. Meanwhile, TNF-α and IL-6 mRNA levels were decreased after administration of Lac-PDMAEMA/miR-146b mimic. So, we made a conclusion that targeted delivering miR-146b mimic to the hepatocytes by, coupling Lac-PDMAEMA nanoparticles could effectively alleviate the hepatic steatosis in NAFLD mice, which maybe bring a new and effective way to intervene and therapy the NAFLD.

  11. Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

    Science.gov (United States)

    Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

    2016-01-01

    We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy.

  12. Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic

    Science.gov (United States)

    Perry, Kay; Hwang, Young; Bushman, Frederic D.; Van Duyne, Gregory D.

    2010-01-01

    Summary Poxviruses encode their own type IB topoisomerases (TopIBs) which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves a novel Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small vs. large enzyme subfamilies. PMID:20152159

  13. Neglected ruptured flexor carpi ulnaris tendon mimics a soft tissue tumor in the wrist.

    Science.gov (United States)

    Rau, Chi-Lun; Yen, Tze-Hsun; Wu, Lien-Chen; Huang, Yi-You; Jaw, Fu-Shan; Liou, Tsan-Hon

    2014-04-01

    A wrist mass is rarely caused by a ruptured tendon in the forearm. The common pathologies are ganglia, tendon tenosynovitis, and giant cell tumors of tendon sheaths. Less common causes are nerve sheath tumors, vascular lesions, or an accessory muscle belly. The authors investigated a case of neglected ruptured flexor carpi ulnaris tendon that mimics a mass in the wrist. To the authors' knowledge, this is the first case report in relevant literature. During investigation, the high-resolution musculoskeletal ultrasound suggested a soft tissue tumor or a ruptured flexor carpi ulnaris tendon. The magnetic resonance imaging scan indicated an accessory flexor carpi ulnaris muscle belly. The diagnosis of ruptured flexor carpi ulnaris tendon was confirmed by surgical exploration. This case indicates that ultrasound may be better suited than magnetic resonance imaging in evaluating a wrist mass for its accuracy, availability, and portability.

  14. Cooperative interplay of let-7 mimic and HuR with MYC RNA.

    Science.gov (United States)

    Gunzburg, Menachem J; Sivakumaran, Andrew; Pendini, Nicole R; Yoon, Je-Hyun; Gorospe, Myriam; Wilce, Matthew C J; Wilce, Jacqueline A

    2015-01-01

    Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites.

  15. Preparation of Artificial Skin that Mimics Human Skin Surface and Mechanical Properties.

    Science.gov (United States)

    Shimizu, Rana; Nonomura, Yoshimune

    2018-01-01

    We have developed an artificial skin that mimics the morphological and mechanical properties of human skin. The artificial skin comprises a polyurethane block possessing a microscopically rough surface. We evaluated the tactile sensations when skin-care cream was applied to the artificial skin. Many subjects perceived smooth, moist, and soft feels during the application process. Cluster analysis showed that these characteristic tactile feels are similar to those when skin-care cream is applied to real human skin. Contact angle analysis showed that an oil droplet spread smoothly on the artificial skin surface, which occurred because there were many grooves several hundred micrometers in width on the skin surface. In addition, when the skin-care cream was applied, the change in frictional force during the dynamic friction process increased. These wetting and frictional properties are important factors controlling the similarity of artificial skin to real human skin.

  16. Enhanced photocatalytic hydrogen production from an MCM-41-immobilized photosensitizer-[Fe-Fe] hydrogenase mimic dyad.

    Science.gov (United States)

    Wang, Wen; Yu, Tianjun; Zeng, Yi; Chen, Jinping; Yang, Guoqiang; Li, Yi

    2014-11-01

    A covalently linked photosensitizer-catalytic center dyad Ps-Hy, consisting of two bis(2-phenylpyridine)(2,2'-bipyridine)iridium(iii) chromophores (Ps) and a diiron hydrogenase mimic (Hy) was constructed by using click reaction. Ps-Hy was incorporated into K(+)-exchanged molecular sieve MCM-41 to form a composite (Ps-Hy@MCM-41), which has been successfully applied to the photochemical production of hydrogen. The catalytic activity of Ps-Hy@MCM-41 is ∼3-fold higher as compared with that of Ps-Hy in the absence of MCM-41. The incorporation of Ps-Hy into MCM-41 stabilizes the catalyst, and consequently, advances the photocatalysis. The present study provides a potential strategy for improving catalytic efficiency of artificial photosynthesis systems using mesoporous molecular sieves.

  17. Systemic lupus erythematosis with antiphospholipid antibody syndrome: A mimic of Buerger′s disease

    Directory of Open Access Journals (Sweden)

    Vasugi Zoya

    2006-01-01

    Full Text Available This case report is about a past smoker who presented with history of recurrent ulcers and digital gangrene with claudication pain of the left foot for the past fifteen years. Clinical examination and angiogram showed disease involving the peripheral vessels of lowervlimb. This patient had been labeled as Buerger′s disease 15 years ago based on clinical and demographic profile of the illness. We felt that the progression of the disease despite the patient having stopped smoking 15 years ago along with the presence of elevated inflammatory markers in the blood with proteinuria was not in keeping with the nature of the disease. Furthur evaluation revealed that the patient had systemic lupus erythematosus with antiphospholipid antibody syndrome. This case highlights the need for a careful search for diseases, which can mimic Buerger′s disease in young smokers who present with peripheral vascular disease and who have an atypical clinical presentation or progression.

  18. Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers.

    Science.gov (United States)

    Graham, Ben; Bailey, Trisha L; Healey, Joseph R J; Marcellini, Moreno; Deville, Sylvain; Gibson, Matthew I

    2017-12-11

    Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent "antifreezes". Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid-phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Hepatitis C virus infection can mimic type 1 (antinuclear antibody positive) autoimmune chronic active hepatitis.

    Science.gov (United States)

    Pawlotsky, J M; Deforges, L; Bretagne, S; André, C; Métreau, J M; Thiers, V; Zafrani, E S; Goossens, M; Duval, J; Mavier, J P

    1993-01-01

    Hepatitis C virus (HCV) has been shown to induce anti-liver-kidney microsomal-1 (LKM1) antibody positive chronic active hepatitis, simulating type 2 autoimmune chronic active hepatitis. The cases of five patients presenting with features of type 1 (antinuclear antibody positive) autoimmune chronic active hepatitis and extrahepatic autoimmune manifestations, in whom immunosuppressive treatment had no effect on liver disease are presented. In these patients, HCV infection could be shown by the presence in serum of anti-HCV antibodies and HCV-RNA detected by polymerase chain reaction. These cases suggest the following: (a) chronic HCV infection can mimic type 1, as well as type 2, autoimmune chronic active hepatitis; (b) HCV infection might be systematically sought in patients presenting with features of type 1 autoimmune chronic active hepatitis, with special care in patients who are unresponsive to immunosuppressive treatment. Images Figure PMID:7686122

  20. Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration

    Science.gov (United States)

    Pearson, Brandon L.; Simon, Jeremy M.; McCoy, Eric S.; Salazar, Gabriela; Fragola, Giulia; Zylka, Mark J.

    2016-01-01

    Environmental factors, including pesticides, have been linked to autism and neurodegeneration risk using retrospective epidemiological studies. Here we sought to prospectively identify chemicals that share transcriptomic signatures with neurological disorders, by exposing mouse cortical neuron-enriched cultures to hundreds of chemicals commonly found in the environment and on food. We find that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer's disease and Huntington's disease). These chemicals stimulate free radical production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane. Our study provides an approach to prospectively identify environmental chemicals that transcriptionally mimic autism and other brain disorders. PMID:27029645

  1. Potent neutralization of hepatitis A virus reveals a receptor mimic mechanism and the receptor recognition site.

    Science.gov (United States)

    Wang, Xiangxi; Zhu, Ling; Dang, Minghao; Hu, Zhongyu; Gao, Qiang; Yuan, Shuai; Sun, Yao; Zhang, Bo; Ren, Jingshan; Kotecha, Abhay; Walter, Thomas S; Wang, Junzhi; Fry, Elizabeth E; Stuart, David I; Rao, Zihe

    2017-01-24

    Hepatitis A virus (HAV) infects ∼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention.

  2. Seminal epithelium in prostate biopsy can mimic malignant and premalignant prostatic lesions.

    Science.gov (United States)

    Arista-Nasr, J; Trolle-Silva, A; Aguilar-Ayala, E; Martínez-Benítez, B

    2016-01-01

    In most prostate biopsies, the seminal epithelium is easily recognised because it meets characteristic histological criteria. However, some biopsies can mimic malignant or premalignant prostatic lesions. The aims of this study were to analyse the histological appearance of the biopsies that mimic adenocarcinomas or preneoplastic prostatic lesions, discuss the differential diagnosis and determine the frequency of seminal epithelia in prostate biopsies. We consecutively reviewed 500 prostate puncture biopsies obtained using the sextant method and selected those cases in which we observed seminal vesicle or ejaculatory duct epithelium. In the biopsies in which the seminal epithelium resembled malignant or premalignant lesions, immunohistochemical studies were conducted that included prostate-specific antigen and MUC6. The most important clinical data were recorded. Thirty-six (7.2%) biopsies showed seminal epithelium, and 7 of them (1.4%) resembled various prostate lesions, including high-grade prostatic intraepithelial neoplasia, atypical acinar proliferations, adenocarcinomas with papillary patterns and poorly differentiated carcinoma. The seminal epithelium resembled prostate lesions when the lipofuscin deposit, the perinuclear vacuoles or the nuclear pseudoinclusions were inconspicuous or missing. Five of the 7 biopsies showed mild to moderate cellular atypia with small and hyperchromatic nuclei, and only 2 showed cellular pleomorphism. The patients were alive and asymptomatic after an average of 6 years of progression. The seminal epithelium resembles prostatic intraepithelial neoplasia, atypical acinar proliferations and various types of prostatic adenocarcinomas in approximately 1.4% of prostate biopsies. Copyright © 2015 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  3. The transport of DDT from chylomicrons to adipocytes does not mimic triacylglycerol transport

    Science.gov (United States)

    Kohan, Alison B.; Vandersall, Abbey E.; Yang, Qing; Xu, Min; Jandacek, Ronald J.; Tso, Patrick

    2012-01-01

    Despite being banned in the U.S., organochlorine toxins such as DDT are frequently detected in human adipose tissue. The main route of exposure is through the consumption of contaminated foods and subsequent intestinal packaging of DDT into chylomicrons. These chylomicrons, which also contain dietary triacylglycerol (TG), are delivered directly to peripheral tissues without first being metabolized by the liver. The physiological process by which these compounds are delivered from chylomicrons to adipose is not well understood, but is clinically relevant since it bypasses first-pass metabolism. Based on its highly lipophilic nature, it has been assumed that DDT is transferred to peripheral tissues similar to TG; however, this has not been measured. Here, we use the lymph fistula rat to isolate chylomicrons containing both DDT and TG. These chylomicrons are the in vivo DDT delivery vehicle. Using 3T3-L1 adipocytes, we investigated the rate at which DDT transfers from chylomicrons to adipocytes, and mediators of this process. This novel approach closely approximates the in vivo DDT exposure route. We show that: 1) DDT repartitions from chylomicrons to adipocytes, 2) this transport does not require hydrolysis of TG within the chylomicron, and is stimulated by the inhibition of LPL, 3) albumin does not inhibit DDT uptake, 4) DDT dissolved in DMSO does not appropriately mimic in vivo DDT transport; and most importantly, 5) DDT uptake from chylomicrons does not mimic the uptake of TG from the same particles. Understanding these factors is important for designing interventions for human populations exposed to DDT. PMID:22885168

  4. A Taylor-Made Design of Phenoxyfuranone-Type Strigolactone Mimic

    Directory of Open Access Journals (Sweden)

    Kosuke Fukui

    2017-06-01

    Full Text Available Strigolactones are a class of plant hormones that inhibit axillary bud outgrowth and are released from plant roots to act as a rhizosphere communication signal. The Orobanchaceae parasitic plant Striga hermonthica perceives strigolactone as its germination signal, indicating host presence. After germination, the Striga plant parasitises the host plant and suppresses host growth by draining photosynthetic products, water and other essential nutrients. Because of this way of life, this parasite threatens crop production in sub-Saharan Africa with infestation in crop fields and crop devastation. Crop protection in such areas is among the most concerning problems to be dealt with as immediately as possible. With respect to crop protection from Striga, many strigolactone agonists have been developed and used in research to reveal Striga biology, and have contributed to development of crop protection methods. However, an effective method has yet to be found. In a previous study, we reported debranones as a group of strigolactone mimics that inhibit axillary buds outgrowth with moderate stimulation activity for Striga germination. Debranones would be accessible because they are simply synthesized from commercially available phenols and bromo butenolide. Taking this advantage of debranones for Striga research, we tried to find the debranones stimulating Striga seed germination. To modulate functional selectivity and to enhance germination inducing activity of debranones, we studied structure–activity relationships. We investigated effects of substituent position and functional group on debranone activity and selectivity as a strigolactone mimic. As a result, we improved stimulation activity of debranones for Striga seed germination by chemical modification, and demonstrated the pharmacophore of debranones for selective modulation of distinct strigolactone responses.

  5. Functional Stroke Mimics: Incidence and Characteristics at a Primary Stroke Center in the Middle East

    Science.gov (United States)

    Wilkins, Stacy Schantz; Bourke, Paula; Salam, Abdul; Akhtar, Naveed; D'Souza, Atlantic; Kamran, Saadat; Bhutta, Zain; Shuaib, Ashfaq

    2018-01-01

    ABSTRACT Objective Approximately 30% of individuals who initially present with stroke are found to be stroke mimics (SM), with functional/psychological SM (FSM) accounting for up to 6.4% of all stroke presentations. Middle Eastern countries may have higher rates of somatization of emotional distress. The aim of this study was to evaluate the incidence and characteristics of FSM at a large general hospital in the Middle East. Methods All patients presenting with an initial diagnosis of stroke from June 2015 to September 2016 were eligible for this study. Clinical and sociodemographic data were obtained from the hospital's stroke database. All SM and strokes were diagnosed by Joint Commission International–certified stroke program neurologists. SM was defined as any discharge diagnosis (other than acute stroke) for symptoms that prompted initial admission for suspected stroke. FSM were compared with medical stroke mimics (MSM) and strokes (ischemic, hemorrhagic, and transient ischemic attacks). Results A total of 1961 patients were identified; 161 FSM (8.2%), 390 MSM (19.9%), and 1410 strokes (71.9%) (985 ischemic strokes, 196 transient ischemic attacks, 229 intracerebral hemorrhages). Admission with FSM was related to patients' nationality, with the highest frequency in Arabic (15.6%) and African (16.8%) patients. FSM patients were younger, more often female, and had fewer cardiovascular risk factors except for smoking compared with the strokes. FSM patients presented with more left-sided weakness and had more magnetic resonance imagings than the stroke and MSM groups. A total of 9.9% of FSM patients received thrombolysis versus only 0.5% of the MSM and 16.4% of ischemic strokes. Conclusions FSM frequencies varied by nationality, with Arab and African nationals being twice as prevalent. Stress, vulnerable status as expats, sociopolitical instability, and exposure to trauma are proposed as potential factors contributing to FSM. PMID:29394187

  6. Lipid bilayer-coated mesoporous silica nanoparticles carrying bovine hemoglobin towards an erythrocyte mimic.

    Science.gov (United States)

    Tu, Jing; Bussmann, Jeroen; Du, Guangsheng; Gao, Yue; Bouwstra, Joke A; Kros, Alexander

    2018-05-30

    Hemoglobin (Hb)-loaded mesoporous silica nanoparticles (MSNs) coated with a lipid bilayer (LB-MSNs) were investigated as an erythrocyte mimic. MSNs with a large average pore size (10 nm) act as a rigid core and provide a protective environment for Hb encapsulated inside the pores. The colloidal stability of Hb-loaded MSNs was enhanced upon the application of a lipid bilayer, through fusion of PEGylated liposomes onto the exterior surface of Hb-loaded MSNs. The morphology and mesostructure of the MSNs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and surface area analysis. The Hb loading capacity (mg/g) in MSNs was studied by thermogravimetric analysis (TGA). UV-Vis absorption spectroscopy revealed that Hb inside MSNs had an identical, but slightly broadened peak in the Soret region compared to free Hb. Furthermore the encapsulated Hb exhibits similar peroxidase-like activity in catalyzing the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with hydrogen peroxide. The introduction of a supported lipid bilayer (LB) demonstrated the potential to prevent premature Hb release (the burst release decreased from 25.50 ± 0.33% to 6.73 ± 0.83%) and increased the colloidal stability of the Hb-loaded MSNs (hydrodynamic diameter remained ∼250 nm for at least one week). The in vivo systemic circulation and biodistribution of LB-MSNs were studied in optically transparent zebrafish embryos, revealing that LB-MSNs have the potential to act as an erythrocyte mimic in transfusion therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. A Multiple Indicators Multiple Causes (MIMIC) model of internal barriers to drug treatment in China.

    Science.gov (United States)

    Qi, Chang; Kelly, Brian C; Liao, Yanhui; He, Haoyu; Luo, Tao; Deng, Huiqiong; Liu, Tieqiao; Hao, Wei; Wang, Jichuan

    2015-03-01

    Although evidence exists for distinct barriers to drug abuse treatment (BDATs), investigations of their inter-relationships and the effect of individual characteristics on the barrier factors have been sparse, especially in China. A Multiple Indicators Multiple Causes (MIMIC) model is applied for this target. A sample of 262 drug users were recruited from three drug rehabilitation centers in Hunan Province, China. We applied a MIMIC approach to investigate the effect of gender, age, marital status, education, primary substance use, duration of primary drug use, and drug treatment experience on the internal barrier factors: absence of problem (AP), negative social support (NSS), fear of treatment (FT), and privacy concerns (PC). Drug users of various characteristics were found to report different internal barrier factors. Younger participants were more likely to report NSS (-0.19, p=0.038) and PC (-0.31, p<0.001). Compared to other drug users, ice users were more likely to report AP (0.44, p<0.001) and NSS (0.25, p=0.010). Drug treatment experiences related to AP (0.20, p=0.012). In addition, differential item functioning (DIF) occurred in three items when participant from groups with different duration of drug use, ice use, or marital status. Individual characteristics had significant effects on internal barriers to drug treatment. On this basis, BDAT perceived by different individuals could be assessed before tactics were utilized to successfully remove perceived barriers to drug treatment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.

    Science.gov (United States)

    Smyth, Danielle J; Harcus, Yvonne; White, Madeleine P J; Gregory, William F; Nahler, Janina; Stephens, Ian; Toke-Bjolgerud, Edward; Hewitson, James P; Ivens, Alasdair; McSorley, Henry J; Maizels, Rick M

    2018-04-01

    We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-β. The Heligmosomoides polygyrus TGF-β Mimic (Hp-TGM) shares no homology to any TGF-β family member, however it binds the mammalian TGF-β receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12-15 amino acids longer than the 60-70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-β activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1-3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation. Copyright © 2018. Published by Elsevier Ltd.

  9. Dynamic Changes in Host Gene Expression following In Vitro Viral Mimic Stimulation in Crocodile Cells

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    Subir Sarker

    2017-11-01

    Full Text Available The initial control of viral infection in a host is dominated by a very well orchestrated early innate immune system; however, very little is known about the ability of a host to control viral infection outside of mammals. The reptiles offer an evolutionary bridge between the fish and mammals, with the crocodile having evolved from the archosauria clade that included the dinosaurs, and being the largest living reptile species. Using an RNA-seq approach, we have defined the dynamic changes of a passaged primary crocodile cell line to stimulation with both RNA and DNA viral mimics. Cells displayed a marked upregulation of many genes known to be involved in the mammalian response to viral infection, including viperin, Mx1, IRF7, IRF1, and RIG-I with approximately 10% of the genes being uncharacterized transcripts. Both pathway and genome analysis suggested that the crocodile may utilize the main known mammalian TLR and cytosolic antiviral RNA signaling pathways, with the pathways being responsible for sensing DNA viruses less clear. Viral mimic stimulation upregulated the type I interferon, IFN-Omega, with many known antiviral interferon-stimulated genes also being upregulated. This work demonstrates for the first time that reptiles show functional regulation of many known and unknown antiviral pathways and effector genes. An enhanced knowledge of these ancient antiviral pathways will not only add to our understanding of the host antiviral innate response in non-mammalian species, but is critical to fully comprehend the complexity of the mammalian innate immune response to viral infection.

  10. Heterologous and endogenous U6 snRNA promoters enable CRISPR/Cas9 mediated genome editing in Aspergillus niger.

    Science.gov (United States)

    Zheng, Xiaomei; Zheng, Ping; Sun, Jibin; Kun, Zhang; Ma, Yanhe

    2018-01-01

    U6 promoters have been used for single guide RNA (sgRNA) transcription in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) genome editing system. However, no available U6 promoters have been identified in Aspergillus niger, which is an important industrial platform for organic acid and protein production. Two CRISPR/Cas9 systems established in A. niger have recourse to the RNA polymerase II promoter or in vitro transcription for sgRNA synthesis, but these approaches generally increase cloning efforts and genetic manipulation. The validation of functional RNA polymerase II promoters is therefore an urgent need for A. niger . Here, we developed a novel CRISPR/Cas9 system in A. niger for sgRNA expression, based on one endogenous U6 promoter and two heterologous U6 promoters. The three tested U6 promoters enabled sgRNA transcription and the disruption of the polyketide synthase albA gene in A. niger . Furthermore, this system enabled highly efficient gene insertion at the targeted genome loci in A. niger using donor DNAs with homologous arms as short as 40-bp. This study demonstrated that both heterologous and endogenous U6 promoters were functional for sgRNA expression in A. niger . Based on this result, a novel and simple CRISPR/Cas9 toolbox was established in A. niger, that will benefit future gene functional analysis and genome editing.

  11. Ectopic expression of MPF2-like protein WSA206 leads to arrest in silique and seed development in heterologous host

    International Nuclear Information System (INIS)

    Khan, M.R.

    2016-01-01

    MPF2-like genes belonging to STMADS11 clade of MADS-box transcription factors are mostly involved in calyx inflation, floral reversion and fertility. However their role in fertility remained enigmatic. In this study we transformed WSA206 gene paralog - originated through genome duplication in a Solanaceous plant Withaniasomnifera - ectopically in a heterologous host Arabidopsis thaliana. Interesting phenotypes in floral organs and fruits were observed. Overexpression of WSA206 leads to arrest in silique development. The siliques were compressed and size was drastically reduced from 34mm to 3mm. Along with siliques, the seed development was also suppressed as revealed by shriveling of seeds and reduction in seed number. In extreme cases the siliques were devoid of any seeds. In cases where seeds developed, these were impaired in viability. Besides, the transgenic Arabidopsis also exhibited exorbitant growth of calyx to an extent that it resembled inflated calyx in Solanaceae. The calyx remained persistent and encapsulated the under-developed siliques containing non-viable seeds inside. Thus, fertility and sepal development are tightly coupled traits that are controlled by WSA206 paralog in heterologous system. (author)

  12. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression.

    Science.gov (United States)

    Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

    2015-12-15

    Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment.

  13. [Tetanus prevention with vaccine and with vaccine plus heterologous immune serum: serum antibody levels in the rabbit].

    Science.gov (United States)

    Bistoni, F; Mosci, L; Vecchiarelli, A; Marconi, P; Pitzurra, M

    1977-01-01

    Haemagglutinating antibodies have been assessed in rabbits undergoing active- passive immunization against tetanus. The animals received 6 injections of horse immune serum, 400 UI/kg, and A1PO4 adsorbed toxoid, 0.35 Lf/kg, every 30th day. One the 5th day, after the first injection, E.A. antibodies appeared, at low levels, as a result of a passive immunization. Thereafter the tests became negative, up to the 70th day, when an active immunization emerged, with a 25 days delay in comparison with controls. Neutralization test in vivo behaved in the same way. The results stress once more the need to give up the use of heterologous immune sera in tetanus prophylaxis, in active-passive immunization as well. Arguments adding force to this point of view are: the sensibilization against heterologous proteins, the very low (if any) passive protective action, and, last not least, the delay in the emergence of active immunization: the only reliable shield against tetanus.

  14. Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

    Science.gov (United States)

    Mendes, Érica Araújo; Fonseca, Flavio G; Casério, Bárbara M; Colina, Janaína P; Gazzinelli, Ricardo Tostes; Caetano, Braulia C

    2013-01-01

    The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

  15. Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

    Directory of Open Access Journals (Sweden)

    Érica Araújo Mendes

    Full Text Available The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1 of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination. Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1, to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

  16. A simple model-based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess.

    Science.gov (United States)

    Cos, Oriol; Ramon, Ramon; Montesinos, José Luis; Valero, Francisco

    2006-09-05

    A predictive control algorithm coupled with a PI feedback controller has been satisfactorily implemented in the heterologous Rhizopus oryzae lipase production by Pichia pastoris methanol utilization slow (Mut(s)) phenotype. This control algorithm has allowed the study of the effect of methanol concentration, ranging from 0.5 to 1.75 g/L, on heterologous protein production. The maximal lipolytic activity (490 UA/mL), specific yield (11,236 UA/g(biomass)), productivity (4,901 UA/L . h), and specific productivity (112 UA/g(biomass)h were reached for a methanol concentration of 1 g/L. These parameters are almost double than those obtained with a manual control at a similar methanol set-point. The study of the specific growth, consumption, and production rates showed different patterns for these rates depending on the methanol concentration set-point. Results obtained have shown the need of implementing a robust control scheme when reproducible quality and productivity are sought. It has been demonstrated that the model-based control proposed here is a very efficient, robust, and easy-to-implement strategy from an industrial application point of view. (c) 2006 Wiley Periodicals, Inc.

  17. CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

    Science.gov (United States)

    Poeschla, Eric M.; Looney, David J.

    1998-01-01

    A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors. PMID:9658135

  18. Introduction of caveolae structural proteins into the protozoan Toxoplasma results in the formation of heterologous caveolae but not caveolar endocytosis.

    Directory of Open Access Journals (Sweden)

    Bao Lige

    Full Text Available Present on the plasma membrane of most metazoans, caveolae are specialized microdomains implicated in several endocytic and trafficking mechanisms. Caveolins and the more recently discovered cavins are the major protein components of caveolae. Previous studies reported that caveolar invaginations can be induced de novo on the surface of caveolae-negative mammalian cells upon heterologous expression of caveolin-1. However, it remains undocumented whether other components in the transfected cells participate in caveolae formation. To address this issue, we have exploited the protozoan Toxoplasma as a heterologous expression system to provide insights into the minimal requirements for caveogenesis and caveolar endocytosis. Upon expression of caveolin-1, Toxoplasma accumulates prototypical exocytic caveolae 'precursors' in the cytoplasm. Toxoplasma expressing caveolin-1 alone, or in conjunction with cavin-1, neither develops surface-located caveolae nor internalizes caveolar ligands. These data suggest that the formation of functional caveolae at the plasma membrane in Toxoplasma and, by inference in all non-mammalian cells, requires effectors other than caveolin-1 and cavin-1. Interestingly, Toxoplasma co-expressing caveolin-1 and cavin-1 displays an impressive spiraled network of membranes containing the two proteins, in the cytoplasm. This suggests a synergistic activity of caveolin-1 and cavin-1 in the morphogenesis and remodeling of membranes, as illustrated for Toxoplasma.

  19. Neonatal BCG vaccination is associated with enhanced T-helper 1 immune responses to heterologous infant vaccines

    Directory of Open Access Journals (Sweden)

    Daniel H. Libraty

    2014-01-01

    Full Text Available Neonatal Bacille Calmette Guérin (BCG vaccination has been reported to have beneficial effects beyond preventing infantile tuberculous meningitis and miliary disease. We hypothesized that BCG vaccine given at birth would enhance T-helper 1 (Th1 immune responses to the first vaccines given later in infancy. We conducted a nested case-control study of neonatal BCG vaccination and its heterologous Th1 immune effects in 2–3 months old infants. BCG vaccination at birth was associated with an increased frequency of interferon-γ (IFN-γ producing spot-forming cells (SFC to tetanus toxoid 2–3 months later. The frequency of IFN-γ producing SFC to polioviruses 1–3 also trended higher among infants who received BCG vaccination at birth. The frequency of IFN-γ+/tumor necrosis factor-α (TNF-α+CD45RO+CD4+ T-cells upon stimulation with phorbol myristate acetate (PMA/Ionomycin was higher in 2–3 months old infants who received BCG vaccination at birth compared to those who did not. The circulating frequency of forkhead box P3 (FoxP3+ CD45RO+ regulatory CD4+ T-cells also trended lower in these infants. Neonatal BCG vaccination is associated with heterologous Th1 immune effects 2–3 months later.

  20. Neonatal BCG vaccination is associated with enhanced T-helper 1 immune responses to heterologous infant vaccines.

    Science.gov (United States)

    Libraty, Daniel H; Zhang, Lei; Woda, Marcia; Acosta, Luz P; Obcena, Anamae; Brion, Job D; Capeding, Rosario Z

    2014-01-01

    Neonatal Bacille Calmette Guérin (BCG) vaccination has been reported to have beneficial effects beyond preventing infantile tuberculous meningitis and miliary disease. We hypothesized that BCG vaccine given at birth would enhance T-helper 1 (Th1) immune responses to the first vaccines given later in infancy. We conducted a nested case-control study of neonatal BCG vaccination and its heterologous Th1 immune effects in 2-3 months old infants. BCG vaccination at birth was associated with an increased frequency of interferon-γ (IFN-γ) producing spot-forming cells (SFC) to tetanus toxoid 2-3 months later. The frequency of IFN-γ producing SFC to polioviruses 1-3 also trended higher among infants who received BCG vaccination at birth. The frequency of IFN-γ+/tumor necrosis factor-α (TNF-α)+CD45RO+CD4+ T-cells upon stimulation with phorbol myristate acetate (PMA)/Ionomycin was higher in 2-3 months old infants who received BCG vaccination at birth compared to those who did not. The circulating frequency of forkhead box P3 (FoxP3)+ CD45RO+ regulatory CD4+ T-cells also trended lower in these infants. Neonatal BCG vaccination is associated with heterologous Th1 immune effects 2-3 months later.

  1. Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production.

    Directory of Open Access Journals (Sweden)

    Adelene Ai-Lian Song

    Full Text Available Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus. A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain's endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR, an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25-1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

  2. Percutaneous CT-Guided Core Needle Biopsy Versus Fine Needle Aspiration in Diagnosing Pneumonia and Mimics of Pneumonia

    International Nuclear Information System (INIS)

    Thanos, Loukas; Galani, Panagiota; Mylona, Sophia; Pomoni, Maria; Mpatakis, Nikolaos

    2004-01-01

    The purpose of this study was to determine the diagnostic value of percutaneous core needle biopsy (CNB) relative to fine needle aspiration (FNA) in patients with pneumonia and pneumonia mimics. In this prospective study we present our experience with 48 thoracic FNAs and CNBs carried out on 48 patients with pneumonia and pneumonia mimics. Samples were obtained from all patients using both CNB (with an automated 18-G core biopsy needle and a gun) and FNA (with a 22-G needle). A specific diagnosis was made in 10/48 cases (20.83%) by FNA and in 42/48 (87.5%) by CNB. The main complications encountered were pneumothorax (n = 4) and hemoptysis (n = 2), yielding a total complication rate of 12.5%. We concluded that CNB using an automated biopsy gun results in a higher diagnostic accuracy for pneumonia and pneumonia mimic biopsies than FNA. Complications should be considered and proper patient observation should follow the procedure

  3. Engineering poly(hydroxy butyrate-co-hydroxy valerate) based vascular scaffolds to mimic native artery.

    Science.gov (United States)

    Deepthi, S; Nivedhitha Sundaram, M; Vijayan, Ponni; Nair, Shantikumar V; Jayakumar, R

    2018-04-01

    Electrospun tri-layered fibrous scaffold incorporating VEGF and Platelet Factor Concentrate (PFC) in multiple layers having different layer architectures was designed to mimic native artery. The scaffold consisted of longitudinally aligned poly(hydroxy butyrate-co-hydroxy valerate) (PHBV) and poly(vinyl alcohol) (PVA) nanofibers (inner layer), radially aligned PHBV-elastin nanofibers (middle layer) to provide the bi-directional alignment and combination of longitudinally aligned PHBV-elastin and random PHBV/PVA multiscale fibers (peripheral layer). Tubular constructs of diameter <6 mm were developed. The developed electrospun fibers were characterised by Scanning Electron Microscope (SEM), Fourier Transform Infrared Spectroscopy and Tensile tests. Further the burst strength, compliance and stiffness index of tri-layered tubular scaffold was evaluated. SEM images of fibrous layers showed the typical longitudinal and radial alignment of fibers in the tubular construct. SEM images showed that the prepared PHBV nanofibers were in the range of 500-800 nm and PHBV microfibers were of 1-2 μm in diameter in the tri-layered electrospun membrane. PVA nanofibers were of size 200-250 nm. The tensile strength, percentage compliance and stiffness index of tri-layered membrane was in accordance with that of native small blood vessels. The developed tri-layered membrane was blood compatible, with hemolysis degree 0.85 ± 0.21% and did not activate platelets. Controlled release of VEGF and PFC was observed from the scaffold. The biocompatibility of the tri-layered scaffold was evaluated using HUVECs, SMCs and MSCs and SMCs infiltration from the outer layer was also evaluated. Specific protein expression for the HUVECs and SMCs was evaluated by flow cytometry and immunocytochemistry. HUVECs and SMCs exhibited good elongation and alignment along the direction of fibers and was found to maintain its CD31, VE-Cadherin and αSMA expression respectively. The results

  4. Orbital interactions and charge redistribution in weak hydrogen bonds: The Watson-Crick AT mimic adenine-2,4-difluorotoluene

    NARCIS (Netherlands)

    Fonseca Guerra, C.; Bickelhaupt, F.M.

    2003-01-01

    An overview is given of results that reestablish hydrogen bonding as an essential factor in DNA replication involving natural bases as well as less polar mimics and they also confirm the importance of steric factors, in line with Kool's experimental work. In addition they show that knowledge of the

  5. High-fidelity in vivo replication of DNA base shape mimics without Watson–Crick hydrogen bonds

    Science.gov (United States)

    Delaney, James C.; Henderson, Paul T.; Helquist, Sandra A.; Morales, Juan C.; Essigmann, John M.; Kool, Eric T.

    2003-01-01

    We report studies testing the importance of Watson–Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells. Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria. Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions. Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity. Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity. The data establish that Watson–Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo. The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism. PMID:12676985

  6. Enzyme active site mimics based on TriAzaCyclophane (TAC)-scaffolded peptides and amino acid residues

    NARCIS (Netherlands)

    Albada, H.B.

    2009-01-01

    This thesis describes the scope and limitations of the application of TriAzaCyclophane (TAC)-scaffolded peptides or amino acid residues as enzyme active site mimics, as ligands in asymmetric catalysis and as hydrolysis catalysts attached to vancomycin. For the mimicry of functional group enzymes, of

  7. Retention of robot-assisted surgical skills in urological surgeons acquired using Mimic dV-Trainer.

    Science.gov (United States)

    Teishima, Jun; Hattori, Minoru; Inoue, Shogo; Ikeda, Kenichiro; Hieda, Keisuke; Ohara, Shinya; Egi, Hiroyuki; Ohdan, Hideki; Matsubara, Akio

    2014-07-01

    We assess the retention of robot-assisted surgical skills among urologic surgeons. The robot-assisted surgery skills of 20 urologic surgeons were assessed using a Mimic dV-Trainer program (Mimic Technologies, Inc., Seattle, WA) consisting of 6 tasks. These 20 surgeons had no previous experience either using the Mimic dV-Trainer or acting as the main surgeon in robot-assisted surgery. The surgeons completed the program 4 times in a row; after 1 year, they completed it again for a fifth time. Performance scores were recorded using the Mimic dV-Trainer's built-in algorithm. For all 6 tasks, there were significant improvements to the scores in the fourth trials compared with those in the first trials. The scores in the fifth trials did not significantly decline compared with those in the fourth trials. There was no significant difference between the fifth trial scores of surgeons with laparoscopic surgery skills/experience and those without. Our results indicate that fundamental robot-assisted surgical skills can be retained in the long-term after they are acquired.

  8. MiR-26b Mimic Inhibits Glioma Proliferation In Vitro and In Vivo Suppressing COX-2 Expression.

    Science.gov (United States)

    Chen, Zheng-Gang; Zheng, Chuan-Yi; Cai, Wang-Qing; Li, Da-Wei; Ye, Fu-Yue; Zhou, Jian; Wu, Ran; Yang, Kun

    2017-08-11

    Glioma is the most common malignant tumor of the nervous system. Studies have shown the microRNA (miR)-26b/cyclooxygenase (COX)-2 axis in the development and progression in many tumor cells. Our study aims to investigate the effect and mechanism of miR-26b/COX-2 axis in glioma. Decreased expression of miR-26b with increased level of COX-2 was found in glioma tissues compared with matched normal tissues. A strong negative correlation was observed between the level of miR-26b and COX-2 in 30 glioma tissues. The miR-26b was then overexpressed by transfecting miR-26b mimic into U-373 cells. The invasive cell number and wounld closing rate were reduced in U-373 cells transfected with miR-26b mimic. Besides, COX2 siRNA enhanced the effect of miR-26b mimic in suppressing the expression of p-ERK1 and p-JNK. Finally, the in vivo experiment revealed that miR-26b mimic transfection strongly reduced the tumor growth, tumor volume and the expression of matrix metalloproteinase (MMP)-2, MMP-9). Taken together, our research indicated a miR-26b/COX-2/ERK/JNK axis in regulating the motility of glioma in vitro and in vivo, providing a new sight for treatment of glioma.

  9. Insights into molecular recognition of LewisX mimics by DC-SIGN using NMR and molecular modelling

    International Nuclear Information System (INIS)

    Guzzi, C.; Angulo, J.; Rojo, J.; Nieto, P.M.; Doro, F.; Bernardi, A.; Thepaut, M.; Fieschi, F.; Thepaut, M.; Fieschi, F.

    2011-01-01

    In this work, we have studied in detail the binding of two α-fucosyl-amide-based mimics of Lewis X to DC-SIGN ECD (ECD = extracellular domain) using STD NMR and docking. We have concluded that the binding mode occurs mainly through the fucose moiety, in the same way as Lewis X . Similarly to other mimics containing mannose or fucose previously studied, we have shown that both compounds bind to DC-SIGN ECD in a multimodal fashion. In this case, the main contact is the interaction of two hydroxyl groups one equatorial and the other one axial (O3 and O4) of the fucose with the Ca 2+ as Lewis X and similarly to mannose-containing mimics (in this case the interacting groups are both in the equatorial position). Finally, we have measured the K D of one mimic that was 0.4 mM. Competitive STD NMR experiments indicate that the aromatic moiety provides additional binding contacts that increase the affinity. (authors)

  10. Mixture Item Response Theory-MIMIC Model: Simultaneous Estimation of Differential Item Functioning for Manifest Groups and Latent Classes

    Science.gov (United States)

    Bilir, Mustafa Kuzey

    2009-01-01

    This study uses a new psychometric model (mixture item response theory-MIMIC model) that simultaneously estimates differential item functioning (DIF) across manifest groups and latent classes. Current DIF detection methods investigate DIF from only one side, either across manifest groups (e.g., gender, ethnicity, etc.), or across latent classes…

  11. Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer.

    Science.gov (United States)

    Daige, Christopher L; Wiggins, Jason F; Priddy, Leslie; Nelligan-Davis, Terri; Zhao, Jane; Brown, David

    2014-10-01

    miR34a is a tumor-suppressor miRNA that functions within the p53 pathway to regulate cell-cycle progression and apoptosis. With apparent roles in metastasis and cancer stem cell development, miR34a provides an interesting opportunity for therapeutic development. A mimic of miR34a was complexed with an amphoteric liposomal formulation and tested in two different orthotopic models of liver cancer. Systemic dosing of the formulated miR34a mimic increased the levels of miR34a in tumors by approximately 1,000-fold and caused statistically significant decreases in the mRNA levels of several miR34a targets. The administration of the formulated miR34a mimic caused significant tumor growth inhibition in both models of liver cancer, and tumor regression was observed in more than one third of the animals. The antitumor activity was observed in the absence of any immunostimulatory effects or dose-limiting toxicities. Accumulation of the formulated miR34a mimic was also noted in the spleen, lung, and kidney, suggesting the potential for therapeutic use in other cancers. ©2014 American Association for Cancer Research.

  12. The pattern recognition molecule deleted in malignant brain tumors 1 (DMBT1) and synthetic mimics inhibit liposomal nucleic acid delivery

    DEFF Research Database (Denmark)

    Lund Hansen, Pernille; Blaich, Stephanie; End, Caroline

    2011-01-01

    Liposomal nucleic acid delivery is a preferred option for therapeutic settings. The cellular pattern recognition molecule DMBT1, secreted at high levels in various diseases, and synthetic mimics efficiently inhibit liposomal nucleic acid delivery to human cells. These findings may have relevance...

  13. Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process.

    Science.gov (United States)

    Yin, Shouliang; Li, Zilong; Wang, Xuefeng; Wang, Huizhuan; Jia, Xiaole; Ai, Guomin; Bai, Zishang; Shi, Mingxin; Yuan, Fang; Liu, Tiejun; Wang, Weishan; Yang, Keqian

    2016-12-01

    Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer Streptomyces rimosus M4018 in 8 days. This work shows that S. venezuelae WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.

  14. Heterologous expression of a rice metallothionein isoform (OsMTI-1b in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance

    Directory of Open Access Journals (Sweden)

    Zahra Ansarypour

    Full Text Available Abstract Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b on the tolerance of Saccharomyces cerevisiae to Cd2+, H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd2+ and accumulated more Cd2+ ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

  15. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of

  16. Comparison of immune responses after intra-typic heterologous and homologous vaccination against foot-and-mouth disease virus infection in pigs

    NARCIS (Netherlands)

    Eble, P.L.; Bruin, de M.G.M.; Bouma, A.; Hemert-Kluitenberg, van F.; Dekker, A.

    2006-01-01

    This study compares the immune responses and protection induced by intra-typic heterologous vaccination with that induced by homologous vaccination against challenge with foot-and-mouth disease virus (FMDV). Humoral and cell-mediated immune responses and protection against challenge with FMDV O

  17. Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae.

    Science.gov (United States)

    Zhu, Lin; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2013-07-01

    The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.

  18. The L polymerase protein of parainfluenza virus 3 forms an oligomer and can interact with the heterologous Sendai virus L, P and C proteins

    International Nuclear Information System (INIS)

    Smallwood, Sherin; Moyer, Sue A.

    2004-01-01

    We recently showed that the L protein of Sendai virus is present as an oligomer in the active P-L polymerase complex [Smallwood et al., Virology 304 (2002) 235]. We now demonstrate using two different epitope tags that the L protein of a second respirovirus, human parainfluenza type 3 virus (PIV3), also forms an L-L complex. L oligomerization requires the coexpression of the differentially epitope tagged L proteins. By exploiting a series of C-terminal truncations the L-L binding site maps to the N-terminal half of L. There is some complex formation between the heterologous PIV3 and Sendai L and P proteins; however, the heterologous L protein does not function in transcription of either the PIV3 or Sendai template. The PIV3 C protein binds PIV3 L and inhibits RNA synthesis in vitro and in vivo. Significant homology exists between the C proteins of PIV3 and Sendai and complex formation occurs between the PIV3 and Sendai heterologous C and L proteins. In addition, the heterologous C proteins can inhibit transcription at ∼50% of the level of the homologous protein. These data suggest that while the C proteins may be functionally somewhat interchangeable, the L and P proteins are specific for each virus

  19. Effect of heterologous expression of acyl-CoA-binding protein on acyl-CoA level and composition in yeast

    DEFF Research Database (Denmark)

    Mandrup, S; Jepsen, R; Skøtt, H

    1993-01-01

    We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovi...

  20. Can f(T) gravity theories mimic ΛCDM cosmic history

    Energy Technology Data Exchange (ETDEWEB)

    Setare, M.R.; Mohammadipour, N., E-mail: rezakord@ipm.ir, E-mail: N.Mohammadipour@uok.ac.ir [Department of Science, University of Kurdistan, Sanandaj (Iran, Islamic Republic of)

    2013-01-01

    Recently the teleparallel Lagrangian density described by the torsion scalar T has been extended to a function of T. The f(T) modified teleparallel gravity has been proposed as the natural gravitational alternative for dark energy to explain the late time acceleration of the universe. In order to reconstruct the function f(T) by demanding a background ΛCDM cosmology we assume that, (i) the background cosmic history provided by the flat ΛCDM (the radiation ere with ω{sub eff} = (1/3), matter and de Sitter eras with ω{sub eff} = 0 and ω{sub eff} = −1, respectively) (ii) the radiation dominate in the radiation era with Ω{sub 0r} = 1 and the matter dominate during the matter phases when Ω{sub 0m} = 1. We find the cosmological dynamical system which can obey the ΛCDM cosmic history. In each era, we find a critical lines that, the radiation dominated and the matter dominated are one points of them in the radiation and matter phases, respectively. Also, we drive the cosmologically viability condition for these models. We investigate the stability condition with respect to the homogeneous scalar perturbations in each era and we obtain the stability conditions for the fixed points in each eras. Finally, we reconstruct the function f(T) which mimics cosmic expansion history.

  1. Three-dimensional culture of buffalo granulosa cells in hanging drop mimics the preovulatory follicle stage.

    Science.gov (United States)

    Yadav, Monica; Agrawal, Himanshu; Pandey, Mamta; Singh, Dheer; Onteru, Suneel K

    2018-03-01

    Granulosa cell (GC) culture models mimicking the intrafollicular environment are limited. Such models have a great potential in reproductive toxicity studies. The buffalo, a monovulatory species like humans, could be a better model than polyovulatory rodents. Therefore, we targeted the development and characterization of three-dimensional (3D) culture systems for buffalo GCs. The GCs from small ovarian follicles (SF) maintained the CYP19 gene expression for 144 hr in a 2D culture system. Hence, GCs from SF were cultured directly in 3D using hanging drop and Poly-([2-hydroxyethyl methacrylate]) (polyHEMA) methods in the DMEM media containing 1 ng/ml FSH and 10 ng/ml IGF-1 for 144 hr. The expression profile of nine GC-specific transcripts; CYP19, TNFAIP6, AMH, PTI, NR4A1, FSHR, RUNX, LHR, and COX2/PTGS2; revealed that 3D-spheroids developed in hanging drop method maintained the GC phenotype of preovulatory follicles. Therefore, hanging drop method is a best method for culturing GCs to mimic the intrafollicular environment. © 2017 Wiley Periodicals, Inc.

  2. Secretory carcinoma of the breast and its histopathological mimics: value of markers for differential diagnosis.

    Science.gov (United States)

    Osako, Tomo; Takeuchi, Kengo; Horii, Rie; Iwase, Takuji; Akiyama, Futoshi

    2013-10-01

    Secretory carcinoma (SC) is a rare histological type of breast cancer, and ETV6-NTRK3 gene fusion is highly specific to it. The differential diagnoses of SC include acinic cell carcinoma (ACCA) and cystic hypersecretory carcinoma (CHC), as well as invasive ductal carcinoma (IDC). For patients with these rare but distinctive histological subtypes, SC and its histopathological mimics should be differentiated from each other. However, differential markers have not yet been assessed systematically, and we aimed to identify and evaluate novel and existing markers. We reviewed 19 cases diagnosed initially as SC using integrated diagnostic techniques, including morphology, immunohistochemistry and molecular pathology, and validated promising markers in 445 breast cancers. We reclassified 19 formerly diagnosed 'SCs' into nine SCs, three ACCAs, three CHCs, three IDCs and one microglandular adenosis. We confirmed that ETV6-NTRK3 gene rearrangement and amylase positivity are good diagnostic markers for SC and ACCA, respectively. Vacuolar staining for adipophilin, positivity for α-lactalbumin and negativity for ETV6 rearrangement are diagnostic markers for CHC. In this study, we propose a panel of four markers (ETV6 rearrangement, amylase, α-lactalbumin and adipophilin) for distinguishing SC, ACCA, CHC and IDC. This simple but robust panel will serve pathologists well as a practical guide for reaching an appropriate diagnosis. © 2013 John Wiley & Sons Ltd.

  3. Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection.

    Science.gov (United States)

    Kolb, Alexander L; Corridon, Peter R; Zhang, Shijun; Xu, Weimin; Witzmann, Frank A; Collett, Jason A; Rhodes, George J; Winfree, Seth; Bready, Devin; Pfeffenberger, Zechariah J; Pomerantz, Jeremy M; Hato, Takashi; Nagami, Glenn T; Molitoris, Bruce A; Basile, David P; Atkinson, Simon J; Bacallao, Robert L

    2018-04-01

    Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine ( P <0.05) observed in controls and increased the mitochondria membrane potential ( P <0.05), maximal respiratory capacity ( P <0.05), and intracellular ATP levels ( P <0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemia-reperfusion injury and mimics ischemic preconditioning. Copyright © 2018 by the American Society of Nephrology.

  4. Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland.

    Science.gov (United States)

    Johnson, Michael D; Kenney, Nicholas; Stoica, Adriana; Hilakivi-Clarke, Leena; Singh, Baljit; Chepko, Gloria; Clarke, Robert; Sholler, Peter F; Lirio, Apolonio A; Foss, Colby; Reiter, Ronald; Trock, Bruce; Paik, Soonmyoung; Martin, Mary Beth

    2003-08-01

    It has been suggested that environmental contaminants that mimic the effects of estrogen contribute to disruption of the reproductive systems of animals in the wild, and to the high incidence of hormone-related cancers and diseases in Western populations. Previous studies have shown that functionally, cadmium acts like steroidal estrogens in breast cancer cells as a result of its ability to form a high-affinity complex with the hormone binding domain of the estrogen receptor. The results of the present study show that cadmium also has potent estrogen-like activity in vivo. Exposure to cadmium increased uterine wet weight, promoted growth and development of the mammary glands and induced hormone-regulated genes in ovariectomized animals. In the uterus, the increase in wet weight was accompanied by proliferation of the endometrium and induction of progesterone receptor (PgR) and complement component C3. In the mammary gland, cadmium promoted an increase in the formation of side branches and alveolar buds and the induction of casein, whey acidic protein, PgR and C3. In utero exposure to the metal also mimicked the effects of estrogens. Female offspring experienced an earlier onset of puberty and an increase in the epithelial area and the number of terminal end buds in the mammary gland.

  5. Cultivation of Human Microvascular Endothelial Cells on Topographical Substrates to Mimic the Human Corneal Endothelium

    Directory of Open Access Journals (Sweden)

    Jie Shi Chua

    2013-03-01

    Full Text Available Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet’s membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+ adenine triphosphatase (ATPase expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.

  6. SIRT1 Gain of Function Does Not Mimic or Enhance the Adaptations to Intermittent Fasting

    Directory of Open Access Journals (Sweden)

    Marie Boutant

    2016-03-01

    Full Text Available Caloric restriction (CR has been shown to prevent the onset of insulin resistance and to delay age-related physiological decline in mammalian organisms. SIRT1, a NAD+-dependent deacetylase enzyme, has been suggested to mediate the adaptive responses to CR, leading to the speculation that SIRT1 activation could be therapeutically used as a CR-mimetic strategy. Here, we used a mouse model of moderate SIRT1 overexpression to test whether SIRT1 gain of function could mimic or boost the metabolic benefits induced by every-other-day feeding (EODF. Our results indicate that SIRT1 transgenesis does not affect the ability of EODF to decrease adiposity and improve insulin sensitivity. Transcriptomic analyses revealed that SIRT1 transgenesis and EODF promote very distinct adaptations in individual tissues, some of which can be even be metabolically opposite, as in brown adipose tissue. Therefore, whereas SIRT1 overexpression and CR both improve glucose metabolism and insulin sensitivity, the etiologies of these benefits are largely different.

  7. SIRT1 Gain of Function Does Not Mimic or Enhance the Adaptations to Intermittent Fasting.

    Science.gov (United States)

    Boutant, Marie; Kulkarni, Sameer S; Joffraud, Magali; Raymond, Frédéric; Métairon, Sylviane; Descombes, Patrick; Cantó, Carles

    2016-03-08

    Caloric restriction (CR) has been shown to prevent the onset of insulin resistance and to delay age-related physiological decline in mammalian organisms. SIRT1, a NAD(+)-dependent deacetylase enzyme, has been suggested to mediate the adaptive responses to CR, leading to the speculation that SIRT1 activation could be therapeutically used as a CR-mimetic strategy. Here, we used a mouse model of moderate SIRT1 overexpression to test whether SIRT1 gain of function could mimic or boost the metabolic benefits induced by every-other-day feeding (EODF). Our results indicate that SIRT1 transgenesis does not affect the ability of EODF to decrease adiposity and improve insulin sensitivity. Transcriptomic analyses revealed that SIRT1 transgenesis and EODF promote very distinct adaptations in individual tissues, some of which can be even be metabolically opposite, as in brown adipose tissue. Therefore, whereas SIRT1 overexpression and CR both improve glucose metabolism and insulin sensitivity, the etiologies of these benefits are largely different. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Gsk-3β Inhibitors Mimic the Cardioprotection Mediated by Ischemic Pre- and Postconditioning in Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    Luisa F. González Arbeláez

    2013-01-01

    Full Text Available The aim of this study was to examine the effects of GSK-3β inhibitors compared with PRE and POS in spontaneously hypertensive rats (SHR. Isolated hearts were submitted to the following protocols: IC: 45 min global ischemia (GI and 1-hour reperfusion (R; PRE: a cycle of 5 min GI and 10 minutes of R prior to 45 min GI; POS: three cycles of 30 sec GI/30 sec R at the start of R. Other hearts received lithium chloride (LiCl or indirubin-3′-monoxime,5-iodo-(IMI as GSK-3β inhibitors. All interventions reduced the infarct size observed in IC group. The expressions of P-GSK-3β and P-Akt decreased in IC and were restored after PRE, POS, and GSK-3β inhibitors treatments. An increase of cytosolic MnSOD activity and lipid peroxidation and a decrease of GSH content observed in IC hearts were attenuated in PRE, POS, and LiCl or IMI treatments. An increase of P-GSK-3β/VDAC physical association and a partial recovery of mitochondrial permeability were also detected after interventions. These data show that, in SHR hearts, GSK-3β inhibitors mimic the cardioprotection afforded by PRE and POS and suggest that a decrease in mitochondrial permeability mediated by P-GSK-3β/VDAC interaction is a crucial event.

  9. A case report of gastric lymphocytic phlebitis, a rare mimic for malignancy.

    Science.gov (United States)

    Chan, Daniel L; Ravindran, Praveen; Chua, Dorothy; Smith, Jason D; Wong, King S; Ghusn, Michael A

    2017-01-01

    Lymphocytic phlebitis is a benign condition characterised by inflammation of the veins and rarely affects the gastrointestinal tract. Reported cases present as acute abdomen and involve the colon or small intestine. We report the fourth case of gastric lymphocytic phlebitis in the literature. A 74-year-old female presented with eight weeks of abdominal pain. Findings at endoscopy were suggestive of a malignant ulcer on the greater curvature of antrum, while biopsies showed chronic gastritis without malignancy. Appearance at diagnostic laparoscopy was consistent with a malignant gastric ulcer with serosal changes. Due to persistent pain and the macroscopic appearance, she proceeded to have an open subtotal gastrectomy and D2 lymph node clearance. Despite macroscopic appearance, the microscopic examination demonstrated no malignancy, and was consistent with lymphocytic phlebitis with overlying ulceration. This case was a mimic for gastric malignancy, with the benign diagnosis only being made after surgical resection. Gastric lymphocytic phlebitis is a rare differential diagnosis for gastric ulcers when biopsies are negative, although preoperative diagnosis is difficult given the lesions do not involve the mucosa. If clinical history and endoscopic findings are suspicious for malignancy, despite normal biopsies, an aggressive surgical resection remains reasonable given the rarity gastric lymphocytic phlebitis. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. A case report of gastric lymphocytic phlebitis, a rare mimic for malignancy

    Directory of Open Access Journals (Sweden)

    Daniel L. Chan

    Full Text Available Introduction: Lymphocytic phlebitis is a benign condition characterised by inflammation of the veins and rarely affects the gastrointestinal tract. Reported cases present as acute abdomen and involve the colon or small intestine. We report the fourth case of gastric lymphocytic phlebitis in the literature. Presentation of case: A 74-year-old female presented with eight weeks of abdominal pain. Findings at endoscopy were suggestive of a malignant ulcer on the greater curvature of antrum, while biopsies showed chronic gastritis without malignancy. Appearance at diagnostic laparoscopy was consistent with a malignant gastric ulcer with serosal changes. Due to persistent pain and the macroscopic appearance, she proceeded to have an open subtotal gastrectomy and D2 lymph node clearance. Despite macroscopic appearance, the microscopic examination demonstrated no malignancy, and was consistent with lymphocytic phlebitis with overlying ulceration. Discussion: This case was a mimic for gastric malignancy, with the benign diagnosis only being made after surgical resection. Gastric lymphocytic phlebitis is a rare differential diagnosis for gastric ulcers when biopsies are negative, although preoperative diagnosis is difficult given the lesions do not involve the mucosa. Conclusion: If clinical history and endoscopic findings are suspicious for malignancy, despite normal biopsies, an aggressive surgical resection remains reasonable given the rarity gastric lymphocytic phlebitis. Keywords: Lymphocytic phlebitis, Gastrectomy, Gastric vasculitis, Gastric ulcer, Case report

  11. Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies

    Directory of Open Access Journals (Sweden)

    Robert Root-Bernstein

    2017-10-01

    Full Text Available Human immunodeficiency virus (HIV hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-β-D-J-β of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR. This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS. Quantitative enzyme-linked immunoadsorption assays (ELISA demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10−8 to 10−9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.

  12. A model system to mimic environmentally active surface film roughness and hydrophobicity.

    Science.gov (United States)

    Grant, Jacob S; Shaw, Scott K

    2017-10-01

    This work presents the development and initial assessment of a laboratory platform to allow quantitative studies on model urban films. The platform consists of stearic acid and eicosane mixtures that are solution deposited from hexanes onto smooth, solid substrates. We show that this model has distinctive capabilities to better mimic a naturally occurring film's morphology and hydrophobicity, two important parameters that have not previously been incorporated into model film systems. The physical and chemical properties of the model films are assessed using a variety of analytical instruments. The film thickness and roughness are probed via atomic force microscopy while the film composition, wettability, and water uptake are analyzed by Fourier transform infrared spectroscopy, contact angle goniometry, and quartz crystal microbalance, respectively. Simulated environmental maturation is achieved by exposing the film to regulated amounts of UV/ozone. Ultimately, oxidation of the film is monitored by the analytical techniques mentioned above and proceeds as expected to produce a utile model film system. Including variable roughness and tunable surface coverage results in several key advantages over prior model systems, and will more accurately represent native urban film behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Unofficial Economy Estimation by the MIMIC Model: the Case of Kenya, Namibia, Ghana and Nigeria

    Directory of Open Access Journals (Sweden)

    Dennis Nchor

    2015-01-01

    Full Text Available This study investigates the size and trend of the underground economies in selected African countries. Underground economies are present in all countries, but they are endemic in developing economies. Their presence is not necessarily bad for the economies, in which they prevail. It could however cause huge losses to government revenue and could also constitute serious violation of Labor regulations. The study uses the Multiple Indicators and Multiple Causes model (MIMIC, a variant of Simultaneous Equations Model (SEM. It involves two sets of variables: the observed variables and the indicator variables. The former include size of government, indirect tax rates, total tax rates, business regulation, interest rate on deposits, unemployment rate, quality of public services, and GDP per capita. The indicator variables were Labor participation rate in the official economy, the amount of cash held outside the banking system and growth in GDP per capita. This study found the average level of underground economies in Kenya, Namibia, Ghana and Nigeria as 33.7%, 29.1%, 36% and 47%, respectively. The estimated results show that the causes of shadow economic activities vary among the countries. The data was obtained from the World Bank country indicators and the International Financial Statistics.

  14. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

    Science.gov (United States)

    Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

    2012-11-01

    The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-β peptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

  15. Peptidoglycan glycosyltransferase substrate mimics as templates for the design of new antibacterial drugs

    Directory of Open Access Journals (Sweden)

    Adeline eDerouaux

    2013-03-01

    Full Text Available Peptidoglycan (PG is an essential net-like macromolecule that surrounds bacteria, gives them their shape, and protects them against their own high osmotic pressure. PG synthesis inhibition leads to bacterial cell lysis, making it an important target for many antibiotics. The final two reactions in PG synthesis are performed by penicillin-binding proteins (PBPs. Their glycosyltransferase (GT activity uses the lipid II precursor to synthesize glycan chains and their transpeptidase (TP activity catalyzes the cross-linking of two glycan chains via the peptide side chains. Inhibition of either of these two reactions leads to bacterial cell death. β-Lactam antibiotics target the transpeptidation reaction while antibiotic therapy based on inhibition of the GTs remains to be developed. Ongoing research is trying to fill this gap by studying the interactions of GTs with inhibitors and substrate mimics and utilizing the latter as templates for the design of new antibiotics. In this mini review we present an updated overview on the GTs and describe the structure-activity relationship of recently developed synthetic ligands.

  16. Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, tempol

    International Nuclear Information System (INIS)

    Mitchell, J.B.; DeGraff, W.; Kaufman, D.; Krishna, M.C.; Samuni, A.; Finkelstein, E.; Ahn, M.S.; Hahn, S.M.; Gamson, J.; Russo, A.

    1991-01-01

    Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress

  17. Medical Textiles as Vascular Implants and Their Success to Mimic Natural Arteries

    Directory of Open Access Journals (Sweden)

    Charanpreet Singh

    2015-06-01

    Full Text Available Vascular implants belong to a specialised class of medical textiles. The basic purpose of a vascular implant (graft and stent is to act as an artificial conduit or substitute for a diseased artery. However, the long-term healing function depends on its ability to mimic the mechanical and biological behaviour of the artery. This requires a thorough understanding of the structure and function of an artery, which can then be translated into a synthetic structure based on the capabilities of the manufacturing method utilised. Common textile manufacturing techniques, such as weaving, knitting, braiding, and electrospinning, are frequently used to design vascular implants for research and commercial purposes for the past decades. However, the ability to match attributes of a vascular substitute to those of a native artery still remains a challenge. The synthetic implants have been found to cause disturbance in biological, biomechanical, and hemodynamic parameters at the implant site, which has been widely attributed to their structural design. In this work, we reviewed the design aspect of textile vascular implants and compared them to the structure of a natural artery as a basis for assessing the level of success as an implant. The outcome of this work is expected to encourage future design strategies for developing improved long lasting vascular implants.

  18. To die or not to die? Lessons from lesion mimic mutants

    KAUST Repository

    Bruggeman, Quentin

    2015-01-30

    Programmed cell death (PCD) is a ubiquitous genetically regulated process consisting in an activation of finely controlled signaling pathways that lead to cellular suicide. Although some aspects of PCD control appear evolutionary conserved between plants, animals and fungi, the extent of conservation remains controversial. Over the last decades, identification and characterization of several lesion mimic mutants (LMM) has been a powerful tool in the quest to unravel PCD pathways in plants. Thanks to progress in molecular genetics, mutations causing the phenotype of a large number of LMM and their related suppressors were mapped, and the identification of the mutated genes shed light on major pathways in the onset of plant PCD such as (i) the involvements of chloroplasts and light energy, (ii) the roles of sphingolipids and fatty acids, (iii) a signal perception at the plasma membrane that requires efficient membrane trafficking, (iv) secondary messengers such as ion fluxes and ROS and (v) the control of gene expression as the last integrator of the signaling pathways.

  19. Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

    Directory of Open Access Journals (Sweden)

    Stefanie Hausammann

    Full Text Available Inhibitory antibodies directed against coagulation factor VIII (FVIII can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

  20. A non-covalent peptide-based carrier for in vivo delivery of DNA mimics.

    Science.gov (United States)

    Morris, May C; Gros, Edwige; Aldrian-Herrada, Gudrun; Choob, Michael; Archdeacon, John; Heitz, Frederic; Divita, Gilles

    2007-01-01

    The dramatic acceleration in identification of new nucleic-acid-based therapeutic molecules has provided new perspectives in pharmaceutical research. However, their development is limited by their poor cellular uptake and inefficient trafficking. Here we describe a short amphipathic peptide, Pep-3, that combines a tryptophan/phenylalanine domain with a lysine/arginine-rich hydrophilic motif. Pep-3 forms stable nano-size complexes with peptide-nucleic acid analogues and promotes their efficient delivery into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. We demonstrate that Pep-3-mediated delivery of antisense-cyclin B1-charged-PNA blocks tumour growth in vivo upon intratumoral and intravenous injection. Moreover, we show that PEGylation of Pep-3 significantly improves complex stability in vivo and consequently the efficiency of antisense cyclin B1 administered intravenously. Given the biological characteristics of these vectors, we believe that peptide-based delivery technologies hold a true promise for therapeutic applications of DNA mimics.

  1. Catalytic nanocrystalline coordination polymers as an efficient peroxidase mimic for labeling and optical immunoassays

    International Nuclear Information System (INIS)

    Čunderlová, Veronika; Hlaváček, Antonín; Horňáková, Veronika; Peterek, Miroslav; Němeček, Daniel; Skládal, Petr; Hampl, Aleš; Eyer, Luděk

    2016-01-01

    We report that nanocrystalline Prussian blue of the type Fe 4 [Fe(CN) 6 ] 3 is a powerful peroxidase mimic for use in labeling of biomolecules. The cubic nanocrystals typically have a diameter of 15 nm and are capable of catalyzing the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine in the presence of H 2 O 2 to form an intensively colored product with an absorption maximum at 662 nm. The determined pseudo turnover number is ∼20,000 s −1 which is the highest value reported for nanoparticles of a size comparable to common proteins. We also present a method for the biotinylation of the surface of these nanocrystals, and show their use in competitive bioaffinity based assays of biotin and human serum albumin. The limits of detection are 0.35 and 0.27 μg mL −1 , respectively. The results prove the applicability of coordination polymers for signal amplification and also their compatibility with the format of enzyme linked immunosorbent assays. (author)

  2. Worm grunting, fiddling, and charming--humans unknowingly mimic a predator to harvest bait.

    Directory of Open Access Journals (Sweden)

    Kenneth C Catania

    Full Text Available BACKGROUND: For generations many families in and around Florida's Apalachicola National Forest have supported themselves by collecting the large endemic earthworms (Diplocardia mississippiensis. This is accomplished by vibrating a wooden stake driven into the soil, a practice called "worm grunting". In response to the vibrations, worms emerge to the surface where thousands can be gathered in a few hours. Why do these earthworms suddenly exit their burrows in response to vibrations, exposing themselves to predation? PRINCIPAL FINDINGS: Here it is shown that a population of eastern American moles (Scalopus aquaticus inhabits the area where worms are collected and that earthworms have a pronounced escape response from moles consisting of rapidly exiting their burrows to flee across the soil surface. Recordings of vibrations generated by bait collectors and moles suggest that "worm grunters" unknowingly mimic digging moles. An alternative possibility, that worms interpret vibrations as rain and surface to avoid drowning is not supported. CONCLUSIONS: Previous investigations have revealed that both wood turtles and herring gulls vibrate the ground to elicit earthworm escapes, indicating that a range of predators may exploit the predator-prey relationship between earthworms and moles. In addition to revealing a novel escape response that may be widespread among soil fauna, the results show that humans have played the role of "rare predators" in exploiting the consequences of a sensory arms race.

  3. To die or not to die? Lessons from lesion mimic mutants

    Directory of Open Access Journals (Sweden)

    Quentin eBruggeman

    2015-01-01

    Full Text Available Programmed cell death (PCD is a ubiquitous genetically regulated process consisting in an activation of finely controlled signaling pathways that lead to cellular suicide. Although some aspects of PCD control appear evolutionary conserved between plants, animals and fungi, the extent of conservation remains controversial. Over the last decades, identification and characterization of several lesion mimic mutants (LMM has been a powerful tool in the quest to unravel PCD pathways in plants. Thanks to progress in molecular genetics, mutations causing the phenotype of a large number of LMM and their related suppressors were mapped, and the identification of the mutated genes shed light on major pathways in the onset of plant PCD such as (i the involvements of chloroplasts and light energy, (ii the roles of sphingolipids and fatty acids, (iii a signal perception at the plasma membrane that requires efficient membrane trafficking, (iv secondary messengers such as ion fluxes and ROS and (v the control of gene expression as the last integrator of the signaling pathways.

  4. Designing Reliable Cohorts of Cardiac Patients across MIMIC and eICU

    Science.gov (United States)

    Chronaki, Catherine; Shahin, Abdullah; Mark, Roger

    2016-01-01

    The design of the patient cohort is an essential and fundamental part of any clinical patient study. Knowledge of the Electronic Health Records, underlying Database Management System, and the relevant clinical workflows are central to an effective cohort design. However, with technical, semantic, and organizational interoperability limitations, the database queries associated with a patient cohort may need to be reconfigured in every participating site. i2b2 and SHRINE advance the notion of patient cohorts as first class objects to be shared, aggregated, and recruited for research purposes across clinical sites. This paper reports on initial efforts to assess the integration of Medical Information Mart for Intensive Care (MIMIC) and Philips eICU, two large-scale anonymized intensive care unit (ICU) databases, using standard terminologies, i.e. LOINC, ICD9-CM and SNOMED-CT. Focus of this work is lab and microbiology observations and key demographics for patients with a primary cardiovascular ICD9-CM diagnosis. Results and discussion reflecting on reference core terminology standards, offer insights on efforts to combine detailed intensive care data from multiple ICUs worldwide. PMID:27774488

  5. BioMimic fabrication of electrospun nanofibers with high-throughput

    International Nuclear Information System (INIS)

    He Jihuan; Liu Yong; Xu Lan; Yu Jianyong; Sun Gang

    2008-01-01

    Spider-spun fiber is of extraordinary strength and toughness comparable to those of electrospun fiber, the later needs a very high voltage (from several thousands voltage to several ten thousands voltages) applied to water-soluble protein 'soup' that was produced by a spider, furthermore, its mechanical strength dramatically decreases comparable to spider silk. A possible mechanism in spider-spinning process is given, the distinct character in spider-spinning is that its spinneret consists of millions of nano scale tubes, and a bubble can be produced at the apex of each nano-tube. The surface tension of each bubble is extremely small such that it can be spun into nanofibers with an awfully small force, either by the spider's body weight or tension created by the rear legs. We mimic the spider-spinning in electrospinning using an aerated solution, which leads to various small bubbles on surface with very small surface tension, as a result the bubble can be easily electrospun into nanofibers with low applied voltage. This fabrication process possesses features of high productivity, versatility, in addition, the minimum diameter of nanofibers produced by this process can reach as small as 50 nm

  6. Fine-Needle Aspiration Cytology of Parathyroid Carcinoma Mimic Hürthle Cell Thyroid Neoplasm

    Directory of Open Access Journals (Sweden)

    Chutintorn Sriphrapradang

    2014-01-01

    Full Text Available Background. Fine-needle aspiration (FNA can cause misdiagnosis of cytomorphological findings between parathyroid and thyroid lesions. Case Presentation. A 31-year-old man presented with a palpable neck mass on the right thyroid lobe. FNA cytology was reported as intrathyroidal lymphoid hyperplasia. After 5 years, repeated FNA was done on the enlarged nodule with result of Hürthle cell lesion. Prior to right lobectomy, laboratories revealed elevated serum calcium and parathyroid hormone (PTH. Careful history taking revealed chronic knee pain and ossifying fibroma at the maxilla. Ultrasonography showed a 2.8 cm mass inferior to right thyroid lobe. Pathology from en bloc resection was parathyroid carcinoma and immunohistochemical study revealed positivity for PTH. Genetic analysis found somatic mutation of CDC73 gene in exon1 (c.70delG which caused premature stop codon in amino acid 26 (p.Glu24Lysfs2*. The final diagnosis was hyperparathyroidism-jaw tumor syndrome. Conclusions. FNA cytology of parathyroid can mimic thyroid lesion. It is important to consider and correlate the entire information from clinical history, laboratory, imaging, and FNA.

  7. Platelet factor 4 activity against P. falciparum and its translation to nonpeptidic mimics as antimalarials.

    Science.gov (United States)

    Love, Melissa S; Millholland, Melanie G; Mishra, Satish; Kulkarni, Swapnil; Freeman, Katie B; Pan, Wenxi; Kavash, Robert W; Costanzo, Michael J; Jo, Hyunil; Daly, Thomas M; Williams, Dewight R; Kowalska, M Anna; Bergman, Lawrence W; Poncz, Mortimer; DeGrado, William F; Sinnis, Photini; Scott, Richard W; Greenbaum, Doron C

    2012-12-13

    Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Intratemporal facial nerve neuromas and their mimics: CT and MR findings

    Energy Technology Data Exchange (ETDEWEB)

    Han, Moon Hee; Chang, Kee Hyun; Lee, Kyung Hwan; Cha, Sang Hoon; Kim, Chong Sun [Seoul National University College of Medicine, Seoul (Korea, Republic of); Kim, Sang Joon [Chungang Gil General Hospital, Seoul (Korea, Republic of)

    1992-05-15

    CT and MR findings of nine cases with intra temporal facial nerve neuromas were described and compared with CT findings of 3 cases with facial nerve palsy and facial nerve canal erosion which may mimic facial nerve neuroma. The tympanic segment of the facial nerve was involved in 8 cases, mastoid segment in 7 cases and labyrinthine segment in 5 cases. The lesions were easily diagnosed with high resolution CT with bone algorithms by showing the expansion of bony structures along the course of the facial nerves. In 4 cases with large vertical segment tumors, extensive destruction of mastoid air cells and external auditory canals posed difficulty in making a diagnosis. Two out of 5 cases with labyrinthine segment involvement were presented as middle cranial fossa masses. MRI with enhancement was performed in 4 cases and was useful in characterizing the lesion as a tumor with its superior sensitivity to enhancement. Three cases of facial neuroma-mimicking lesion including post-inflammatory peri neural thickening, peri neural extension from parotid adenoid cystic carcinoma, and congenita; cholesteatoma showed irregular erosion or mild expansion of the facial nerve canal which may be helpful for differential diagnosis from neuromas.

  9. Improving methyl ketone production in Escherichia coli by heterologous expression of NADH-dependent FabG

    DEFF Research Database (Denmark)

    Goh, Ee Been; Chen, Yan; Petzold, Christopher J.

    2018-01-01

    balance, as fatty acid-derived pathways face the systematic metabolic challenge of net NADPH consumption (in large part, resulting from the key fatty acid biosynthetic enzyme FabG [β-ketoacyl-ACP reductase]) and net NADH production. In this study, we attempted to mitigate cofactor imbalance...... by heterologously expressing NADH-dependent, rather than NADPH-dependent, versions of FabG identified in previous studies. Of the four NADH-dependent versions of FabG tested in our previously best-reported methyl ketone-producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed...... for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed-batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain...

  10. A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

    DEFF Research Database (Denmark)

    de Vries, R.P.; Burgers, K.; van de Vondervoort, P.J.I

    2004-01-01

    A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces...... very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted....... The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A...

  11. Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja

    2002-01-01

    whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non......It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid...... DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens...

  12. Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach

    DEFF Research Database (Denmark)

    Nielsen, Morten Thrane; Nielsen, Jakob Blæsbjerg; Anyaogu, Dianna Chinyere

    2013-01-01

    was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to ransformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were...... of solid methodology for genetic manipulation of most species severely hampers pathway haracterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus...... successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor...

  13. Multi-omic profiling of EPO-producing CHO cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we...... discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed...... a multi-omic physiological characterization including metabolic foot printing of amino acids, metabolite fingerprinting of glycolytic intermediates, NAD(P)H-/NAD(P)+ and adenosine nucleotide phosphates. We used qPCR, qRT-PCR, western blots and Affymetrix CHO microarrays to assess EPO gene copy numbers...

  14. Homologous and heterologous antibody responses of mice immunized with purified feline herpesvirus type 1 and canine herpesvirus glycoproteins.

    Science.gov (United States)

    Limcumpao, J A; Horimoto, T; Xuan, X N; Tohya, Y; Azetaka, M; Takahashi, E; Mikami, T

    1991-06-01

    The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.

  15. Enhancement of crystallinity of cellulose produced by Escherichia coli through heterologous expression of bcsD gene from Gluconacetobacter xylinus.

    Science.gov (United States)

    Sajadi, Elaheh; Babaipour, Valiollah; Deldar, Ali Asghar; Yakhchali, Bagher; Fatemi, Seyed Safa-Ali

    2017-09-01

    To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001. The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD. The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

  16. Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

    International Nuclear Information System (INIS)

    Joo Lee, Pom; Ahn, Ji-Young; Kim, Yang-Hoon; Wook Kim, Seung; Kim, Ji-Yeon; Park, Jae-Sung; Lee, Jeewon

    2004-01-01

    We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus (AJ308438), Photorhabdus luminescens W14 (AF346499) P. luminescens TTO1 (BX571873), and Yersinia pestis CO92 (NC 0 03143). The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity

  17. Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening

    Directory of Open Access Journals (Sweden)

    Sansavini Silviero

    2010-10-01

    Full Text Available Abstract Background Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-Methylcyclopropene. Results To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies, we utilized both homologous and heterologous (tomato microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated. The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. Conclusion Our combined strategy based on microarray hybridization enabled transcriptome characterization

  18. Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening.

    Science.gov (United States)

    Costa, Fabrizio; Alba, Rob; Schouten, Henk; Soglio, Valeria; Gianfranceschi, Luca; Serra, Sara; Musacchi, Stefano; Sansavini, Silviero; Costa, Guglielmo; Fei, Zhangjun; Giovannoni, James

    2010-10-25

    Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-methylcyclopropene. To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies), we utilized both homologous and heterologous (tomato) microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated.The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database) and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato) microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. Our combined strategy based on microarray hybridization enabled transcriptome characterization during normal climacteric apple ripening, as well as

  19. Heterologous production of a ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs the vegetative and reproductive growth.

    Science.gov (United States)

    Gwak, Yu Shin; Han, Jung Yeon; Adhikari, Prakash Babu; Ahn, Chang Ho; Choi, Yong Eui

    2017-06-01

    Production of compound K (a ginsenoside saponin) and its precursors in transgenic tobacco resulted in stunted growth and seed set failure, which may be caused by strong autotoxicity of heterologously produced phytochemicals against the tobacco itself. Panax ginseng roots contain various saponins (ginsenosides), which are major bioactive compounds. A monoglucosylated saponin, compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol), has high medicinal and cosmetic values but is present in undetectable amounts in naturally grown ginseng roots. The production of compound K (CK) requires complicated deglycosylation of ginsenosides using physicochemical and/or enzymatic degradation. In this work, we report the production of CK in transgenic tobacco by co-overexpressing three genes (PgDDS, CYP716A47 and UGT71A28) isolated from P. ginseng. Introduction and expression of the transgenes in tobacco lines were confirmed by genomic PCR and RT-PCR. All the lines of transgenic tobacco produced CK including its precursors, protopanaxadiol and dammarenediol-II (DD). The concentrations of CK in the leaves ranged from 1.55 to 2.64 µg/g dry weight, depending on the transgenic line. Interestingly, production of CK in tobacco brought stunted plant growth and gave rise to seed set failure. This seed set failure was caused by both long-styled flowers and abnormal pollen development in transgenic tobacco. Both CK and DD treatments highly suppressed in vitro germination and tube growth in wild-type pollens. Based on these results, metabolic engineering for CK production in transgenic tobacco was successfully achieved, but the production of CK and its precursors in tobacco severely affects vegetative and reproductive growth due to the cytotoxicity of phytochemicals that are heterologously produced in transgenic tobacco.

  20. Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds.

    Science.gov (United States)

    Clarkson, Sonya M; Giannone, Richard J; Kridelbaugh, Donna M; Elkins, James G; Guss, Adam M; Michener, Joshua K

    2017-09-15

    The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of lignin-derived aromatics. IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related

  1. Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

    Directory of Open Access Journals (Sweden)

    Yuhan Kong

    2013-11-01

    Full Text Available Background and Aims: Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3βmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP. In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.

  2. Protection of macaques with diverse MHC genotypes against a heterologous SIV by vaccination with a deglycosylated live-attenuated SIV.

    Directory of Open Access Journals (Sweden)

    Chie Sugimoto

    Full Text Available HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc, following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a

  3. A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes

    Science.gov (United States)

    Capra, Valérie; Accomazzo, Maria Rosa; Gardoni, Fabrizio; Barbieri, Silvia; Rovati, G. Enrico

    2010-01-01

    Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage–like U937 cells, extracellular nucleotides heterologously desensitize CysLT1 receptor (CysLT1R)-induced Ca2+ transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating Gi-coupled receptor (FPR1, BLT1) desensitize CysLT1R-induced Ca2+ response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to Gq, cross-desensitizes CysLT1R without the apparent involvement of any kinase. Interestingly, Gs-coupled receptors (β2AR, H1/2R, EP2/4R) are also able to desensitize CysLT1R response through activation of PKA. Heterologous desensitization seems to affect mostly the Gi-mediated signaling of the CysLT1R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals. PMID:19965602

  4. Orbital interactions and charge redistribution in weak hydrogen bonds: Watson-Crick GC mimic involving C-H proton donor and F proton acceptor groups

    NARCIS (Netherlands)

    Fonseca Guerra, C.; Baerends, E.J.; Bickelhaupt, F.M.

    2006-01-01

    The discovery by Kool and coworkers that 2,4-difluorotoluene (F) mimics thymine (T) in DNA replication has led to controversy regarding the question of whether this mimic has the capability of forming hydrogen bonds with adenine (A). Recently, we have provided evidence for an important role of both

  5. An athymic mouse model to mimic cobalt-60 cutaneous radiation injury

    Energy Technology Data Exchange (ETDEWEB)

    Mosca, Rodrigo Crespo; Ferreira, Danilo Cardenuto; Napolitano, Celia Marina; Santin, Stefany Plumeri; Dornelles, Leonardo Dalla Porta; Alvarenga, Eluara Ortigoso; Mathor, Monica Beatriz, E-mail: rcmosca@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    Propose: Cutaneous wound from irradiation is the most common complication in radiotherapy treatment, and can be lead to mortality. We describe an athymic mouse model to mimic cutaneous radiation injury by Cobalt-60. Methods: A protocol was including dosimetry with silicon diodes,10x10x5 cm arrangement made by four lead bricks and PVC pipe designed to immobilize the athymic mouse in order to irradiate one clamped back skin point that was subdivided in four parts. To get the measurements of dose rates on the arrangement in Panoramic Irradiator, it was used a silicon diode encased in an opaque protection for ambient light and connected to an electric cable, forming a dosing probe. The currents generated in diode sensitive volume as a function of time of exposure to gamma radiation coming from the radiator, with dose rate of 0,015 Gy/min in positions 1, 0,021 Gy/min in position 2, 0,55 Gy/min in position 3 and 1,45 Gy/min in position four. After the dosimetry, each athymic mouse was anesthetized using Xylazine and Ketamine dilution and entered into a PVC pipe and a small portion of skin (1 cm{sup 3}) was clamped. This tube was then fixed to arrangement and the athymic mouse was irradiate for 60 min, than it was being returned to its cage. Results: The wound was visualized in all animals and photographed after 5 days of irradiation, with the emergence of ulceration after 9 days. No systemic or lethal sequelae occurred or visualized in any animals. Late clinical signs included a wound healing after 22 days. Conclusion: While still being a baseline study, we created a new functional preclinical animal model that can be used for new therapies and may improve radiotherapy management. (author)

  6. An athymic mouse model to mimic cobalt-60 cutaneous radiation injury

    International Nuclear Information System (INIS)

    Mosca, Rodrigo Crespo; Ferreira, Danilo Cardenuto; Napolitano, Celia Marina; Santin, Stefany Plumeri; Dornelles, Leonardo Dalla Porta; Alvarenga, Eluara Ortigoso; Mathor, Monica Beatriz

    2013-01-01

    Propose: Cutaneous wound from irradiation is the most common complication in radiotherapy treatment, and can be lead to mortality. We describe an athymic mouse model to mimic cutaneous radiation injury by Cobalt-60. Methods: A protocol was including dosimetry with silicon diodes,10x10x5 cm arrangement made by four lead bricks and PVC pipe designed to immobilize the athymic mouse in order to irradiate one clamped back skin point that was subdivided in four parts. To get the measurements of dose rates on the arrangement in Panoramic Irradiator, it was used a silicon diode encased in an opaque protection for ambient light and connected to an electric cable, forming a dosing probe. The currents generated in diode sensitive volume as a function of time of exposure to gamma radiation coming from the radiator, with dose rate of 0,015 Gy/min in positions 1, 0,021 Gy/min in position 2, 0,55 Gy/min in position 3 and 1,45 Gy/min in position four. After the dosimetry, each athymic mouse was anesthetized using Xylazine and Ketamine dilution and entered into a PVC pipe and a small portion of skin (1 cm 3 ) was clamped. This tube was then fixed to arrangement and the athymic mouse was irradiate for 60 min, than it was being returned to its cage. Results: The wound was visualized in all animals and photographed after 5 days of irradiation, with the emergence of ulceration after 9 days. No systemic or lethal sequelae occurred or visualized in any animals. Late clinical signs included a wound healing after 22 days. Conclusion: While still being a baseline study, we created a new functional preclinical animal model that can be used for new therapies and may improve radiotherapy management. (author)

  7. Sulfinylated Azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

    Science.gov (United States)

    Qin, Yuan; Wysocki, Ronald J; Somogyi, Arpad; Feinstein, Yelena; Franco, Jessica Y; Tsukamoto, Tatsuya; Dunatunga, Damayanthi; Levy, Clara; Smith, Steven; Simpson, Robert; Gang, David; Johnson, Mark A; Palanivelu, Ravishankar

    2011-01-01

    SUMMARY Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultrahigh resolution ESI FT-ICR and MS/MS techniques to accurately determine the mass (202.126 daltons) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C10H19NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N-Methanesulfinyl 1- and 2-azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 µM stimulated ~50% germination) and elicit accession-specific response. Although N-Methanesulfinyl 2-azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin-like molecules to ensure rapid fertilization by the appropriate pollen. PMID:21801250

  8. Shape-specific nanostructured protein mimics from de novo designed chimeric peptides.

    Science.gov (United States)

    Jiang, Linhai; Yang, Su; Lund, Reidar; Dong, He

    2018-01-30

    Natural proteins self-assemble into highly-ordered nanoscaled architectures to perform specific functions. The intricate functions of proteins have provided great impetus for researchers to develop strategies for designing and engineering synthetic nanostructures as protein mimics. Compared to the success in engineering fibrous protein mimetics, the design of discrete globular protein-like nanostructures has been challenging mainly due to the lack of precise control over geometric packing and intermolecular interactions among synthetic building blocks. In this contribution, we report an effective strategy to construct shape-specific nanostructures based on the self-assembly of chimeric peptides consisting of a coiled coil dimer and a collagen triple helix folding motif. Under salt-free conditions, we showed spontaneous self-assembly of the chimeric peptides into monodisperse, trigonal bipyramidal-like nanoparticles with precise control over the stoichiometry of two folding motifs and the geometrical arrangements relative to one another. Three coiled coil dimers are interdigitated on the equatorial plane while the two collagen triple helices are located in the axial position, perpendicular to the coiled coil plane. A detailed molecular model was proposed and further validated by small angle X-ray scattering experiments and molecular dynamics (MD) simulation. The results from this study indicated that the molecular folding of each motif within the chimeric peptides and their geometric packing played important roles in the formation of discrete protein-like nanoparticles. The peptide design and self-assembly mechanism may open up new routes for the construction of highly organized, discrete self-assembling protein-like nanostructures with greater levels of control over assembly accuracy.

  9. Cadmium mimics estrogen-driven cell proliferation and prolactin secretion from anterior pituitary cells.

    Directory of Open Access Journals (Sweden)

    Sonia A Ronchetti

    Full Text Available Cadmium (Cd is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2 involving E2 receptor (ER activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health.

  10. Functionalized hybrid nanofibers to mimic native ECM for tissue engineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Karuppuswamy, Priyadharsini [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore (Singapore); Department Physics and Nanotechnology, SRM University, Kattankulathur, Chennai (India); Department of Engineering and System Science, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Venugopal, Jayarama Reddy, E-mail: nnijrv@nus.edu.sg [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore (Singapore); Navaneethan, Balchandar [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore (Singapore); Department Physics and Nanotechnology, SRM University, Kattankulathur, Chennai (India); Laiva, Ashang Luwang; Sridhar, Sreepathy; Ramakrishna, Seeram [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, Singapore (Singapore)

    2014-12-15

    Highlights: • Functionalized hybrid polymer mats fabricated for tissue engineering. • Hybrid polymer mats showed high surface area, high porosity and good wettability. • Incorporation of natural polymers modified the properties of nanofiber mats more biologically favorable for biomedical applications. - Abstract: Nanotechnology being one of the most promising technologies today shows an extremely huge potential in the field of tissue engineering to mimic the porous topography of natural extracellular matrix (ECM). Natural polymers are incorporated into the synthetic polymers to fabricate functionalized hybrid nanofibrous scaffolds, which improve cell and tissue compatibility. The present study identified the biopolymers – aloe vera, silk fibroin and curcumin incorporated into polycaprolactone (PCL) as suitable substrates for tissue engineering. Different combinations of PCL with natural polymers – PCL/aloe vera, PCL/silk fibroin, PCL/aloe vera/silk fibroin, PCL/aloe vera/silk fibroin/curcumin were electrospun into nanofibrous scaffolds. The fabricated two dimensional nanofibrous scaffolds showed high surface area, appropriate mechanical properties, hydrophilicity and porosity, required for the regeneration of diseased tissues. The nanofibrous scaffolds were characterized by Scanning electron microscope (SEM), porometry, Instron tensile tester, VCA optima contact angle measurement and FTIR to analyze the fiber diameter and morphology, porosity and pore size distribution, mechanical strength, wettability, chemical bonds and functional groups, respectively. The average fiber diameter of obtained fibers ranged from 250 nm to 350 nm and the tensile strength of PCL scaffolds at 4.49 MPa increased upto 8.3 MPa for PCL/silk fibroin scaffolds. Hydrophobicity of PCL decreased with the incorporation of natural polymers, especially for PCL/aloe vera scaffolds. The properties of as-spun nanofiber scaffolds showed their potential as promising scaffold materials in

  11. Earwigs ( Labidura riparia) mimic rotting-flesh odor to deceive vertebrate predators

    Science.gov (United States)

    Byers, John A.

    2015-08-01

    Many insects repel predators with caustic chemicals, while insects mimicking odors of wastes/dead insects to fool predators have not been documented. We found that the shore earwig, Labidura riparia (Dermaptera: Labiduridae) when bitten by anole lizards, Anolis carolinenesus, spits a rotting-flesh odor that deceives these insectivores into rejecting prey. Once a lizard attacked and rejected an earwig, the lizard did not attack another earwig during several weeks despite consuming other prey, indicating associative learning after one trial. The fetid odor was found in the head-prothorax containing salivary glands of both male and female earwigs and was comprised of ˜100 ng dimethyl disulfide and ˜600 ng dimethyl trisulfide. Nymphs had odorous sulfides after prolonged attacks by harvester ants, Pogonomyrmex rugosus, who were only deterred by the earwig's forceps. Sulfides released by the earwig are similar to odors of carrion/feces, which may be innately repulsive to some vertebrate predators. The mean initial discharge percentage (IDP) of sulfides from a cohort of earwigs was 62 %; however, IDPs of individuals were highly variable (3-99 %; mean 57 %). The discharge refill time (DRT) to refill 50 % of the earwig's allomone reservoir was estimated at 13 h. A positive relationship in sulfide amounts with body weight was found only in females in 2009, suggesting metabolic cost tradeoffs were revealed when sulfide content was half that in 2010. This is the first report of insects releasing sulfur-containing compounds that may mimic carrion-fecal odors as a deceptive defense against vertebrate predators.

  12. Functionalized hybrid nanofibers to mimic native ECM for tissue engineering applications

    International Nuclear Information System (INIS)

    Karuppuswamy, Priyadharsini; Venugopal, Jayarama Reddy; Navaneethan, Balchandar; Laiva, Ashang Luwang; Sridhar, Sreepathy; Ramakrishna, Seeram

    2014-01-01

    Highlights: • Functionalized hybrid polymer mats fabricated for tissue engineering. • Hybrid polymer mats showed high surface area, high porosity and good wettability. • Incorporation of natural polymers modified the properties of nanofiber mats more biologically favorable for biomedical applications. - Abstract: Nanotechnology being one of the most promising technologies today shows an extremely huge potential in the field of tissue engineering to mimic the porous topography of natural extracellular matrix (ECM). Natural polymers are incorporated into the synthetic polymers to fabricate functionalized hybrid nanofibrous scaffolds, which improve cell and tissue compatibility. The present study identified the biopolymers – aloe vera, silk fibroin and curcumin incorporated into polycaprolactone (PCL) as suitable substrates for tissue engineering. Different combinations of PCL with natural polymers – PCL/aloe vera, PCL/silk fibroin, PCL/aloe vera/silk fibroin, PCL/aloe vera/silk fibroin/curcumin were electrospun into nanofibrous scaffolds. The fabricated two dimensional nanofibrous scaffolds showed high surface area, appropriate mechanical properties, hydrophilicity and porosity, required for the regeneration of diseased tissues. The nanofibrous scaffolds were characterized by Scanning electron microscope (SEM), porometry, Instron tensile tester, VCA optima contact angle measurement and FTIR to analyze the fiber diameter and morphology, porosity and pore size distribution, mechanical strength, wettability, chemical bonds and functional groups, respectively. The average fiber diameter of obtained fibers ranged from 250 nm to 350 nm and the tensile strength of PCL scaffolds at 4.49 MPa increased upto 8.3 MPa for PCL/silk fibroin scaffolds. Hydrophobicity of PCL decreased with the incorporation of natural polymers, especially for PCL/aloe vera scaffolds. The properties of as-spun nanofiber scaffolds showed their potential as promising scaffold materials in

  13. AICAR stimulation metabolome widely mimics electrical contraction in isolated rat epitrochlearis muscle.

    Science.gov (United States)

    Miyamoto, Licht; Egawa, Tatsuro; Oshima, Rieko; Kurogi, Eriko; Tomida, Yosuke; Tsuchiya, Koichiro; Hayashi, Tatsuya

    2013-12-15

    Physical exercise has potent therapeutic and preventive effects against metabolic disorders. A number of studies have suggested that 5'-AMP-activated protein kinase (AMPK) plays a pivotal role in regulating carbohydrate and lipid metabolism in contracting skeletal muscles, while several genetically manipulated animal models revealed the significance of AMPK-independent pathways. To elucidate significance of AMPK and AMPK-independent signals in contracting skeletal muscles, we conducted a metabolomic analysis that compared the metabolic effects of 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) stimulation with the electrical contraction ex vivo in isolated rat epitrochlearis muscles, in which both α1- and α2-isoforms of AMPK and glucose uptake were equally activated. The metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry detected 184 peaks and successfully annotated 132 small molecules. AICAR stimulation exhibited high similarity to the electrical contraction in overall metabolites. Principal component analysis (PCA) demonstrated that the major principal component characterized common effects whereas the minor principal component distinguished the difference. PCA and a factor analysis suggested a substantial change in redox status as a result of AMPK activation. We also found a decrease in reduced glutathione levels in both AICAR-stimulated and contracting muscles. The muscle contraction-evoked influences related to the metabolism of amino acids, in particular, aspartate, alanine, or lysine, are supposed to be independent of AMPK activation. Our results substantiate the significance of AMPK activation in contracting skeletal muscles and provide novel evidence that AICAR stimulation closely mimics the metabolomic changes in the contracting skeletal muscles.

  14. A molecular docking study of phytochemical estrogen mimics from dietary herbal supplements.

    Science.gov (United States)

    Powers, Chelsea N; Setzer, William N

    2015-01-01

    The purpose of this study is to use a molecular docking approach to identify potential estrogen mimics or anti-estrogens in phytochemicals found in popular dietary herbal supplements. In this study, 568 phytochemicals found in 17 of the most popular herbal supplements sold in the United States were built and docked with two isoforms of the estrogen receptor, ERα and ERβ (a total of 27 different protein crystal structures). The docking results revealed six strongly docking compounds in Echinacea, three from milk thistle (Silybum marianum), three from Gingko biloba, one from Sambucus nigra, none from maca (Lepidium meyenii), five from chaste tree (Vitex agnus-castus), two from fenugreek (Trigonella foenum-graecum), and two from Rhodiola rosea. Notably, of the most popular herbal supplements for women, there were numerous compounds that docked strongly with the estrogen receptor: Licorice (Glycyrrhiza glabra) had a total of 26 compounds strongly docking to the estrogen receptor, 15 with wild yam (Dioscorea villosa), 11 from black cohosh (Actaea racemosa), eight from muira puama (Ptychopetalum olacoides or P. uncinatum), eight from red clover (Trifolium pratense), three from damiana (Turnera aphrodisiaca or T. diffusa), and three from dong quai (Angelica sinensis). Of possible concern were the compounds from men's herbal supplements that exhibited strong docking to the estrogen receptor: Gingko biloba had three compounds, gotu kola (Centella asiatica) had two, muira puama (Ptychopetalum olacoides or P. uncinatum) had eight, and Tribulus terrestris had six compounds. This molecular docking study has revealed that almost all popular herbal supplements contain phytochemical components that may bind to the human estrogen receptor and exhibit selective estrogen receptor modulation. As such, these herbal supplements may cause unwanted side effects related to estrogenic activity.

  15. 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.

  16. A mimic of sexually-motivated homicide: insect stings and heat exhaustion in a forest.

    Science.gov (United States)

    Liu, Nancy X; Pollanen, Michael S

    2017-06-01

    We report the case a woman who was found dead in a forest. The body was nude and the position of the body suggested a sexually motivated homicide. We concluded that death was not related to homicide, but was related to the conjunction of environmental factors, including insect stings, and acute psychosis. A medicolegal death investigation with postmortem examination was undertaken to determine cause of death. At the scene, the body was supine with legs spread apart and the knees flexed, exposing the external genitalia. There were multiple apparent bruises on the body and neck. At autopsy, based on macroscopic and microscopic examination, the apparent bruises were found to be hemorrhagic insect bites. No significant injuries were present and no semen was found. Death appeared to be related to heat exhaustion and innumerable insect stings. Investigation of the medical history revealed longstanding schizoaffective disorder with episodic psychotic decompensations. In the past, during an acute psychotic episode the decedent removed her clothing and ran wildly in a forest, until she was rescued in a state of exhaustion and marked agitation, and taken to hospital for treatment. We concluded that the same circumstances had been repeated but with a fatal outcome. This case is an example of a mimic of sexually-motivated homicide and is a reminder to forensic pathologists to avoid tunnel vision. We need to be skeptical of the allure of common sense based on first impressions of the scene and the body. Forensic pathologists must be unafraid to scientifically explore improbable, but true, alternate explanations.

  17. ceRNAs in plants: computational approaches and associated challenges for target mimic research.

    Science.gov (United States)

    Paschoal, Alexandre Rossi; Lozada-Chávez, Irma; Domingues, Douglas Silva; Stadler, Peter F

    2017-05-30

    The competing endogenous RNA hypothesis has gained increasing attention as a potential global regulatory mechanism of microRNAs (miRNAs), and as a powerful tool to predict the function of many noncoding RNAs, including miRNAs themselves. Most studies have been focused on animals, although target mimic (TMs) discovery as well as important computational and experimental advances has been developed in plants over the past decade. Thus, our contribution summarizes recent progresses in computational approaches for research of miRNA:TM interactions. We divided this article in three main contributions. First, a general overview of research on TMs in plants is presented with practical descriptions of the available literature, tools, data, databases and computational reports. Second, we describe a common protocol for the computational and experimental analyses of TM. Third, we provide a bioinformatics approach for the prediction of TM motifs potentially cross-targeting both members within the same or from different miRNA families, based on the identification of consensus miRNA-binding sites from known TMs across sequenced genomes, transcriptomes and known miRNAs. This computational approach is promising because, in contrast to animals, miRNA families in plants are large with identical or similar members, several of which are also highly conserved. From the three consensus TM motifs found with our approach: MIM166, MIM171 and MIM159/319, the last one has found strong support on the recent experimental work by Reichel and Millar [Specificity of plant microRNA TMs: cross-targeting of mir159 and mir319. J Plant Physiol 2015;180:45-8]. Finally, we stress the discussion on the major computational and associated experimental challenges that have to be faced in future ceRNA studies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Caloric restriction in C57BL/6J mice mimics therapeutic fasting in humans

    Directory of Open Access Journals (Sweden)

    Denny Christine A

    2006-05-01

    Full Text Available Abstract Background Caloric restriction (CR has long been recognized as a dietary therapy that improves health and increases longevity. Little is known about the persistent effects of CR on plasma biomarkers (glucose, ketone bodies, and lipids following re-feeding in mice. It is also unclear how these biomarker changes in calorically restricted mice relate to those observed previously in calorically restricted humans. Results Three groups of individually housed adult female C57BL/6J (B6 mice (n = 4/group were fed a standard rodent chow diet either: (1 unrestricted (UR; (2 restricted for three weeks to reduce body weight by approximately 15–20% (R; or (3 restricted for three weeks and then re-fed unrestricted (ad libitum for an additional three weeks (R-RF. Body weight and food intake were measured throughout the study, while plasma lipids and levels of glucose and ketone bodies (β-hydroxybutyrate were measured at the termination of the study. Plasma glucose, phosphatidylcholine, cholesterol, and triglycerides were significantly lower in the R mice than in the UR mice. In contrast, plasma fatty acids and β-hydroxybutyrate were significantly higher in the R mice than in the UR mice. CR had no effect on plasma phosphatidylinositol levels. While body weight and plasma lipids of the R-RF mice returned to unrestricted levels upon re-feeding, food intake and glucose levels remained significantly lower than those prior to the initiation of CR. Conclusion CR establishes a new homeostatic state in B6 mice that persists for at least three weeks following ad libitum re-feeding. Moreover, the plasma biomarker changes observed in B6 mice during CR mimic those reported in humans on very low calorie diets or during therapeutic fasting.

  19. Somatostatin receptor 1 and 5 double knockout mice mimic neurochemical changes of Huntington's disease transgenic mice.

    Directory of Open Access Journals (Sweden)

    Padmesh S Rajput

    Full Text Available Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD. However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5.To delineate subtype selective biological responses we have here investigated changes in SSTR1 and 5 double knockout mice brain and compared with HD transgenic mouse model (R6/2. Our study revealed significant loss of dopamine and cAMP regulated phosphoprotein of 32 kDa (DARPP-32 and comparable changes in SST, N-methyl-D-aspartic acid receptors subtypes, calbindin and brain nitric oxide synthase expression as well as in key signaling proteins including calpain, phospho-extracellular-signal-regulated kinases1/2, synapsin-IIa, protein kinase C-α and calcineurin in SSTR1/5(-/- and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice.This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the pathophysiology of Huntington's disease.

  20. Migrainous aura as stroke-mimic: The role of perfusion-computed tomography.

    Science.gov (United States)

    Ridolfi, Mariana; Granato, Antonio; Polverino, Paola; Furlanis, Giovanni; Ukmar, Maja; Zorzenon, Irene; Manganotti, Paolo

    2018-03-01

    The acute-onset of migrainuos aura (MA) can be erroneously diagnosed in Emergency Department (ED) as acute stroke (AS) and it can be classified as "stroke mimic" (SM). Perfusion computer tomography (PCT) may be useful to improve detection of infarcts. The aim of the study was to investigate the role in ED of PCT in improving diagnosis of migrainous aura. Data were compared with the well-defined perfusion patterns in patients with acute ischemic stroke. A standardized Stroke Protocol was planned. The protocol consisted in centralizing in ED all the patients with acute-onset of neurological symptoms compatible with cerebrovascular disease and in performing a general and neurological examination, hematological tests, brain non-contrast computed tomography (NCCT), CT angiography (CTA) of the supra-aortic and intracranial arteries and cerebral PCT. Patients with diagnosis of definite or probable acute stroke were hospitalized in Stroke Unit (SU). A six-months retrospective analysis of all the patients included in the Stroke Protocol and discharged from ED or from SU with a diagnosis of migraine with aura was performed. 172 patients were included in the Stroke Protocol and 6 patients were enrolled. NCCT, CTA and PCT were performed after 60-90 min from symptoms onset and revealed normal perfusion. Intravenous thrombolysis was performed only in one patient. Patients with acute-onset of neurological symptoms, who have rapid progressive improvement of symptoms, normal neuroimaging, in particular PCT, and preceding episodes of migraine with aura, may be considered as suffering from MA. In these cases, even if thrombolysis is safe, clinicians may defer a prompt aggressive treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Cirrus cloud mimic surfaces in the laboratory: organic acids, bases and NOx heterogeneous reactions

    Science.gov (United States)

    Sodeau, J.; Oriordan, B.

    2003-04-01

    CIRRUS CLOUD MIMIC SURFACES IN THE LABORATORY:ORGANIC ACIDS, BASES AND NOX HETEROGENEOUS REACTIONS. B. ORiordan, J. Sodeau Department of Chemistry and Environment Research Institute, University College Cork, Ireland j.sodeau@ucc.ie /Fax: +353-21-4902680 There are a variety of biogenic and anthropogenic sources for the simple carboxylic acids to be found in the troposphere giving rise to levels as high as 45 ppb in certain urban areas. In this regard it is of note that ants of genus Formica produce some 10Tg of formic acid each year; some ten times that produced by industry. The expected sinks are those generally associated with tropospheric chemistry: the major routes studied, to date, being wet and dry deposition. No studies have been carried out hitherto on the role of water-ice surfaces in the atmospheric chemistry of carboxylic acids and the purpose of this paper is to indicate their potential function in the heterogeneous release of atmospheric species such as HONO. The deposition of formic acid on a water-ice surface was studied using FT-RAIR spectroscopy over a range of temperatures between 100 and 165K. In all cases ionization to the formate (and oxonium) ions was observed. The results were confirmed by TPD (Temperature Programmed Desorption) measurements, which indicated that two distinct surface species adsorb to the ice. Potential reactions between the formic acid/formate ion surface and nitrogen dioxide were subsequently investigated by FT-RAIRS. Co-deposition experiments showed that N2O3 and the NO+ ion (associated with water) were formed as products. A mechanism is proposed to explain these results, which involves direct reaction between the organic acid and nitrogen dioxide. Similar experiments involving acetic acid also indicate ionization on a water-ice surface. The results are put into the context of atmospheric chemistry potentially occuring on cirrus cloud surfaces.

  2. Flavonols Protect Against UV Radiation-Induced Thymine Dimer Formation in an Artificial Skin Mimic.

    Science.gov (United States)

    Maini, Sabia; Fahlman, Brian M; Krol, Ed S

    2015-01-01

    Exposure of skin to ultraviolet light has been shown to have a number of deleterious effects including photoaging, photoimmunosuppression and photoinduced DNA damage which can lead to the development of skin cancer. In this paper we present a study on the ability of three flavonols to protect EpiDerm™, an artificial skin mimic, against UV-induced damage. EpiDerm™ samples were treated with flavonol in acetone and exposed to UVA (100 kJ/m(2) at 365 nm) and UVB (9000 J/m(2) at 310 nm) radiation. Secretion of matrix metalloproteinase-1 (MMP-1) and tumor necrosis factor-α (TNF-a) were determined by ELISA, cyclobutane pyrimidine dimers were quantified using LC-APCI-MS. EpiDerm™ treated topically with quercetin significantly decreased MMP-1 secretion induced by UVA (100 µM) or UVB (200 µM) and TNF-a secretion was significantly reduced at 100 µM quercetin for both UVA and UVB radiation. In addition, topically applied quercetin was found to be photostable over the duration of the experiment. EpiDerm™ samples were treated topically with quercetin, kaempferol or galangin (52 µM) immediately prior to UVA or UVB exposure, and the cyclobutane thymine dimers (T-T (CPD)) were quantified using an HPLC-APCI MS/MS method. All three flavonols significantly decreased T-T (CPD) formation in UVB irradiated EpiDerm™, however no effect could be observed for the UVA irradiation experiments as thymine dimer formation was below the limit of quantitation. Our results suggest that flavonols can provide protection against UV radiation-induced skin damage through both antioxidant activity and direct photo-absorption. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  3. Optimized animal model to mimic the reality of stress-induced depression in the clinic.

    Science.gov (United States)

    Zhang, Yi; Wang, Yuting; Lei, Hui; Wang, Lei; Xue, Liang; Wang, Xin; Zhu, Xiongzhao

    2017-05-06

    Animal models are useful tools for verifying the relationship between stress and depression; however, an operational criterion for excluding the resilient animals from the analysis has not been established yet, which hinders the model's ability to more accurately mimic the scenario in humans. To induce depression-like symptoms, rats received maternal deprivation (MD) during PND1-14, and/or chronic unpredictable stress (CUS) exposure. The latent profile analysis (LPA) was used to determine latent subgroups in treatment naive adult rats. The percentile method was used to distinguish sensitive and non-sensitive behaviors in rats. The sucrose preference rate of treatment naive adult rats was fit using a Beta distribution, while immobility time was fit using a Gamma distribution. Indexes of behavioral tests revealed the 4-class model as the best fit for treatment naive adult rats. The incidence of stress-resilience in MD rats was significantly higher than that in CUS rats and MD + CUS rats. There was a significantly higher incidence of stress-resilience in CUS rats compared with MD + CUS rats. Recovery rate of anhedonia-like and sub anhedonia-like behaviors in CUS rats was significantly higher than that in MD and MD + CUS rats. There was a significantly higher recovery rate of anhedonia-like behaviors in MD rats compared to MD + CUS rats. The percentile method is suitable for setting up an operational cutoff to classify depression-like, sub depression-like, and resilient behaviors in rats exposed to MD and CUS.

  4. The glutathione mimic ebselen inhibits oxidative stress but not endoplasmic reticulum stress in endothelial cells.

    Science.gov (United States)

    Ahwach, Salma Makhoul; Thomas, Melanie; Onstead-Haas, Luisa; Mooradian, Arshag D; Haas, Michael J

    2015-08-01

    Reactive oxygen species are associated with cardiovascular disease, diabetes, and atherosclerosis, yet the use of antioxidants in clinical trials has been ineffective at improving outcomes. In endothelial cells, high-dextrose-induced oxidative stress and endoplasmic reticulum stress promote endothelial dysfunction leading to the recruitment and activation of peripheral blood lymphocytes and the breakdown of barrier function. Ebselen, a glutathione peroxidase 1 (GPX1) mimic, has been shown to improve β-cell function in diabetes and prevent atherosclerosis. To determine if ebselen inhibits both oxidative stress and endoplasmic reticulum (ER) stress in endothelial cells, we examined its effects in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) with and without high-dextrose. Oxidative stress and ER stress were measured by 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence and ER stress alkaline phosphatase assays, respectively. GPX1 over-expression and knockdown were performed by transfecting cells with a GPX1 expression construct or a GPX1-specific siRNA, respectively. Ebselen inhibited dextrose-induced oxidative stress but not ER stress in both HUVEC and HCAEC. Ebselen also had no effect on tunicamycin-induced ER stress in HCAEC. Furthermore, augmentation of GPX1 activity directly by sodium selenite supplementation or transfection of a GPX1 expression plasmid decreased dextrose-induced oxidative stress but not ER stress, while GPX1 knockout enhanced oxidative stress but had no effect on ER stress. These results suggest that ebselen targets only oxidative stress but not ER stress. Copyright © 2015. Published by Elsevier Inc.

  5. Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures.

    Science.gov (United States)

    Schwalbe, E C; Hicks, D; Rafiee, G; Bashton, M; Gohlke, H; Enshaei, A; Potluri, S; Matthiesen, J; Mather, M; Taleongpong, P; Chaston, R; Silmon, A; Curtis, A; Lindsey, J C; Crosier, S; Smith, A J; Goschzik, T; Doz, F; Rutkowski, S; Lannering, B; Pietsch, T; Bailey, S; Williamson, D; Clifford, S C

    2017-10-18

    Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

  6. Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

    Science.gov (United States)

    Ziach, Krzysztof; Chollet, Céline; Parissi, Vincent; Prabhakaran, Panchami; Marchivie, Mathieu; Corvaglia, Valentina; Bose, Partha Pratim; Laxmi-Reddy, Katta; Godde, Frédéric; Schmitter, Jean-Marie; Chaignepain, Stéphane; Pourquier, Philippe; Huc, Ivan

    2018-05-01

    Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

  7. Phenol degradation catalyzed by a peroxidase mimic constructed through the grafting of heme onto metal-organic frameworks.

    Science.gov (United States)

    Jiang, Wei; Yang, Jiebing; Wang, Xinghuo; Han, Haobo; Yang, Yan; Tang, Jun; Li, Quanshun

    2018-01-01

    The aim of this work was to construct a peroxidase mimic for achieving the phenol degradation through Fenton reaction. The enzyme mimic was synthesized through the conjugation of heme with the amino group of 2-amino-1,4-benzene dicarboxylate in UiO-66-NH 2 (ZrMOF), namely Heme-ZrMOF. Compared to free heme, the composite Heme-ZrMOF exhibited an obviously enhanced ability for phenol degradation with up to 97.3% of phenol removal after 2h. Meanwhile, it could achieve the easy separation of catalyst from the system and the elimination of iron residues in the process of phenol degradation. Finally, the catalyst Heme-ZrMOF was observed to possess good recyclability in the phenol degradation with still 76.2% of phenol removal after 4 cycles. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Biocompatible Polymer Nanoformulation To Improve the Release and Safety of a Drug Mimic Molecule Detectable via ICP-MS.

    Science.gov (United States)

    Ferrari, Raffaele; Talamini, Laura; Violatto, Martina Bruna; Giangregorio, Paola; Sponchioni, Mattia; Morbidelli, Massimo; Salmona, Mario; Bigini, Paolo; Moscatelli, Davide

    2017-01-03

    Fluorescent poly(ε-caprolactone)-based nanoparticles (NPs) have been synthesized and successfully loaded with a titanium organometallic compound as a mimic of a water-insoluble drug. The nature of this nanovector enabled us to combine the quantification of the metal in tissues after systemic administration in healthy immunocompetent mice by inductively coupled plasma mass spectroscopy (ICP-MS) followed by the visualization of NPs in organ sections by confocal microscopy. This innovative method of nanodrug screening has enabled us to elucidate the crucial parameters of their kinetics. The organometallic compound is a good mimic of most anticancer drugs, and this approach is an interesting starting point to design the relevance of a broad range of nanoformulations in terms of safety and targeted delivery of the cargoes.

  9. Simultaneous Vascular Targeting and Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody

    Science.gov (United States)

    2014-05-01

    in May 2013, the difference between nude mice (which lack T- cells , but still have a partially functional adaptive and innate immune system) and NSG...Mangada J, Greiner DL, Handgretinger R. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human...Targeting of Cerebral Breast Cancer Metastases Using a T- Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel

  10. Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism

    OpenAIRE

    Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

    2015-01-01

    Background Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. Methods We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and rel...

  11. A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

    Science.gov (United States)

    Mollet, Björne B; Bogaerts, Iven L J; van Almen, Geert C; Dankers, Patricia Y W

    2017-06-01

    Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  12. A latex agglutination test for the field determination of abnormal vitellogenin production in male fishes contaminated by estrogen mimics

    International Nuclear Information System (INIS)

    Magalhaes, Ilizabete; Pihan, Jean-Claude; Falla, Jairo

    2004-01-01

    Estrogen mimics are pollutants present in the aquatic environment. These compounds induce abnormalities in the reproductive system of male fishes, which lead to a total or partial male feminization, or to their demasculinization. Ultimately, these alterations could lead to a disappearance of the total contaminated fish population. Moreover, these toxic substances possess the capacity to mimic endogenous estrogens and to induce the abnormal production of vitellogenin (VTG) in male and immature fishes. The purpose of this research was to develop an easy, specific, cheap and fast method for diagnosing the contamination of male fishes by estrogen mimics, using VTG as biomarker. The selected method is based on a reverse latex agglutination test (rLAT), developed with monoclonal antibodies specific of this biomarker. The development of this VTG-rLAT has involved, firstly, the purification of carp VTG to produce monoclonal antibodies, specifics of this protein. One of these antibodies was selected to recover latex particles (diameter: 1 μm). Finally, the immunoreactivity of the VTG-rLAT was verified with different fish plasma samples from males treated with 17β-estradiol and non-treated males or females in vitellogenesis

  13. Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death

    Directory of Open Access Journals (Sweden)

    Sijia Liu

    2017-06-01

    Full Text Available Switchgrass (Panicum virgatum has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT, a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

  14. Skin lesions over the pocket area that may mimic cardiac implantable electronic device infection: A case series.

    Science.gov (United States)

    Korantzopoulos, Panagiotis; Plakoutsi, Sofia; Florou, Elizabeth; Bechlioulis, Aris

    2018-05-21

    The early and correct diagnosis of cardiac implantable electronic device (CIED) infections is critical given that early aggressive treatment with complete removal of the system along with antimicrobial therapy dramatically improves outcomes. Pocket infection manifested by local signs of inflammation is the most common form of CIED infections. Conditions mimicking pocket infection have been described in the literature. These include various types of malignancy and rarely allergic reactions/contact dermatitis to pacemaker compounds. We aimed to describe skin lesions and disorders over the pocket area that mimic CIED infection. We present a series of 5 cases with skin lesions that mimic pocket infection. We document these cases with corresponding photographs. Most of them have not been described in this setting. We report the following cases of conditions that proved not to be CIED infection: One case of superficial cellulitis, one case of herpes zoster over the pocket area, one case of spontaneous bruising over the pocket a long time after implantation in a patient taking oral anticoagulation, and 2 cases of contact dermatitis due to prolonged postoperative application of povidone iodine. All cases had favorable outcome after conservative treatment and no CIED infection was developed during follow-up. Clinicians should be aware of rare conditions that mimic CIED infection. Incorrect diagnosis of these disorders may falsely lead to CIED extraction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  15. Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death.

    Science.gov (United States)

    Liu, Sijia; Fu, Chunxiang; Gou, Jiqing; Sun, Liang; Huhman, David; Zhang, Yunwei; Wang, Zeng-Yu

    2017-01-01

    Switchgrass ( Panicum virgatum ) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O -methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT -deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

  16. Characteristics and genetic mapping of a lesion mimic mutant pl(t) in japonica rice variety zhejing 22

    International Nuclear Information System (INIS)

    Chen Pingping; Zhang Xiaoming; Ye Shenghai; Zhao Ningchun; Lu Yanting; Liu Heqin; Jin Qingsheng; Yang Ling

    2010-01-01

    A lesion mimic mutant,obtained by radiation mutagenesis on the seeds of a japonica rice variety Zhejing 22, exhibited a lesion mimic phenotype during the whole growth stage under different environments. Genetic analysis indicated that the mutant trait was controlled by a single recessive gene named spl (t). Relying on simple sequence repeat (SSR) and recessive class analysis method to map the spl (t) gene with a F 2 population was constructed by crossing the mutant spl (t) with Zhenshan 97B.spl (t) was mapped in the interval of 0.8cM between RM7195 and RM27929 near centromere region on the short arm of chromosome 12.Blue trypan dye analyses indicated that the lesion mimic trait of the mutant was caused by the programmer cell death. Further study showed that the programmer cell death was caused by H 2 O 2 oxidative burst. By inoculation of bacterial leaf blight and blast strains, the resistances of the mutant were similar to the wild variety Zhejing 22. (authors)

  17. A peroxidase mimic with atom transfer radical polymerization activity constructed through the grafting of heme onto metal-organic frameworks.

    Science.gov (United States)

    Jiang, Wei; Pan, Yue; Yang, Jiebing; Liu, Yong; Yang, Yan; Tang, Jun; Li, Quanshun

    2018-07-01

    Atom transfer radical polymerization (ATRP) has been considered to be an efficient strategy for constructing functional macromolecules owing to its simple operation and versatile monomers, and thus it is of great significance to develop ideal catalysts with higher activity and perfect reusability. We constructed a peroxidase mimic through the grafting of heme onto metal-organic frameworks UiO-66-NH 2 (ZrMOF), namely Heme-ZrMOF. After the systematic characterization of structure, the composite Heme-ZrMOF was demonstrated to possess high peroxidase activity using 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) and 3,3',5,5'-tetramethylbenzidine as substrates. The enzyme mimic was then used as catalysts in the ATRP reactions of different monomers, in which favorable monomer conversion (44.6-98.0%) and product molecular weight (8600-25,600 g/mol) could be obtained. Compared to free heme, Heme-ZrMOF could efficiently achieve the easy separation of heme from the catalytic system and facilitate the ATRP reaction in an aqueous environment to avoid the utilization of organic solvents. In conclusion, the enzyme mimic Heme-ZrMOF could be potentially used as an effective catalyst for preparing well-defined polymers with biomedical applications. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Antioxidant activity of selenenamide-based mimic as a function of the aromatic thiols nucleophilicity, a DFT-SAPE model.

    Science.gov (United States)

    Kheirabadi, Ramesh; Izadyar, Mohammad

    2018-05-18

    The mechanism of action of the selenenamide 1 as a mimic of the glutathione peroxidase (GPx) was investigated by the density functional theory. The solvent-assisted proton exchange procedure was applied to model the catalytic behavior and antioxidant activity of this mimic. To have an insight into the charge transfer effect, different aromatic thiols, including electron donating substituents on the phenyl ring were considered. The catalytic behavior of the selenenamide was modeled in a four-step mechanism, described by the oxidation of the mimic, the reduction of the obtained product, selenoxide, the reduction of the selenenylsulfide and dehydration of selenenic acid. On the basis of the activation parameters, the final step of the proposed mechanism is the rate determining states of the catalytic cycle. Turnover frequency (TOF) analysis showed that the electron donating groups at the para-position of the phenyl ring of the PhSH do not affect the catalytic activity of the selenenamide in contrast to p-methyl thiophenol which indicates the highest nucleophilicity. The evaluation of the electronic contribution of the various donating groups on the phenyl ring of the aromatic thiols shows that the antioxidant activity of the selenenamide sufficiently increases in the presence of the electron-donating substitutions. Finally, the charge transfer process at the rate-determining state was investigated based on the natural bond orbital analysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Ghrelin mimics fasting to enhance human hedonic, orbitofrontal cortex, and hippocampal responses to food.

    Science.gov (United States)

    Goldstone, Anthony P; Prechtl, Christina G; Scholtz, Samantha; Miras, Alexander D; Chhina, Navpreet; Durighel, Giuliana; Deliran, Seyedeh S; Beckmann, Christian; Ghatei, Mohammad A; Ashby, Damien R; Waldman, Adam D; Gaylinn, Bruce D; Thorner, Michael O; Frost, Gary S; Bloom, Stephen R; Bell, Jimmy D

    2014-06-01

    Ghrelin, which is a stomach-derived hormone, increases with fasting and energy restriction and may influence eating behaviors through brain hedonic reward-cognitive systems. Therefore, changes in plasma ghrelin might mediate counter-regulatory responses to a negative energy balance through changes in food hedonics. We investigated whether ghrelin administration (exogenous hyperghrelinemia) mimics effects of fasting (endogenous hyperghrelinemia) on the hedonic response and activation of brain-reward systems to food. In a crossover design, 22 healthy, nonobese adults (17 men) underwent a functional magnetic resonance imaging (fMRI) food-picture evaluation task after a 16-h overnight fast (Fasted-Saline) or after eating breakfast 95 min before scanning (730 kcal, 14% protein, 31% fat, and 55% carbohydrate) and receiving a saline (Fed-Saline) or acyl ghrelin (Fed-Ghrelin) subcutaneous injection before scanning. One male subject was excluded from the fMRI analysis because of excess head motion, which left 21 subjects with brain-activation data. Compared with the Fed-Saline visit, both ghrelin administration to fed subjects (Fed-Ghrelin) and fasting (Fasted-Saline) significantly increased the appeal of high-energy foods and associated orbitofrontal cortex activation. Both fasting and ghrelin administration also increased hippocampus activation to high-energy- and low-energy-food pictures. These similar effects of endogenous and exogenous hyperghrelinemia were not explicable by consistent changes in glucose, insulin, peptide YY, and glucagon-like peptide-1. Neither ghrelin administration nor fasting had any significant effect on nucleus accumbens, caudate, anterior insula, or amygdala activation during the food-evaluation task or on auditory, motor, or visual cortex activation during a control task. Ghrelin administration and fasting have similar acute stimulatory effects on hedonic responses and the activation of corticolimbic reward-cognitive systems during food

  20. Various clinical conditions can mimic Crimean-Congo hemorrhagic fever in pediatric patients in endemic regions

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    Soner S. Kara

    2016-09-01

    Full Text Available Summary: Crimean-Congo hemorrhagic fever (CCHF is a tick-borne disease with high mortality. Many disorders can mimic CCHF. It is important to recognize the condition and to perform differential diagnosis in endemic countries. Twenty-one children aged 18 years or less with a preliminary diagnosis of CCHF were retrospectively evaluated. Real-time PCR and a confirmatory indirect immunofluorescence assay for negative results were performed. The diagnoses determined that 9 patients had (42.9% CCHF; 7 patients had (33.3% viral upper respiratory tract infections (URTI; 2 patients had (9.5% brucellosis; 1 patients had (4.7% periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA syndrome episode; 1 patient had (4.7% cerebral palsy, diabetes insipidus, acute gastroenteritis, and hypernatremic dehydration; and 1 patient had (4.7% cellulitis after a tick bite. The mean age of patients with CCHF was greater than that of the other patients (116.1 ± 53.6 vs. 94.1 ± 52.1 months, p = 0.02. Seventeen (81% of the children included had a history of tick bites, 2 (9.5% had a history of contact with a patient with CCHF, and 2 (9.5% had no exposure, but were living in an endemic region. Three patients had an underlying disorder: cerebral palsy and diabetes insipidus, epilepsy, or PFAPA. All of the children experienced fever. Other frequent symptoms were malaise, diarrhea, vomiting, and abdominal pain, but none of these differed statistically between the patient groups. CCHF patients had a longer mean duration of symptoms (10.56 ± 1.42 vs. 6.75 ± 3.62 days, p = 0.008 and a longer mean length of hospitalization (8.00 ± 2.08 vs. 3.58 ± 1.56 days, p < 0.001 than the other patients. At laboratory examination, patients with CCHF had statistically significant lower leukocyte and platelet counts, more prolonged coagulation parameters, and greater AST, ALT, LDH, and CK levels than the other patients. No mortality or complications occurred