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Sample records for cells reveal complex

  1. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  3. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  4. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.

    Science.gov (United States)

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F

    2013-06-18

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells. PMID:23733942

  5. Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging.

    Science.gov (United States)

    Kuznetsov, Andrey V; Hermann, Martin; Troppmair, Jakob; Margreiter, Raimund; Hengster, Paul

    2010-01-01

    Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. PMID:19382913

  6. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2011-03-01

    Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

  7. Mapping enteroendocrine cell populations in transgenic mice reveals an unexpected degree of complexity in cellular differentiation within the gastrointestinal tract

    OpenAIRE

    1990-01-01

    The gastrointestinal tract is lined with a monolayer of cells that undergo perpetual and rapid renewal. Four principal, terminally differentiated cell types populate the monolayer, enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. This epithelium exhibits complex patterns of regional differentiation, both from crypt- to-villus and from duodenum-to-colon. The "liver" fatty acid binding protein (L-FABP) gene represents a useful model for analyzing the molecular basis for intes...

  8. Amyloid-β-Anti-Amyloid-β Complex Structure Reveals an Extended Conformation in the Immunodominant B-Cell Epitope

    Energy Technology Data Exchange (ETDEWEB)

    Miles, Luke A; Wun, Kwok S; Crespi, Gabriela A.N.; Fodero-Tavoletti, Michelle T; Galatis, Denise; Bagley, Christopher J; Beyreuther, Konrad; Masters, Colin L; Cappai, Roberto; McKinstry, William J; Barnham, Kevin J; Parker, Michael W [SVIMR-A; (Hanson); (Heidelberg); (Melbourne)

    2012-04-17

    Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.

  9. The Plant Cell Wall: A Complex and Dynamic Structure As Revealed by the Responses of Genes under Stress Conditions

    Science.gov (United States)

    Houston, Kelly; Tucker, Matthew R.; Chowdhury, Jamil; Shirley, Neil; Little, Alan

    2016-01-01

    The plant cell wall has a diversity of functions. It provides a structural framework to support plant growth and acts as the first line of defense when the plant encounters pathogens. The cell wall must also retain some flexibility, such that when subjected to developmental, biotic, or abiotic stimuli it can be rapidly remodeled in response. Genes encoding enzymes capable of synthesizing or hydrolyzing components of the plant cell wall show differential expression when subjected to different stresses, suggesting they may facilitate stress tolerance through changes in cell wall composition. In this review we summarize recent genetic and transcriptomic data from the literature supporting a role for specific cell wall-related genes in stress responses, in both dicot and monocot systems. These studies highlight that the molecular signatures of cell wall modification are often complex and dynamic, with multiple genes appearing to respond to a given stimulus. Despite this, comparisons between publically available datasets indicate that in many instances cell wall-related genes respond similarly to different pathogens and abiotic stresses, even across the monocot-dicot boundary. We propose that the emerging picture of cell wall remodeling during stress is one that utilizes a common toolkit of cell wall-related genes, multiple modifications to cell wall structure, and a defined set of stress-responsive transcription factors that regulate them. PMID:27559336

  10. Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas

    OpenAIRE

    Gerlinger, Marco; Quezada, Sergio A; Peggs, Karl S.; Furness, Andrew JS; Fisher, Rosalie; Marafioti, Teresa; Shende, Vishvesh H.; McGranahan, Nicholas; Rowan, Andrew J.; Hazell, Steven; Hamm, David; Robins, Harlan S; Pickering, Lisa; Gore, Martin; Nicol, David L.

    2013-01-01

    The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and β-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples...

  11. Cross-Platform Assessment of Genomic Imbalance Confirms the Clinical Relevance of Genomic Complexity and Reveals Loci with Potential Pathogenic Roles in Diffuse Large B-Cell Lymphoma

    Science.gov (United States)

    Dias, Lizalynn M.; Thodima, Venkata; Friedman, Julia; Ma, Charles; Guttapalli, Asha; Mendiratta, Geetu; Siddiqi, Imran N.; Syrbu, Sergei; Chaganti, R. S. K.; Houldsworth, Jane

    2016-01-01

    Genomic copy number alterations (CNAs) in diffuse large B-cell lymphoma (DLBCL) have roles in disease pathogenesis but overall clinical relevance remains unclear. Herein, an unbiased algorithm was uniformly applied across three genome profiling datasets comprising 392 newly-diagnosed DLBCL specimens that defined 32 overlapping CNAs, involving 36 minimal common regions (MCRs). Scoring criteria were established for 50 aberrations within the MCRs while considering peak gains/losses. Application of these criteria to independent datasets revealed novel candidate genes with coordinated expression, such as CNOT2, potentially with pathogenic roles. No one single aberration significantly associated with patient outcome across datasets, but genomic complexity, defined by imbalance in more than one MCR, significantly portended adverse outcome in two of three independent datasets. Thus, the standardized scoring of CNAs currently developed can be uniformly applied across platforms, affording robust validation of genomic imbalance and complexity in DLBCL and overall clinical utility as biomarkers of patient outcome. PMID:26294112

  12. Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas.

    Science.gov (United States)

    Gerlinger, Marco; Quezada, Sergio A; Peggs, Karl S; Furness, Andrew J S; Fisher, Rosalie; Marafioti, Teresa; Shende, Vishvesh H; McGranahan, Nicholas; Rowan, Andrew J; Hazell, Steven; Hamm, David; Robins, Harlan S; Pickering, Lisa; Gore, Martin; Nicol, David L; Larkin, James; Swanton, Charles

    2013-12-01

    The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and β-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra-deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR-sequencing data. A polyclonal T cell repertoire with 367-16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, -0.218 to 0.465). 3-93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra-deep TCR-sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches. PMID:24122851

  13. Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells

    OpenAIRE

    Hulme, Amy E.; Kelley, Z; Foley, Deirdre; Hope, Thomas J.

    2015-01-01

    During uncoating, the conical capsid of HIV disassembles by dissociation of the p24 capsid protein (CA). Uncoating is known to be required for HIV replication, but the mechanism is poorly defined. Here, we examined the timing and effect of two capsid binding drugs (PF74 and BI2) on infectivity and capsid integrity in HIV-1-infected cells. The virus remained susceptible to the action of PF74 and BI2 for hours after uncoating as defined in parallel drug addition and cyclosporine (CsA) washout a...

  14. Adaptation to High Ethanol Reveals Complex Evolutionary Pathways

    Science.gov (United States)

    Das, Anupam; Espinosa-Cantú, Adriana; De Maeyer, Dries; Arslan, Ahmed; Van Pee, Michiel; van der Zande, Elisa; Meert, Wim; Yang, Yudi; Zhu, Bo; Marchal, Kathleen; DeLuna, Alexander; Van Noort, Vera; Jelier, Rob; Verstrepen, Kevin J.

    2015-01-01

    Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts. PMID:26545090

  15. Structures of MART-126/27-35Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Borbulevych, Oleg Y; Insaidoo, Francis K; Baxter, Tiffany K; Powell, Jr., Daniel J.; Johnson, Laura A; Restifo, Nicholas P; Baker, Brian M [NIH; (Notre)

    2008-09-17

    Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26-35 decamer, although only the 27-35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26-35 and 27-35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1{sub 26/27-35}-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27-35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.

  16. Analysis of the Rab GTPase Interactome in Dendritic Cells Reveals Anti-microbial Functions of the Rab32 Complex in Bacterial Containment.

    Science.gov (United States)

    Li, Yuanyuan; Wang, Yu; Zou, Liyun; Tang, Xiangyu; Yang, Yi; Ma, Li; Jia, Qingzhu; Ni, Qingshan; Liu, Siqi; Tang, Lizhang; Lin, Regina; Wong, Elizabeth; Sun, Wei; Wang, Liting; Wei, Quanfang; Ran, Haiying; Zhang, Liqun; Lian, Hengning; Huang, Wei; Wu, Yuzhang; Li, Qi-Jing; Wan, Ying

    2016-02-16

    Dendritic cells (DCs) orchestrate complex membrane trafficking through an interconnected transportation network linked together by Rab GTPases. Through a tandem affinity purification strategy and mass spectrometry, we depicted an interactomic landscape of major members of the mammalian Rab GTPase family. When complemented with imaging tools, this proteomic analysis provided a global view of intracellular membrane organization. Driven by this analysis, we investigated dynamic changes to the Rab32 subnetwork in DCs induced by L. monocytogenes infection and uncovered an essential role of this subnetwork in controlling the intracellular proliferation of L. monocytogenes. Mechanistically, Rab32 formed a persistent complex with two interacting proteins, PHB and PHB2, to encompass bacteria both during early phagosome formation and after L. monocytogenes escaped the original containment vacuole. Collectively, we have provided a functional compartmentalization overview and an organizational framework of intracellular Rab-mediated vesicle trafficking that can serve as a resource for future investigations. PMID:26885862

  17. Visualization by BiFC of different C/EBPβ dimers and their interaction with HP1α reveals a differential subnuclear distribution of complexes in living cells

    International Nuclear Information System (INIS)

    How the co-ordinated events of gene activation and silencing during cellular differentiation are influenced by spatial organization of the cell nucleus is still poorly understood. Little is known about the molecular mechanisms controlling subnuclear distribution of transcription factors, and their interplay with nuclear proteins that shape chromatin structure. Here we show that C/EBPβ not only associates with pericentromeric heterochromatin but also interacts with the nucleoskeleton upon induction of adipocyte differentiation of 3T3-L1 cells. Different C/EBPβ dimers localize in different nuclear domains. Using BiFC in living cells, we show that LAP (Liver Activating Protein) homodimers localize in euchromatin and heterochromatin. In contrast, LIP (Liver Inhibitory Protein) homodimers localize exclusively in heterochromatin. Importantly, their differential subnuclear distribution mirrors the site for interaction with HP1α. HP1α inhibits LAP transcriptional capacity and occupies the promoter of the C/EBPβ-dependent gene c/ebpα in 3T3-L1 preadipocytes. When adipogenesis is induced, HP1α binding decreases from c/ebpα promoter, allowing transcription. Thus, the equilibrium among different pools of C/EBPβ associated with chromatin or nucleoskeleton, and dynamic changes in their interaction with HP1α, play key roles in the regulation of C/EBP target genes during adipogenesis.

  18. Beyond Contagion: Reality Mining Reveals Complex Patterns of Social Influence.

    Directory of Open Access Journals (Sweden)

    Aamena Alshamsi

    Full Text Available Contagion, a concept from epidemiology, has long been used to characterize social influence on people's behavior and affective (emotional states. While it has revealed many useful insights, it is not clear whether the contagion metaphor is sufficient to fully characterize the complex dynamics of psychological states in a social context. Using wearable sensors that capture daily face-to-face interaction, combined with three daily experience sampling surveys, we collected the most comprehensive data set of personality and emotion dynamics of an entire community of work. From this high-resolution data about actual (rather than self-reported face-to-face interaction, a complex picture emerges where contagion (that can be seen as adaptation of behavioral responses to the behavior of other people cannot fully capture the dynamics of transitory states. We found that social influence has two opposing effects on states: adaptation effects that go beyond mere contagion, and complementarity effects whereby individuals' behaviors tend to complement the behaviors of others. Surprisingly, these effects can exhibit completely different directions depending on the stable personality or emotional dispositions (stable traits of target individuals. Our findings provide a foundation for richer models of social dynamics, and have implications on organizational engineering and workplace well-being.

  19. Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.

    Directory of Open Access Journals (Sweden)

    Mariya M Kucherenko

    Full Text Available The Dystroglycan-Dystrophin (Dg-Dys complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.

  20. Geometric Mechanics Reveals Optimal Complex Terrestrial Undulation Patterns

    Science.gov (United States)

    Gong, Chaohui; Astley, Henry; Schiebel, Perrin; Dai, Jin; Travers, Matthew; Goldman, Daniel; Choset, Howie; CMU Team; GT Team

    Geometric mechanics offers useful tools for intuitively analyzing biological and robotic locomotion. However, utility of these tools were previously restricted to systems that have only two internal degrees of freedom and in uniform media. We show kinematics of complex locomotors that make intermittent contacts with substrates can be approximated as a linear combination of two shape bases, and can be represented using two variables. Therefore, the tools of geometric mechanics can be used to analyze motions of locomotors with many degrees of freedom. To demonstrate the proposed technique, we present studies on two different types of snake gaits which utilize combinations of waves in the horizontal and vertical planes: sidewinding (in the sidewinder rattlesnake C. cerastes) and lateral undulation (in the desert specialist snake C. occipitalis). C. cerastes moves by generating posteriorly traveling body waves in the horizontal and vertical directions, with a relative phase offset equal to +/-π/2 while C. occipitalismaintains a π/2 offset of a frequency doubled vertical wave. Geometric analysis reveals these coordination patterns enable optimal movement in the two different styles of undulatory terrestrial locomotion. More broadly, these examples demonstrate the utility of geometric mechanics in analyzing realistic biological and robotic locomotion.

  1. Communities in Neuronal Complex Networks Revealed by Activation Patterns

    CERN Document Server

    Costa, Luciano da Fontoura

    2008-01-01

    Recently, it has been shown that the communities in neuronal networks of the integrate-and-fire type can be identified by considering patterns containing the beginning times for each cell to receive the first non-zero activation. The received activity was integrated in order to facilitate the spiking of each neuron and to constrain the activation inside the communities, but no time decay of such activation was considered. The present article shows that, by taking into account exponential decays of the stored activation, it is possible to identify the communities also in terms of the patterns of activation along the initial steps of the transient dynamics. The potential of this method is illustrated with respect to complex neuronal networks involving four communities, each of a different type (Erd\\H{o}s-R\\'eny, Barab\\'asi-Albert, Watts-Strogatz as well as a simple geographical model). Though the consideration of activation decay has been found to enhance the communities separation, too intense decays tend to y...

  2. Single cell transcriptional analysis reveals novel innate immune cell types

    Directory of Open Access Journals (Sweden)

    Linda E. Kippner

    2014-06-01

    Full Text Available Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription

  3. Intersubject information mapping: revealing canonical representations of complex natural stimuli

    Directory of Open Access Journals (Sweden)

    Nikolaus Kriegeskorte

    2015-03-01

    Full Text Available Real-world time-continuous stimuli such as video promise greater naturalism for studies of brain function. However, modeling the stimulus variation is challenging and introduces a bias in favor of particular descriptive dimensions. Alternatively, we can look for brain regions whose signal is correlated between subjects, essentially using one subject to model another. Intersubject correlation mapping (ICM allows us to find brain regions driven in a canonical manner across subjects by a complex natural stimulus. However, it requires a direct voxel-to-voxel match between the spatiotemporal activity patterns and is thus only sensitive to common activations sufficiently extended to match up in Talairach space (or in an alternative, e.g. cortical-surface-based, common brain space. Here we introduce the more general approach of intersubject information mapping (IIM. For each brain region, IIM determines how much information is shared between the subjects' local spatiotemporal activity patterns. We estimate the intersubject mutual information using canonical correlation analysis applied to voxels within a spherical searchlight centered on each voxel in turn. The intersubject information estimate is invariant to linear transforms including spatial rearrangement of the voxels within the searchlight. This invariance to local encoding will be crucial in exploring fine-grained brain representations, which cannot be matched up in a common space and, more fundamentally, might be unique to each individual – like fingerprints. IIM yields a continuous brain map, which reflects intersubject information in fine-grained patterns. Performed on data from functional magnetic resonance imaging (fMRI of subjects viewing the same television show, IIM and ICM both highlighted sensory representations, including primary visual and auditory cortices. However, IIM revealed additional regions in higher association cortices, namely temporal pole and orbitofrontal cortex. These

  4. Principles of assembly reveal a periodic table of protein complexes.

    Science.gov (United States)

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  5. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    Science.gov (United States)

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation. PMID:26000486

  6. Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors

    Science.gov (United States)

    Sala, Adrien; Shoaib, Muhammad; Anufrieva, Olga; Mutharasu, Gnanavel; Jahan Hoque, Rawnak; Yli-Harja, Olli; Kandhavelu, Meenakshisundaram

    2015-05-01

    In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

  7. 454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

    Directory of Open Access Journals (Sweden)

    Gustafsson Lars

    2010-12-01

    Full Text Available Abstract Background Because of their functional significance, the Major Histocompatibility Complex (MHC class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. Results Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. Conclusions 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective

  8. Revealing the Complexity of Community-Campus Interactions

    Science.gov (United States)

    Nichols, Naomi Elizabeth; Phipps, David; Gaetz, Stephen; Fisher, Alison L.; Tanguay, Nancy

    2014-01-01

    In this paper, four qualitative case studies capture the complex interplay between the social and structural relations that shape community - academic partnerships. Collaborations begin as relationships among people. They are sustained by institutional structures that recognize and support these relationships. Productive collaborations centralize…

  9. Gastrin release: Antrum microdialysis reveals a complex neural control

    DEFF Research Database (Denmark)

    Ericsson, P; Håkanson, R; Rehfeld, Jens F.;

    2010-01-01

    serum regardless of the prandial state. The rats were conscious during microdialysis except when subjected to electrical vagal stimulation. Acid blockade (omeprazole treatment of freely fed rats for 4 days), or bilateral sectioning of the abdominal vagal trunks (fasted, 3 days post-op.), raised the...... gastrin concentration in blood as well as microdialysate. The high gastrin concentration following omeprazole treatment was not affected by vagotomy. Vagal excitation stimulated the G cells: electrical vagal stimulation and pylorus ligation (fasted rats) raised the gastrin concentration transiently in...... microdialysate gastrin concentration in omeprazole-treated rats by 65%. We conclude that activated gastrin release, unlike basal gastrin release, is highly dependent on a neural input: 1) Vagal excitation has a transient stimulating effect on the G cells. The transient nature of the response suggests that the...

  10. Phylogenetic analyses of melanoma reveal complex patterns of metastatic dissemination

    OpenAIRE

    Sanborn, JZ; Chung, J.; Purdom, E; Wang, NJ; Kakavand, H; Wilmott, JS; Butler, T.; Thompson, JF; Mann, GJ; Haydu, LE; Saw, RPM; Busam, KJ; Lo, RS; Collisson, EA; Hur, JS

    2015-01-01

    © 2015, National Academy of Sciences. All rights reserved. Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized...

  11. Phylogenetic analyses of melanoma reveal complex patterns of metastatic dissemination.

    Science.gov (United States)

    Sanborn, J Zachary; Chung, Jongsuk; Purdom, Elizabeth; Wang, Nicholas J; Kakavand, Hojabr; Wilmott, James S; Butler, Timothy; Thompson, John F; Mann, Graham J; Haydu, Lauren E; Saw, Robyn P M; Busam, Klaus J; Lo, Roger S; Collisson, Eric A; Hur, Joe S; Spellman, Paul T; Cleaver, James E; Gray, Joe W; Huh, Nam; Murali, Rajmohan; Scolyer, Richard A; Bastian, Boris C; Cho, Raymond J

    2015-09-01

    Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models. PMID:26286987

  12. Gastrin release: Antrum microdialysis reveals a complex neural control

    DEFF Research Database (Denmark)

    Ericsson, P; Håkanson, R; Rehfeld, Jens F.;

    2010-01-01

    vagus has not only a prompt stimulatory but also a slow inhibitory effect on gastrin release. 2) Although vagal denervation did not affect the gastrin response to anacidity, the TTX experiments revealed that both food-evoked and anacidity-evoked gastrin release depends on neural input.......We used microdialysis to monitor local gastrin release in response to food, acid blockade and acute vagal excitation. For the first time, gastrin release has been monitored continuously in intact conscious rats in a physiologically relevant experimental setting in a fashion that minimizes...... serum regardless of the prandial state. The rats were conscious during microdialysis except when subjected to electrical vagal stimulation. Acid blockade (omeprazole treatment of freely fed rats for 4 days), or bilateral sectioning of the abdominal vagal trunks (fasted, 3 days post-op.), raised the...

  13. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners

    Science.gov (United States)

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W. Thomas; Bush, Nicole R.; Adler, Nancy; Levine, Joel D.

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of “reciprocal” interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  14. Revealing the complex conduction heat transfer mechanism of nanofluids.

    Science.gov (United States)

    Sergis, A; Hardalupas, Y

    2015-12-01

    Nanofluids are two-phase mixtures consisting of small percentages of nanoparticles (sub 1-10 %vol) inside a carrier fluid. The typical size of nanoparticles is less than 100 nm. These fluids have been exhibiting experimentally a significant increase of thermal performance compared to the corresponding carrier fluids, which cannot be explained using the classical thermodynamic theory. This study deciphers the thermal heat transfer mechanism for the conductive heat transfer mode via a molecular dynamics simulation code. The current findings are the first of their kind and conflict with the proposed theories for heat transfer propagation through micron-sized slurries and pure matter. The authors provide evidence of a complex new type of heat transfer mechanism, which explains the observed abnormal heat transfer augmentation. The new mechanism appears to unite a number of popular speculations for the thermal heat transfer mechanism employed by nanofluids as predicted by the majority of the researchers of the field into a single one. The constituents of the increased diffusivity of the nanoparticle can be attributed to mismatching of the local temperature profiles between parts of the surface of the solid and the fluid resulting in increased local thermophoretic effects. These effects affect the region surrounding the solid manifesting interfacial layer phenomena (Kapitza resistance). In this region, the activity of the fluid and the interactions between the fluid and the nanoparticle are elevated. Isotropic increased nanoparticle mobility is manifested as enhanced Brownian motion and diffusion effects. PMID:26058515

  15. Laser altimetry reveals complex pattern of Greenland Ice Sheet dynamics.

    Science.gov (United States)

    Csatho, Beata M; Schenk, Anton F; van der Veen, Cornelis J; Babonis, Gregory; Duncan, Kyle; Rezvanbehbahani, Soroush; van den Broeke, Michiel R; Simonsen, Sebastian B; Nagarajan, Sudhagar; van Angelen, Jan H

    2014-12-30

    We present a new record of ice thickness change, reconstructed at nearly 100,000 sites on the Greenland Ice Sheet (GrIS) from laser altimetry measurements spanning the period 1993-2012, partitioned into changes due to surface mass balance (SMB) and ice dynamics. We estimate a mean annual GrIS mass loss of 243 ± 18 Gt ⋅ y(-1), equivalent to 0.68 mm ⋅ y(-1) sea level rise (SLR) for 2003-2009. Dynamic thinning contributed 48%, with the largest rates occurring in 2004-2006, followed by a gradual decrease balanced by accelerating SMB loss. The spatial pattern of dynamic mass loss changed over this time as dynamic thinning rapidly decreased in southeast Greenland but slowly increased in the southwest, north, and northeast regions. Most outlet glaciers have been thinning during the last two decades, interrupted by episodes of decreasing thinning or even thickening. Dynamics of the major outlet glaciers dominated the mass loss from larger drainage basins, and simultaneous changes over distances up to 500 km are detected, indicating climate control. However, the intricate spatiotemporal pattern of dynamic thickness change suggests that, regardless of the forcing responsible for initial glacier acceleration and thinning, the response of individual glaciers is modulated by local conditions. Recent projections of dynamic contributions from the entire GrIS to SLR have been based on the extrapolation of four major outlet glaciers. Considering the observed complexity, we question how well these four glaciers represent all of Greenland's outlet glaciers. PMID:25512537

  16. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners.

    Science.gov (United States)

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W Thomas; Bush, Nicole R; Adler, Nancy; Levine, Joel D

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of "reciprocal" interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  17. Revealing the complex conduction heat transfer mechanism of nanofluids

    Science.gov (United States)

    Sergis, A.; Hardalupas, Y.

    2015-06-01

    Nanofluids are two-phase mixtures consisting of small percentages of nanoparticles (sub 1-10 %vol) inside a carrier fluid. The typical size of nanoparticles is less than 100 nm. These fluids have been exhibiting experimentally a significant increase of thermal performance compared to the corresponding carrier fluids, which cannot be explained using the classical thermodynamic theory. This study deciphers the thermal heat transfer mechanism for the conductive heat transfer mode via a molecular dynamics simulation code. The current findings are the first of their kind and conflict with the proposed theories for heat transfer propagation through micron-sized slurries and pure matter. The authors provide evidence of a complex new type of heat transfer mechanism, which explains the observed abnormal heat transfer augmentation. The new mechanism appears to unite a number of popular speculations for the thermal heat transfer mechanism employed by nanofluids as predicted by the majority of the researchers of the field into a single one. The constituents of the increased diffusivity of the nanoparticle can be attributed to mismatching of the local temperature profiles between parts of the surface of the solid and the fluid resulting in increased local thermophoretic effects. These effects affect the region surrounding the solid manifesting interfacial layer phenomena (Kapitza resistance). In this region, the activity of the fluid and the interactions between the fluid and the nanoparticle are elevated. Isotropic increased nanoparticle mobility is manifested as enhanced Brownian motion and diffusion effects

  18. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

    OpenAIRE

    Ellen, Albert F.; Albers, Sonja-Verena; Huibers, Wim; Pitcher, Angela; Hobel, Cedric F. V.; Schwarz, Heinz; Folea, Mihaela; Schouten, Stefan; Boekema, Egbert J.; Poolman, Bert; Driessen, Arnold J. M.

    2009-01-01

    The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles ...

  19. Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Heidi Koldsø

    2014-10-01

    Full Text Available Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2, in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

  20. 454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

    OpenAIRE

    Gustafsson Lars; Stuglik Michał; Babik Wiesław; Zagalska-Neubauer Magdalena; Cichoń Mariusz; Radwan Jacek

    2010-01-01

    Abstract Background Because of their functional significance, the Major Histocompatibility Complex (MHC) class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by ge...

  1. Comparative analyses of developmental transcription factor repertoires in sponges reveal unexpected complexity of the earliest animals.

    Science.gov (United States)

    Fortunato, Sofia A V; Adamski, Marcin; Adamska, Maja

    2015-12-01

    Developmental transcription factors (DTFs) control development of animals by affecting expression of target genes, some of which are transcription factors themselves. In bilaterians and cnidarians, conserved DTFs are involved in homologous processes such as gastrulation or specification of neurons. The genome of Amphimedon queenslandica, the first sponge to be sequenced, revealed that only a fraction of these conserved DTF families are present in demosponges. This finding was in line with the view that morphological complexity in the animal lineage correlates with developmental toolkit complexity. However, as the phylum Porifera is very diverse, Amphimedon's genome may not be representative of all sponges. The recently sequenced genomes of calcareous sponges Sycon ciliatum and Leucosolenia complicata allowed investigations of DTFs in a sponge lineage evolutionarily distant from demosponges. Surprisingly, the phylogenetic analyses of identified DTFs revealed striking differences between the calcareous sponges and Amphimedon. As these differences appear to be a result of independent gene loss events in the two sponge lineages, the last common ancestor of sponges had to possess a much more diverse repertoire of DTFs than extant sponges. Developmental expression of sponge homologs of genes involved in specification of the Bilaterian endomesoderm and the neurosensory cells suggests that roles of many DTFs date back to the last common ancestor of all animals. Strikingly, even DTFs displaying apparent pan-metazoan conservation of sequence and function are not immune to being lost from individual species genomes. The quest for a comprehensive picture of the developmental toolkit in the last common metazoan ancestor is thus greatly benefitting from the increasing accessibility of sequencing, allowing comparisons of multiple genomes within each phylum. PMID:26253310

  2. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.

    Science.gov (United States)

    Gokce, Ozgun; Stanley, Geoffrey M; Treutlein, Barbara; Neff, Norma F; Camp, J Gray; Malenka, Robert C; Rothwell, Patrick E; Fuccillo, Marc V; Südhof, Thomas C; Quake, Stephen R

    2016-07-26

    The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states. PMID:27425622

  3. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq

    Directory of Open Access Journals (Sweden)

    Ozgun Gokce

    2016-07-01

    Full Text Available The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

  4. Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes.

    Science.gov (United States)

    Ohta, Shinya; Montaño-Gutierrez, Luis F; de Lima Alves, Flavia; Ogawa, Hiromi; Toramoto, Iyo; Sato, Nobuko; Morrison, Ciaran G; Takeda, Shunichi; Hudson, Damien F; Rappsilber, Juri; Earnshaw, William C

    2016-08-01

    Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. PMID:27231315

  5. Homophilic Protocadherin Cell-Cell Interactions Promote Dendrite Complexity.

    Science.gov (United States)

    Molumby, Michael J; Keeler, Austin B; Weiner, Joshua A

    2016-05-01

    Growth of a properly complex dendrite arbor is a key step in neuronal differentiation and a prerequisite for neural circuit formation. Diverse cell surface molecules, such as the clustered protocadherins (Pcdhs), have long been proposed to regulate circuit formation through specific cell-cell interactions. Here, using transgenic and conditional knockout mice to manipulate γ-Pcdh repertoire in the cerebral cortex, we show that the complexity of a neuron's dendritic arbor is determined by homophilic interactions with other cells. Neurons expressing only one of the 22 γ-Pcdhs can exhibit either exuberant or minimal dendrite complexity, depending only on whether surrounding cells express the same isoform. Furthermore, loss of astrocytic γ-Pcdhs, or disruption of astrocyte-neuron homophilic matching, reduces dendrite complexity cell non-autonomously. Our data indicate that γ-Pcdhs act locally to promote dendrite arborization via homophilic matching, and they confirm that connectivity in vivo depends on molecular interactions between neurons and between neurons and astrocytes. PMID:27117416

  6. Homophilic Protocadherin Cell-Cell Interactions Promote Dendrite Complexity

    Directory of Open Access Journals (Sweden)

    Michael J. Molumby

    2016-05-01

    Full Text Available Growth of a properly complex dendrite arbor is a key step in neuronal differentiation and a prerequisite for neural circuit formation. Diverse cell surface molecules, such as the clustered protocadherins (Pcdhs, have long been proposed to regulate circuit formation through specific cell-cell interactions. Here, using transgenic and conditional knockout mice to manipulate γ-Pcdh repertoire in the cerebral cortex, we show that the complexity of a neuron’s dendritic arbor is determined by homophilic interactions with other cells. Neurons expressing only one of the 22 γ-Pcdhs can exhibit either exuberant or minimal dendrite complexity, depending only on whether surrounding cells express the same isoform. Furthermore, loss of astrocytic γ-Pcdhs, or disruption of astrocyte-neuron homophilic matching, reduces dendrite complexity cell non-autonomously. Our data indicate that γ-Pcdhs act locally to promote dendrite arborization via homophilic matching, and they confirm that connectivity in vivo depends on molecular interactions between neurons and between neurons and astrocytes.

  7. Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Lincan [Department of Thoracic Surgery, The Third Affiliated Hospital of Kunming Medical University, Kunming (China); Shen, Hongmei [Cancer Center of Integrative Medicine, The Third Affiliated Hospital of Kunming Medical University, Kunming (China); Zhao, Guangqiang [Department of Thoracic Surgery, The Third Affiliated Hospital of Kunming Medical University, Kunming (China); Yang, Runxiang [Cancer Chemotherapy Center, The Third Affiliated Hospital of Kunming Medical University, Kunming (China); Cai, Xinyi [Colorectal Cancer Center, The Third Affiliated Hospital of Kunming Medical University, Kunming (China); Zhang, Lijuan [Department of Pathology, The Third Affiliated Hospital of Kunming Medical University, Kunming (China); Jin, Congguo [Cancer Institute, The Third Affiliated Hospital of Kunming Medical University, Kunming (China); Huang, Yunchao, E-mail: daliduanlincan@163.com [Department of Thoracic Surgery, The Third Affiliated Hospital of Kunming Medical University, Kunming (China)

    2014-04-18

    Highlights: • Disulfiram and copper synergistically inhibit lung cancer cell proliferation. • Lung cancer cell colony formation ability is inhibited by Disulfiram/copper. • Disulfiram/copper increases the sensitivity of cisplatin to lung cancer cells. • Lung cancer stem cells are specifically targeted by Disulfiram/copper complex. - Abstract: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.

  8. Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    Highlights: • Disulfiram and copper synergistically inhibit lung cancer cell proliferation. • Lung cancer cell colony formation ability is inhibited by Disulfiram/copper. • Disulfiram/copper increases the sensitivity of cisplatin to lung cancer cells. • Lung cancer stem cells are specifically targeted by Disulfiram/copper complex. - Abstract: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients

  9. Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

    International Nuclear Information System (INIS)

    The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1. The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (Rfree = 23.6%) and 20.9 (Rfree = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound

  10. Functional Features of Trans-differentiated Hair Cells Mediated by Atoh1 Reveals a Primordial Mechanism

    OpenAIRE

    Yang, Juanmei; Bouvron, Sonia; Lv, Ping; Chi, Fanglu; Yamoah, Ebenezer N.

    2012-01-01

    Evolution has transformed a simple ear with few vestibular maculae into a complex 3-dimensional structure consisting of nine distinct endorgans. It is debatable whether the sensory epithelia underwent progressive segregation or emerged from distinct sensory patches. To address these uncertainties we examined the morphological and functional phenotype of trans-differentiated rat hair cells to reveal their primitive or endorgan-specific origins. Additionally, it is uncertain how Atoh1-mediated ...

  11. A phosphorylated pseudokinase complex controls cell wall synthesis in mycobacteria.

    Science.gov (United States)

    Gee, Christine L; Papavinasasundaram, Kadamba G; Blair, Sloane R; Baer, Christina E; Falick, Arnold M; King, David S; Griffin, Jennifer E; Venghatakrishnan, Harene; Zukauskas, Andrew; Wei, Jun-Rong; Dhiman, Rakesh K; Crick, Dean C; Rubin, Eric J; Sassetti, Christopher M; Alber, Tom

    2012-01-24

    Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase-FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks. PMID:22275220

  12. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    Science.gov (United States)

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  13. Single-Cell Transcriptome Analyses Reveal Signals to Activate Dormant Neural Stem Cells

    OpenAIRE

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary

    2015-01-01

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133+/GFAP− ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133+/GFAP− quiescent cells were enriched...

  14. Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes.

    Science.gov (United States)

    Dean, Samuel; Moreira-Leite, Flavia; Varga, Vladimir; Gull, Keith

    2016-08-30

    The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics. PMID:27519801

  15. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  16. Contrast Adaptation Decreases Complexity in Retinal Ganglion Cell Spike Train

    Institute of Scientific and Technical Information of China (English)

    WANG Guang-Li; HUANG Shi-Yong; ZHANG Ying-Ying; LIANG Pei-Ji

    2007-01-01

    @@ The difference in temporal structures of retinal ganglion cell spike trains between spontaneous activity and firing activity after contrast adaptation is investigated. The Lempel-Ziv complexity analysis reveals that the complexity of the neural spike train decreases after contrast adaptation. This implies that the behaviour of the neuron becomes ordered, which may carry relevant information about the external stimulus. Thus, during the neuron activity after contrast adaptation, external information could be encoded in forms of some certain patterns in the temporal structure of spike train that is significantly different, compared to that of the spike train during spontaneous activity, although the firing rates in spontaneous activity and firing activity after contrast adaptation are sometime similar.

  17. Principles of bacterial cell-size determination revealed by cell wall synthesis perturbations

    OpenAIRE

    Carolina Tropini; Timothy K. Lee; Jen Hsin; Samantha M. Desmarais; Tristan Ursell; Russell D. Monds; Kerwyn Casey Huang

    2014-01-01

    Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cyto...

  18. Optical tweezers reveal a dynamic mechanical response of cationic peptide-DNA complexes

    Science.gov (United States)

    Lee, Amy; Zheng, Tai; Sucayan, Sarah; Chou, Szu-Ting; Tricoli, Lucas; Hustedt, Jason; Kahn, Jason; Mixson, A. James; Seog, Joonil

    2013-03-01

    Nonviral carriers have been developed to deliver nucleic acids by forming nanoscale complexes; however, there has been limited success in achieving high transfection efficiency. Our hypothesis is that a factor affecting gene delivery efficiency is the mechanical response of the condensed complex. To begin to test this hypothesis, we directly measured the mechanical properties of DNA-carrier complexes using optical tweezers. Histidine-lysine (HK) polymer, Asparagine-lysine (NK) polymer and poly-L-lysine were used to form complexes with a single DNA molecule. As carriers were introduced, a sudden decrease in DNA extension occurrs at a force level which is defined as critical force (Fc). Fc is carrier and concentration dependent. Pulling revealed reduction in DNA extension length for HK-DNA complexes. The characteristics of force profiles vary by agent and can be dynamically manipulated by changes in environmental conditions such as ionic strength of the buffer as well as pH. Heparin can remove cationic reagents which are otherwise irreversibly bound to DNA. The implications for optimizing molecular interactions to enhance transfection efficiency will be discussed.

  19. Mechanics of Cellulose Synthase Complexes in Living Plant Cells

    Science.gov (United States)

    Zehfroosh, Nina; Liu, Derui; Ramos, Kieran P.; Yang, Xiaoli; Goldner, Lori S.; Baskin, Tobias I.

    The polymer cellulose is one of the major components of the world's biomass with unique and fascinating characteristics such as its high tensile strength, renewability, biodegradability, and biocompatibility. Because of these distinctive aspects, cellulose has been the subject of enormous scientific and industrial interest, yet there are still fundamental open questions about cellulose biosynthesis. Cellulose is synthesized by a complex of transmembrane proteins called ``Cellulose Synthase A'' (CESA) in the plasma membrane. Studying the dynamics and kinematics of the CESA complex will help reveal the mechanism of cellulose synthesis and permit the development and validation of models of CESA motility. To understand what drives these complexes through the cell membrane, we used total internal reflection fluorescence microscopy (TIRFM) and variable angle epi-fluorescence microscopy to track individual, fluorescently-labeled CESA complexes as they move in the hypocotyl and root of living plants. A mean square displacement analysis will be applied to distinguish ballistic, diffusional, and other forms of motion. We report on the results of these tracking experiments. This work was funded by NSF/PHY-1205989.

  20. Deep seismic reflection profiling revealing the complex crustal structure of the Tongling ore district

    Institute of Scientific and Technical Information of China (English)

    L(U); Qingtian; HOU; Zengqian; ZHAO; Jinhua; SHI; Danian

    2004-01-01

    The deep seismic profiling across the Tongling ore district reveals a complex crustal structure. Strong contrasting dipping layered reflections (4-11 s, TWT),which dominate the lower crust of the northern part of the profile, are interpreted as the underplating of the basalt related to extensional tectonism. In the south of the profile, the Yangtze craton is characterized by strong reflections in the middle crust, showing a distinct two-layer crustal structure. Over the region of the Tongling uplift, there appear the complex arc shape reflections, suggesting folded, faulted and intruded structures, and the transparent zone below them reveals the existence of batholith. The south dipping strong reflections between the upper crust and lower crust (4-7 s, TWT) suggest a detachment between them. The detachment provided space for the magma intrusion, and caused the formation of the batholith. The Yangtze craton has a clear Moho reflection, while the Tongling uplift has a weak Moho, whereas below the reflective lower crust in the northern part of the profile, there are sub-Moho reflections. The abrupt variation of the Moho characteristics within a short distance indicates the complexity of magmatic activity.

  1. Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells.

    Directory of Open Access Journals (Sweden)

    Sarita Raghunayakula

    Full Text Available Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological

  2. Survey of large protein complexes D. vulgaris reveals great structural diversity

    Energy Technology Data Exchange (ETDEWEB)

    Han, B.-G.; Dong, M.; Liu, H.; Camp, L.; Geller, J.; Singer, M.; Hazen, T. C.; Choi, M.; Witkowska, H. E.; Ball, D. A.; Typke, D.; Downing, K. H.; Shatsky, M.; Brenner, S. E.; Chandonia, J.-M.; Biggin, M. D.; Glaeser, R. M.

    2009-08-15

    An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr {approx} 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate {approx}10 times greater than that of previous 'proteomic' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

  3. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

    Science.gov (United States)

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

    2012-01-01

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans. PMID:23012415

  4. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    Science.gov (United States)

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-01

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy. PMID:25556991

  5. Chromatin Repressive Complexes in Stem Cells, Development, and Cancer

    DEFF Research Database (Denmark)

    Laugesen, Anne; Helin, Kristian

    2014-01-01

    The chromatin environment is essential for the correct specification and preservation of cell identity through modulation and maintenance of transcription patterns. Many chromatin regulators are required for development, stem cell maintenance, and differentiation. Here, we review the roles of the...... polycomb repressive complexes, PRC1 and PRC2, and the HDAC1- and HDAC2-containing complexes, NuRD, Sin3, and CoREST, in stem cells, development, and cancer, as well as the ongoing efforts to develop therapies targeting these complexes in human cancer. Furthermore, we discuss the role of repressive...... complexes in modulating thresholds for gene activation and their importance for specification and maintenance of cell fate....

  6. Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ

    Science.gov (United States)

    Pertsinidis, Alexandros; Mukherjee, Konark; Sharma, Manu; Pang, Zhiping P.; Park, Sang Ryul; Zhang, Yunxiang; Brunger, Axel T.; Südhof, Thomas C.; Chu, Steven

    2013-01-01

    Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ. PMID:23821748

  7. Cell-to-Cell Diversity in a Synchronized Chlamydomonas Culture As Revealed by Single-Cell Analyses

    OpenAIRE

    Garz, Andreas; Sandmann, Michael; Rading, Michael; Ramm, Sascha; Menzel, Ralf; Steup, Martin

    2012-01-01

    In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cel...

  8. Neisseria gonorrhoeae induced disruption of cell junction complexes in epithelial cells of the human genital tract.

    Science.gov (United States)

    Rodríguez-Tirado, Carolina; Maisey, Kevin; Rodríguez, Felipe E; Reyes-Cerpa, Sebastián; Reyes-López, Felipe E; Imarai, Mónica

    2012-03-01

    Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epithelial barriers in order to reach subepithelial tissues for host colonization. The aim of this study was to examine the effects of gonococci on cell junction complexes of genital epithelial cells of women. Polarized Ishikawa cells, a cell line derived from endometrial epithelium, were used for experimental infection. Infected cells displayed a spindle-like shape with an irregular distribution, indicating potential alteration of cell-cell contacts. Accordingly, analysis by confocal microscopy and cellular fractionation revealed that gonococci induced redistribution of the adherens junction proteins E-cadherin and its adapter protein β-catenin from the membrane to a cytoplasmic pool, with no significant differences in protein levels. In contrast, gonococcal infection did not induce modification of either expression or distribution of the tight junction proteins Occludin and ZO-1. Similar results were observed for Fallopian tube epithelia. Interestingly, infected Ishikawa cells also showed an altered pattern of actin cytoskeleton, observed in the form of stress fibers across the cytoplasm, which in turn matched a strong alteration on the expression of fibronectin, an adhesive glycoprotein component of extracellular matrix. Interestingly, using western blotting, activation of the ERK pathway was detected after gonococcal infection while p38 pathway was not activated. All effects were pili and Opa independent. Altogether, results indicated that gonococcus, as a mechanism of pathogenesis, induced disruption of junction complexes with early detaching of E-cadherin and β-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix. PMID:22146107

  9. A multi-gene approach reveals a complex evolutionary history in the Cyanistes species group.

    Science.gov (United States)

    Illera, Juan Carlos; Koivula, Kari; Broggi, Juli; Päckert, Martin; Martens, Jochen; Kvist, Laura

    2011-10-01

    Quaternary climatic oscillations have been considered decisive in shaping much of the phylogeographic structure around the Mediterranean Basin. Within this paradigm, peripheral islands are usually considered as the endpoints of the colonization processes. Here, we use nuclear and mitochondrial markers to investigate the phylogeography of the blue tit complex (blue tit Cyanistes caeruleus, Canary blue tit C. teneriffae and azure tit C. cyanus), and assess the role of the Canary Islands for the geographic structuring of genetic variation. The Canary blue tit exhibits strong genetic differentiation within the Canary Islands and, in combination with other related continental species, provides an ideal model in which to examine recent differentiation within a closely related group of continental and oceanic island avian species. We analysed DNA sequences from 51 breeding populations and more than 400 individuals in the blue tit complex. Discrepancies in the nuclear and mitochondrial gene trees provided evidence of a complex evolutionary process around the Mediterranean Basin. Coalescent analyses revealed gene flow between C. caeruleus and C. teneriffae suggesting a dynamic process with multiple phases of colonization and geographic overlapping ranges. Microsatellite data indicated strong genetic differentiation among the Canary Islands and between the Canary archipelago and the close continental areas, indicating limited contemporary gene flow. Diversification of the blue tit complex is estimated to have started during the early Pliocene (≈ 5 Ma), coincident with the end of Messinian salinity crisis. Phylogenetic analyses indicated that the North African blue tit is derived from the Canary blue tits, a pattern is avian 'back colonization' that contrasts with more traditionally held views of islands being sinks rather than sources. PMID:21880092

  10. Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response.

    Science.gov (United States)

    Gurley, Kyle A; Elliott, Sarah A; Simakov, Oleg; Schmidt, Heiko A; Holstein, Thomas W; Sánchez Alvarado, Alejandro

    2010-11-01

    Regeneration is widespread throughout the animal kingdom, but our molecular understanding of this process in adult animals remains poorly understood. Wnt/β-catenin signaling plays crucial roles throughout animal life from early development to adulthood. In intact and regenerating planarians, the regulation of Wnt/β-catenin signaling functions to maintain and specify anterior/posterior (A/P) identity. Here, we explore the expression kinetics and RNAi phenotypes for secreted members of the Wnt signaling pathway in the planarian Schmidtea mediterranea. Smed-wnt and sFRP expression during regeneration is surprisingly dynamic and reveals fundamental aspects of planarian biology that have been previously unappreciated. We show that after amputation, a wounding response precedes rapid re-organization of the A/P axis. Furthermore, cells throughout the body plan can mount this response and reassess their new A/P location in the complete absence of stem cells. While initial stages of the amputation response are stem cell independent, tissue remodeling and the integration of a new A/P address with anatomy are stem cell dependent. We also show that WNT5 functions in a reciprocal manner with SLIT to pattern the planarian mediolateral axis, while WNT11-2 patterns the posterior midline. Moreover, we perform an extensive phylogenetic analysis on the Smed-wnt genes using a method that combines and integrates both sequence and structural alignments, enabling us to place all nine genes into Wnt subfamilies for the first time. PMID:20707997

  11. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  12. Catalytic nanomedicine technology: copper complexes loaded on titania nanomaterials as cytotoxic agents of cancer cell.

    Science.gov (United States)

    Lopez, Tessy; Ortiz-Islas, Emma; Guevara, Patricia; Gómez, Esteban

    2013-01-01

    The anticancer properties of pure copper (II) acetate and copper (II) acetylacetonate, alone and loaded on functionalized sol-gel titania (TiO(2)), were determined in four different cancer cell lines (C6, RG2, B16, and U373), using increasing concentrations of these compounds. The copper complexes were loaded onto the TiO(2) network during its preparation by the solgel process. Once copper-TiO(2) materials were obtained, these were characterized by several physical-chemical techniques. An in vitro copper complex-release test was developed in an aqueous medium at room temperature and monitored by ultraviolet spectroscopy. The toxic effect of the copper complexes, alone and loaded on TiO(2), was determined using a cell viability 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, when cancer cells were treated with increasing concentrations (15.75-1000 mg/mL) of these. Characterization studies revealed that the addition of copper complexes to the TiO(2) sol-gel network during its preparation, did not generate changes in the molecular structure of the complexes. The surface area, pore volume, and pore diameter were affected by the copper complex additions and by the crystalline phases obtained. The kinetic profiles of both copper complexes released indicated two different stages of release: The first one was governed by first-order kinetics and the second was governed by zero-order kinetics. The cell viability assay revealed a cytotoxic effect of copper complexes, copper-TiO(2), and cisplatin in a dose-dependent response for all the cell lines; however, the copper complexes exhibited a better cytotoxic effect than the cisplatin compound. TiO(2) alone presented a minor cytotoxicity for C6 and B16 cells; however, it did not cause any toxic effect on the RG2 and U373 cells, which indicates its high biocompatibility with these cells. PMID:23413123

  13. Genome sequence of the pattern forming Paenibacillus vortex bacterium reveals potential for thriving in complex environments

    Directory of Open Access Journals (Sweden)

    Leshkowitz Dena

    2010-12-01

    Full Text Available Abstract Background The pattern-forming bacterium Paenibacillus vortex is notable for its advanced social behavior, which is reflected in development of colonies with highly intricate architectures. Prior to this study, only two other Paenibacillus species (Paenibacillus sp. JDR-2 and Paenibacillus larvae have been sequenced. However, no genomic data is available on the Paenibacillus species with pattern-forming and complex social motility. Here we report the de novo genome sequence of this Gram-positive, soil-dwelling, sporulating bacterium. Results The complete P. vortex genome was sequenced by a hybrid approach using 454 Life Sciences and Illumina, achieving a total of 289× coverage, with 99.8% sequence identity between the two methods. The sequencing results were validated using a custom designed Agilent microarray expression chip which represented the coding and the non-coding regions. Analysis of the P. vortex genome revealed 6,437 open reading frames (ORFs and 73 non-coding RNA genes. Comparative genomic analysis with 500 complete bacterial genomes revealed exceptionally high number of two-component system (TCS genes, transcription factors (TFs, transport and defense related genes. Additionally, we have identified genes involved in the production of antimicrobial compounds and extracellular degrading enzymes. Conclusions These findings suggest that P. vortex has advanced faculties to perceive and react to a wide range of signaling molecules and environmental conditions, which could be associated with its ability to reconfigure and replicate complex colony architectures. Additionally, P. vortex is likely to serve as a rich source of genes important for agricultural, medical and industrial applications and it has the potential to advance the study of social microbiology within Gram-positive bacteria.

  14. Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

    Science.gov (United States)

    Dizhe, E B; Ignatovich, I A; Burov, S V; Pohvoscheva, A V; Akifiev, B N; Efremov, A M; Perevozchikov, A P; Orlov, S V

    2006-12-01

    This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene. PMID:17223788

  15. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  16. Brain responses in humans reveal ideal observer-like sensitivity to complex acoustic patterns.

    Science.gov (United States)

    Barascud, Nicolas; Pearce, Marcus T; Griffiths, Timothy D; Friston, Karl J; Chait, Maria

    2016-02-01

    We use behavioral methods, magnetoencephalography, and functional MRI to investigate how human listeners discover temporal patterns and statistical regularities in complex sound sequences. Sensitivity to patterns is fundamental to sensory processing, in particular in the auditory system, because most auditory signals only have meaning as successions over time. Previous evidence suggests that the brain is tuned to the statistics of sensory stimulation. However, the process through which this arises has been elusive. We demonstrate that listeners are remarkably sensitive to the emergence of complex patterns within rapidly evolving sound sequences, performing on par with an ideal observer model. Brain responses reveal online processes of evidence accumulation--dynamic changes in tonic activity precisely correlate with the expected precision or predictability of ongoing auditory input--both in terms of deterministic (first-order) structure and the entropy of random sequences. Source analysis demonstrates an interaction between primary auditory cortex, hippocampus, and inferior frontal gyrus in the process of discovering the regularity within the ongoing sound sequence. The results are consistent with precision based predictive coding accounts of perceptual inference and provide compelling neurophysiological evidence of the brain's capacity to encode high-order temporal structure in sensory signals. PMID:26787854

  17. Phase sensitivity of complex cells in primary visual cortex.

    Science.gov (United States)

    Hietanen, M A; Cloherty, S L; van Kleef, J P; Wang, C; Dreher, B; Ibbotson, M R

    2013-05-01

    Neurons in the primary visual cortex are often classified as either simple or complex based on the linearity (or otherwise) of their response to spatial luminance contrast. In practice, classification is typically based on Fourier analysis of a cell's response to an optimal drifting sine-wave grating. Simple cells are generally considered to be linear and produce responses modulated at the fundamental frequency of the stimulus grating. In contrast, complex cells exhibit significant nonlinearities that reduce the response at the fundamental frequency. Cells can therefore be easily and objectively classified based on the relative modulation of their responses - the ratio of the phase-sensitive response at the fundamental frequency of the stimulus (F₁) to the phase-invariant sustained response (F₀). Cells are classified as simple if F₁/F₀>1 and complex if F₁/F₀<1. This classification is broadly consistent with criteria based on the spatial organisation of cells' receptive fields and is accordingly presumed to reflect disparate functional roles of simple and complex cells in coding visual information. However, Fourier analysis of spiking responses is sensitive to the number of spikes available - F₁/F₀ increases as the number of spikes is reduced, even for phase-invariant complex cells. Moreover, many complex cells encountered in the laboratory exhibit some phase sensitivity, evident as modulation of their responses at the fundamental frequency. There currently exists no objective quantitative means of assessing the significance or otherwise of these modulations. Here we derive a statistical basis for objectively assessing whether the modulation of neuronal responses is reliable, thereby adding a level of statistical certainty to measures of phase sensitivity. We apply our statistical analysis to neuronal responses to moving sine-wave gratings recorded from 367 cells in cat primary visual cortex. We find that approximately 60% of complex cells exhibit

  18. Biomimetic emulsions reveal the effect of homeostatic pressure on cell-cell adhesion

    CERN Document Server

    Pontani, Lea-Laetitia; Viasnoff, Virgile; Brujic, Jasna

    2012-01-01

    Cell-cell contacts in tissues are continuously subject to mechanical forces due to homeostatic pressure and active cytoskeleton dynamics. While much is known about the molecular pathways of adhesion, the role of mechanics is less well understood. To isolate the role of pressure we present a dense packing of functionalized emulsion droplets in which surface interactions are tuned to mimic those of real cells. By visualizing the microstructure in 3D we find that a threshold compression force is necessary to overcome electrostatic repulsion and surface elasticity and establish protein-mediated adhesion. Varying the droplet interaction potential maps out a phase diagram for adhesion as a function of force and salt concentration. Remarkably, fitting the data with our theoretical model predicts binder concentrations in the adhesion areas that are similar to those found in real cells. Moreover, we quantify the adhesion size dependence on the applied force and thus reveal adhesion strengthening with increasing homeos...

  19. Labile complexes facilitate cadmium uptake by Caco-2 cells

    International Nuclear Information System (INIS)

    The Free Ion Activity Model (FIAM) predicts that metal uptake in biota is related to the free ion activity in the external solution and that metal complexes do not contribute. However, studies with plants have shown that labile metal complexes enhance metal bioavailability when the uptake is rate-limited by transport of the free ion in solution to the uptake site. Here, the role of labile complexes of Cd on metal bioavailability was assessed using Caco-2 cells, the cell model for intestinal absorption. At low Cd2+ concentration (1 nM), the CdCln2−n complexes contributed to the uptake almost to the same extent as the free ion. At large Cd2+ concentration (10 μM), the contribution of the complexes was much smaller. At constant Cd2+ concentration, Cd intake in the cells from solutions containing synthetic ligands such as EDTA increased as the dissociation rate of the cadmium complexes increased, and correlated well with the Cd diffusion flux in solution measured with the Diffusive Gradient in Thin Films technique (DGT). The Cd intake fluxes in the cells were well predicted assuming that the specific uptake is limited by diffusion of the free Cd2+ ion to the cell surface. Our results underline that speciation of Cd has a major effect on its uptake by intestinal cells, but the availability is not simply related to the free ion concentration. Labile complexes of Cd enhance metal bioavailability in these cells, likely by alleviating diffusive limitations. - Highlights: ► We examined the role of labile complexes on the uptake of Cd and Zn by Caco-2 cells. ► The availability of Cd and Zn is not simply related to the free ion activity. ► Labile complexes of Cd enhance metal bioavailability in the Caco-2 cells at low free ion activities. ► The active uptake is limited by diffusion of the free Cd2+ ion to the cell surface.

  20. High-resolution crystal structure of Streptococcus pyogenes β-NAD+ glycohydrolase in complex with its endogenous inhibitor IFS reveals a highly water-rich interface

    International Nuclear Information System (INIS)

    The crystal structure of the complex between the C-terminal domain of Streptococcus pyogenes β-NAD+ glycohydrolase and an endogenous inhibitor for SPN was determined at 1.70 Å. It reveals that the interface between the two proteins is highly rich in water molecules. One of the virulence factors produced by Streptococcus pyogenes is β-NAD+ glycohydrolase (SPN). S. pyogenes injects SPN into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. As SPN is toxic to bacterial cells themselves, S. pyogenes possesses the ifs gene that encodes an endogenous inhibitor for SPN (IFS). IFS is localized intracellularly and forms a complex with SPN. This intracellular complex must be dissociated during export through the cell envelope. To provide a structural basis for understanding the interactions between SPN and IFS, the complex was overexpressed between the mature SPN (residues 38–451) and the full-length IFS (residues 1–161), but it could not be crystallized. Therefore, limited proteolysis was used to isolate a crystallizable SPNct–IFS complex, which consists of the SPN C-terminal domain (SPNct; residues 193–451) and the full-length IFS. Its crystal structure has been determined by single anomalous diffraction and the model refined at 1.70 Å resolution. Interestingly, our high-resolution structure of the complex reveals that the interface between SPNct and IFS is highly rich in water molecules and many of the interactions are water-mediated. The wet interface may facilitate the dissociation of the complex for translocation across the cell envelope

  1. High-resolution crystal structure of Streptococcus pyogenes β-NAD{sup +} glycohydrolase in complex with its endogenous inhibitor IFS reveals a highly water-rich interface

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Ji Young; An, Doo Ri; Yoon, Hye-Jin [Seoul National University, Seoul 151-747 (Korea, Republic of); Kim, Hyoun Sook [Seoul National University, Seoul 151-747 (Korea, Republic of); Seoul National University, Seoul 151-742 (Korea, Republic of); Lee, Sang Jae [Seoul National University, Seoul 151-742 (Korea, Republic of); Im, Ha Na; Jang, Jun Young [Seoul National University, Seoul 151-747 (Korea, Republic of); Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Seoul National University, Seoul 151-747 (Korea, Republic of); Seoul National University, Seoul 151-747 (Korea, Republic of)

    2013-11-01

    The crystal structure of the complex between the C-terminal domain of Streptococcus pyogenes β-NAD{sup +} glycohydrolase and an endogenous inhibitor for SPN was determined at 1.70 Å. It reveals that the interface between the two proteins is highly rich in water molecules. One of the virulence factors produced by Streptococcus pyogenes is β-NAD{sup +} glycohydrolase (SPN). S. pyogenes injects SPN into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. As SPN is toxic to bacterial cells themselves, S. pyogenes possesses the ifs gene that encodes an endogenous inhibitor for SPN (IFS). IFS is localized intracellularly and forms a complex with SPN. This intracellular complex must be dissociated during export through the cell envelope. To provide a structural basis for understanding the interactions between SPN and IFS, the complex was overexpressed between the mature SPN (residues 38–451) and the full-length IFS (residues 1–161), but it could not be crystallized. Therefore, limited proteolysis was used to isolate a crystallizable SPN{sub ct}–IFS complex, which consists of the SPN C-terminal domain (SPN{sub ct}; residues 193–451) and the full-length IFS. Its crystal structure has been determined by single anomalous diffraction and the model refined at 1.70 Å resolution. Interestingly, our high-resolution structure of the complex reveals that the interface between SPN{sub ct} and IFS is highly rich in water molecules and many of the interactions are water-mediated. The wet interface may facilitate the dissociation of the complex for translocation across the cell envelope.

  2. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W. (SVIMR-A); (Hanson)

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  3. RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

    Science.gov (United States)

    Lescale, Chloé; Abramowski, Vincent; Bedora-Faure, Marie; Murigneux, Valentine; Vera, Gabriella; Roth, David B; Revy, Patrick; de Villartay, Jean-Pierre; Deriano, Ludovic

    2016-01-01

    XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly. PMID:26833222

  4. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Athanasia D Panopoulos; Margaret Lutz; W Travis Berggren; Kun Zhang; Ronald M Evans; Gary Siuzdak; Juan Carlos Izpisua Belmonte; Oscar Yanes; SergioRuiz; Yasuyuki S Kida; Dinh Diep; Ralf Tautenhahn; Aida Herrerias; Erika M Batchelder; Nongluk Plongthongkum

    2012-01-01

    Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs)remains largely unexplored.Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration.Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency.Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies.We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming.Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence.

  5. Molecular analysis of endothelial progenitor cell (EPC subtypes reveals two distinct cell populations with different identities

    Directory of Open Access Journals (Sweden)

    Simpson David A

    2010-05-01

    Full Text Available Abstract Background The term endothelial progenitor cells (EPCs is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs and outgrowth endothelial cells (OECs. Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN with links to immunity and inflammation (TLRs, CD14, HLAs, whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

  6. Robustness and adaptation reveal plausible cell cycle controlling subnetwork in Saccharomyces cerevisiae.

    Science.gov (United States)

    Huang, Jiun-Yan; Huang, Chi-Wei; Kao, Kuo-Ching; Lai, Pik-Yin

    2013-04-10

    Biological systems are often organized spatially and temporally by multi-scale functional subsystems (modules). A specific subcellular process often corresponds to a subsystem composed of some of these interconnected modules. Accurate identification of system-level modularity organization from the large scale networks can provide valuable information on subsystem models of subcellular processes or physiological phenomena. Computational identification of functional modules from the large scale network is the key approach to solve the complexity of modularity in the past decade, but the overlapping and multi-scale nature of modules often renders unsatisfactory results in these methods. Most current methods for modularity detection are optimization-based and suffered from the drawback of size resolution limit. It is difficult to trace the origin of the unsatisfactory results, which may be due to poor data, inappropriate objective function selection or simply resulted from natural evolution, and hence no system-level accurate modular models for subcellular processes can be offered. Motivated by the idea of evolution with robustness and adaption as guiding principles, we propose a novel approach that can identify significant multi-scale overlapping modules that are sufficiently accurate at the system and subsystem levels, giving biological insights for subcellular processes. The success of our evolution strategy method is demonstrated by applying to the yeast protein-protein interaction network. Functional subsystems of important physiological phenomena can be revealed. In particular, the cell cycle controlling network is selected for detailed discussion. The cell cycle subcellular processes in yeast can be successfully dissected into functional modules of cell cycle control, cell size check point, spindle assembly checkpoint, and DNA damage check point in G2/M and S phases. The interconnections between check points and cell cycle control modules provide clues on the

  7. Capillary hydrodynamic chromatography reveals temporal profiles of cell aggregates.

    Science.gov (United States)

    Tang, Ya-Ru; Huang, Hsin-Yi; Hu, Jie-Bi; Rattinam, Rajesh; Li, Chun-Hsien; Chen, Yu-Chie; Urban, Pawel L

    2016-03-01

    Microbial cells are known to form aggregates. Such aggregates can be found in various matrices; for example, functional drinks. Capillary hydrodynamic chromatography (HDC) enables separation of particles by size using nanoliter-scale volumes of samples. Here we propose an approach based on HDC for characterisation of real samples containing aggregated and non-aggregated bacterial and fungal cells. Separation of cells and cell aggregates in HDC arises from the parabolic flow profile under laminar flow conditions. In the presented protocol, hydrodynamic separation is coupled with different on-line and off-line detectors (light absorption/scattering and microscopy). The method has successfully been applied in the monitoring of dynamic changes in the microbiome of probiotic drinks. Chromatographic profiles of yogurt and kefir samples obtained at different times during fermentation are in a good agreement with microscopic images. Moreover, thanks to the implementation of an area imaging detector, capillary HDC could be multiplexed and used to profile spatial gradients in cell suspensions, which arise in the course of sedimentation of cells and cell aggregates. This result shows compatibility of sedimentation analysis and capillary HDC. We believe that the approach may find applications in the profiling of functional foods and other matrices containing aggregated bioparticles. PMID:26873471

  8. Gene-disease network analysis reveals functional modules in mendelian, complex and environmental diseases.

    Directory of Open Access Journals (Sweden)

    Anna Bauer-Mehren

    Full Text Available BACKGROUND: Scientists have been trying to understand the molecular mechanisms of diseases to design preventive and therapeutic strategies for a long time. For some diseases, it has become evident that it is not enough to obtain a catalogue of the disease-related genes but to uncover how disruptions of molecular networks in the cell give rise to disease phenotypes. Moreover, with the unprecedented wealth of information available, even obtaining such catalogue is extremely difficult. PRINCIPAL FINDINGS: We developed a comprehensive gene-disease association database by integrating associations from several sources that cover different biomedical aspects of diseases. In particular, we focus on the current knowledge of human genetic diseases including mendelian, complex and environmental diseases. To assess the concept of modularity of human diseases, we performed a systematic study of the emergent properties of human gene-disease networks by means of network topology and functional annotation analysis. The results indicate a highly shared genetic origin of human diseases and show that for most diseases, including mendelian, complex and environmental diseases, functional modules exist. Moreover, a core set of biological pathways is found to be associated with most human diseases. We obtained similar results when studying clusters of diseases, suggesting that related diseases might arise due to dysfunction of common biological processes in the cell. CONCLUSIONS: For the first time, we include mendelian, complex and environmental diseases in an integrated gene-disease association database and show that the concept of modularity applies for all of them. We furthermore provide a functional analysis of disease-related modules providing important new biological insights, which might not be discovered when considering each of the gene-disease association repositories independently. Hence, we present a suitable framework for the study of how genetic and

  9. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  10. Transcriptome network analysis reveals candidate genes for renal cell carcinoma

    OpenAIRE

    Wei Zhai; Yun-Fei Xu; Min Liu; Jun-Hua Zheng

    2012-01-01

    Context: Renal cell carcinoma (RCC) is a kidney cancer that originates in renal parenchyma and it is the most common type of kidney cancer with approximately 80% lethal cases. Aims: To interpret the mechanism, explore the regulation of TF-target genes and TF-pathway, and identify the potential key genes of renal cell carcinoma. Settings and Design: After constructing a regulation network from differently expressed genes and transcription factors, pathway regulation network and gene onto...

  11. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    OpenAIRE

    Laura-Roxana Stingaciu; Hugh O’Neill; Michelle Liberton; Urban, Volker S.; Himadri B. Pakrasi; Michael Ohl

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membran...

  12. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    Energy Technology Data Exchange (ETDEWEB)

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M. (UCSC); (LBNL)

    2012-07-18

    Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible 'pocket' domain loop such that it mimics and directly blocks E2F transactivation domain (E2F{sup TD}) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2F{sup TD}-pocket association and protein binding to the pocket 'LxCxE' site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

  13. Crystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding Cofactor

    Energy Technology Data Exchange (ETDEWEB)

    Mirza, I.; Yachnin, B; Wang, S; Grosse, S; Bergeron, H; Imura, A; Iwaki, H; Hasegawa, Y; Lau, P; Berghuis, A

    2009-01-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O{sub 2} as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP+ in two distinct states, to resolutions of 2.3 and 2.2 {angstrom}. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  14. Microbial dark matter ecogenomics reveals complex synergistic networks in a methanogenic bioreactor.

    Science.gov (United States)

    Nobu, Masaru K; Narihiro, Takashi; Rinke, Christian; Kamagata, Yoichi; Tringe, Susannah G; Woyke, Tanja; Liu, Wen-Tso

    2015-08-01

    Ecogenomic investigation of a methanogenic bioreactor degrading terephthalate (TA) allowed elucidation of complex synergistic networks of uncultivated microorganisms, including those from candidate phyla with no cultivated representatives. Our previous metagenomic investigation proposed that Pelotomaculum and methanogens may interact with uncultivated organisms to degrade TA; however, many members of the community remained unaddressed because of past technological limitations. In further pursuit, this study employed state-of-the-art omics tools to generate draft genomes and transcriptomes for uncultivated organisms spanning 15 phyla and reports the first genomic insight into candidate phyla Atribacteria, Hydrogenedentes and Marinimicrobia in methanogenic environments. Metabolic reconstruction revealed that these organisms perform fermentative, syntrophic and acetogenic catabolism facilitated by energy conservation revolving around H2 metabolism. Several of these organisms could degrade TA catabolism by-products (acetate, butyrate and H2) and syntrophically support Pelotomaculum. Other taxa could scavenge anabolic products (protein and lipids) presumably derived from detrital biomass produced by the TA-degrading community. The protein scavengers expressed complementary metabolic pathways indicating syntrophic and fermentative step-wise protein degradation through amino acids, branched-chain fatty acids and propionate. Thus, the uncultivated organisms may interact to form an intricate syntrophy-supported food web with Pelotomaculum and methanogens to metabolize catabolic by-products and detritus, whereby facilitating holistic TA mineralization to CO2 and CH4. PMID:25615435

  15. Complex Contact-Based Dynamics of Microsphere Monolayers Revealed by Resonant Attenuation of Surface Acoustic Waves

    Science.gov (United States)

    Hiraiwa, M.; Abi Ghanem, M.; Wallen, S. P.; Khanolkar, A.; Maznev, A. A.; Boechler, N.

    2016-05-01

    Contact-based vibrations play an essential role in the dynamics of granular materials. Significant insights into vibrational granular dynamics have previously been obtained with reduced-dimensional systems containing macroscale particles. We study contact-based vibrations of a two-dimensional monolayer of micron-sized spheres on a solid substrate that forms a microscale granular crystal. Measurements of the resonant attenuation of laser-generated surface acoustic waves reveal three collective vibrational modes that involve displacements and rotations of the microspheres, as well as interparticle and particle-substrate interactions. To identify the modes, we tune the interparticle stiffness, which shifts the frequency of the horizontal-rotational resonances while leaving the vertical resonance unaffected. From the measured contact resonance frequencies we determine both particle-substrate and interparticle contact stiffnesses and find that the former is an order of magnitude larger than the latter. This study paves the way for investigating complex contact-based dynamics of microscale granular crystals and yields a new approach to studying micro- to nanoscale contact mechanics in multiparticle networks.

  16. Meditation effects within the hippocampal complex revealed by voxel-based morphometry and cytoarchitectonic probabilistic mapping

    Directory of Open Access Journals (Sweden)

    Eileen eLuders

    2013-07-01

    Full Text Available Scientific studies addressing anatomical variations in meditators’ brains have emerged rapidly over the last few years, where significant links are most frequently reported with respect to gray matter (GM. To advance prior work, this study examined GM characteristics in a large sample of 100 subjects (50 meditators, 50 controls, where meditators have been practicing close to twenty years, on average. A standard, whole-brain voxel-based morphometry approach was applied and revealed significant meditation effects in the vicinity of the hippocampus, showing more GM in meditators than in controls as well as positive correlations with the number of years practiced. However, the hippocampal complex is regionally segregated by architecture, connectivity, and functional relevance. Thus, to establish differential effects within the hippocampal formation (cornu ammonis, fascia dentate, entorhinal cortex, subiculum as well as the hippocampal-amygdaloid transition area, we utilized refined cytoarchitectonic probabilistic maps of (peri- hippocampal subsections. Significant meditation effects were observed within the subiculum specifically. Since the subiculum is known to play a key role in stress regulation and meditation is an established form of stress reduction, these GM findings may reflect neuronal preservation in long-term meditators – perhaps due to an attenuated release of stress hormones and decreased neurotoxicity.

  17. Meditation effects within the hippocampal complex revealed by voxel-based morphometry and cytoarchitectonic probabilistic mapping.

    Science.gov (United States)

    Luders, Eileen; Kurth, Florian; Toga, Arthur W; Narr, Katherine L; Gaser, Christian

    2013-01-01

    Scientific studies addressing anatomical variations in meditators' brains have emerged rapidly over the last few years, where significant links are most frequently reported with respect to gray matter (GM). To advance prior work, this study examined GM characteristics in a large sample of 100 subjects (50 meditators, 50 controls), where meditators have been practicing close to 20 years, on average. A standard, whole-brain voxel-based morphometry approach was applied and revealed significant meditation effects in the vicinity of the hippocampus, showing more GM in meditators than in controls as well as positive correlations with the number of years practiced. However, the hippocampal complex is regionally segregated by architecture, connectivity, and functional relevance. Thus, to establish differential effects within the hippocampal formation (cornu ammonis, fascia dentata, entorhinal cortex, subiculum) as well as the hippocampal-amygdaloid transition area, we utilized refined cytoarchitectonic probabilistic maps of (peri-) hippocampal subsections. Significant meditation effects were observed within the subiculum specifically. Since the subiculum is known to play a key role in stress regulation and meditation is an established form of stress reduction, these GM findings may reflect neuronal preservation in long-term meditators-perhaps due to an attenuated release of stress hormones and decreased neurotoxicity. PMID:23847572

  18. Dynamic localization of electronic excitation in photosynthetic complexes revealed with chiral two-dimensional spectroscopy

    Science.gov (United States)

    Fidler, Andrew F.; Singh, Ved P.; Long, Phillip D.; Dahlberg, Peter D.; Engel, Gregory S.

    2014-02-01

    Time-resolved ultrafast optical probes of chiral dynamics provide a new window allowing us to explore how interactions with such structured environments drive electronic dynamics. Incorporating optical activity into time-resolved spectroscopies has proven challenging because of the small signal and large achiral background. Here we demonstrate that two-dimensional electronic spectroscopy can be adapted to detect chiral signals and that these signals reveal how excitations delocalize and contract following excitation. We dynamically probe the evolution of chiral electronic structure in the light-harvesting complex 2 of purple bacteria following photoexcitation by creating a chiral two-dimensional mapping. The dynamics of the chiral two-dimensional signal directly reports on changes in the degree of delocalization of the excitonic states following photoexcitation. The mechanism of energy transfer in this system may enhance transfer probability because of the coherent coupling among chromophores while suppressing fluorescence that arises from populating delocalized states. This generally applicable spectroscopy will provide an incisive tool to probe ultrafast transient molecular fluctuations that are obscured in non-chiral experiments.

  19. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  20. Secret Chambers: The Insider Story of Cells and Complex Life

    OpenAIRE

    Farid Pazhoohi

    2014-01-01

    Review of Secret Chambers: The Insider Story of Cells and Complex Life. Martin Brasier. 2012. Oxford University Press, UK. Pp. 320 with 15 black and white illustrations and 8 pages of color plates. £16.99 (hardcover). ISBN 9780199644001.

  1. Radiosensitization of EMT6 cells by four platinum complexes.

    Science.gov (United States)

    Teicher, B A; Rockwell, S; Lee, J B

    1985-05-01

    The greatest research effort in producing radiation sensitizers has been directed toward organic compounds. However, platinum complexes also have radiosensitizing capabilities, perhaps because they bind to DNA. The compound described here are dichloro complexes of bivalent platinum with one or two potentially radiosensitizing ligands. The radiosensitization of oxygenated and hypoxic exponentially growing EMT6 cells in vitro was measured. The dose modifying factors obtained with 200 microM and 400 microM trans-bis(2-nitroimidazole)dichloroplatinum II (NIPt) in hypoxic cells were 1.5 and 2.1, respectively. For trans-bis(2-amino-5-nitrothiazole)dichloroplatinum II (Plant) under the same conditions, the dose modifying factor was 1.5 at 200 microM and 1.8 at 400 microM. Neither compound sensitized oxygenated cells when tested similar protocols. Unlike the trans complexes, (1,2-diamino-4-nitrobenzene)dichloroplatinum II (Plato) was cytotoxic toward the hypoxic cells in the absence of X rays. The time course of cytotoxicity for 100 microM Plato in exponentially growing cells showed rapid killing of hypoxic cells, and much less toxicity toward oxygenated cells. In radiosensitization studies, dose modifying factors of 1.6 and 2.0 were found with 200 microM and 400 microM Plato in hypoxic cells. The compound did not sensitize aerobic cells. The well-known platinum complex cis-dipyridinedichloroplatinum II (PyPt) represents a cis-platinum heterocyclic aromatic complex that does not have a nitro-functionality. The dose modifying factor obtained with 400 microM PyPt in hypoxic cells was 1.7. On a molar basis, the nitro-functional platinum complexes appear to be more effective as hypoxic cell radiosensitizers than the corresponding free ligands. PMID:4039304

  2. A Phosphorylated Pseudokinase Complex Controls Cell Wall Synthesis in Mycobacteria

    OpenAIRE

    Gee, Christine L.; Papavinasasundaram, Kadamba G.; Blair, Sloane R.; Baer, Christina E.; Falick, Arnold M.; King, David S.; Griffin, Jennifer E.; Venghatakrishnan, Harene; Zukauskas, Andrew; Wei, Jun-Rong; Dhiman, Rakesh K.; Crick, Dean C.; Rubin, Eric J.; Sassetti, Christopher M.; Alber, Tom

    2012-01-01

    Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structura...

  3. Dynamics of ribosome scanning and recycling revealed by translation complex profiling.

    Science.gov (United States)

    Archer, Stuart K; Shirokikh, Nikolay E; Beilharz, Traude H; Preiss, Thomas

    2016-07-28

    Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5′ UTRs. Scanning SSU have 5′-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5′ start site but differ at the 3′ end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences

  4. Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System.

    Science.gov (United States)

    Wang, Zining; Ji, Jingyun; Peng, Di; Ma, Feng; Cheng, Genhong; Qin, F Xiao-Feng

    2016-05-15

    Induction of type I IFN (IFN-I) is essential for host antiviral immune responses. However, IFN-I also plays divergent roles in antibacterial immunity, persistent viral infections, autoimmune diseases, and tumorigenesis. IFN regulatory factor 3 (IRF3) is the master transcription factor that controls IFN-I production via phosphorylation-dependent dimerization in most cell types in response to viral infections and various innate stimuli by pathogen-associated molecular patterns (PAMPs). To monitor the dynamic process of IRF3 activation, we developed a novel IRF3 dimerization reporter based on bimolecular luminescence complementation (BiLC) techniques, termed the IRF3-BiLC reporter. Robust induction of luciferase activity of the IRF3-BiLC reporter was observed upon viral infection and PAMP stimulation with a broad dynamic range. Knockout of TANK-binding kinase 1, the critical upstream kinase of IRF3, as well as the mutation of serine 386, the essential phosphorylation site of IRF3, completely abolished the luciferase activity of IRF3-BiLC reporter, confirming the authenticity of IRF3 activation. Taken together, these results demonstrated that the IRF3-BiLC reporter is a highly specific, reliable, and sensitive system to measure IRF3 activity. Using this reporter system, we further observed that the temporal pattern and magnitude of IRF3 activation induced by various PAMPs are highly complex with distinct cell type-specific characteristics, and IRF3 dimerization is a direct regulatory node for IFN-α/β receptor-mediated feed-forward regulation and crosstalk with other pathways. Therefore, the IRF3-BiLC reporter has multiple potential applications, including mechanistic studies as well as the identification of novel compounds that can modulate IRF3 activation. PMID:27045107

  5. Genome-Wide Analysis Revealed the Complex Regulatory Network of Brassinosteroid Effects in Photomorphogenesis

    Institute of Scientific and Technical Information of China (English)

    Li Song; Xiao-Yi Zhou; Li Li; Liang-Jiao Xue; Xi Yang; Hong-Wei Xue

    2009-01-01

    Light and brassinosteroids (BRs) have been proved to be crucial in regulating plant growth and development;however,the mechanism of how they synergistically function is still largely unknown.To explore the underlying mechanisms in photomorphogenesis,genome-wide analyses were carried out through examining the gene expressions of the dark-grown WT or BR biosynthesis-defective mutant det2 seedlings in the presence of light stimuli or exogenous Brassinolide (BL).Results showed that BR deficiency stimulates,while BL treatment suppresses,the expressions of lightresponsive genes and photomorphogenesis,confirming the negative effects of BR in photomorphogenesis.This is consistent with the specific effects of BR on the expression of genes involved in cell wall modification,cellular metabolism and energy utilization during dark-light transition.Further analysis revealed that hormone biosynthesis and signaling-related genes,especially those of auxin,were altered under BL treatment or light stimuli,indicating that BR may modulate photomorphogenesis through synergetic regulation with other hormones.Additionally,suppressed ubiquitin-cycle pathway during light-dark transition hinted the presence of a complicated network among light,hormone,and protein degradation.The study provides the direct evidence of BR effects in photomorphogenesis and identified the genes involved in BR and light signaling pathway,which will help to elucidate the molecular mechanism of plant photomorphogenesis.

  6. Markov numbers and Lagrangian cell complexes in the complex projective plane

    OpenAIRE

    Evans, Jonathan David; Smith, Ivan

    2016-01-01

    We study Lagrangian embeddings of a class of two-dimensional cell complexes $L_{p,q}$ into the complex projective plane. These cell complexes, which we call pinwheels, arise naturally in algebraic geometry as vanishing cycles for quotient singularities of type $\\frac{1}{p^2}(pq-1,1)$ (Wahl singularities). We show that if a pinwheel admits a Lagrangian embedding into $\\mathbf{CP}^2$ then $p$ is a Markov number and we completely characterise $q$. We also show that a collection of Lagrangian pin...

  7. Dynamic Modelling Reveals 'Hotspots' on the Pathway to Enzyme-Substrate Complex Formation.

    Directory of Open Access Journals (Sweden)

    Shane E Gordon

    2016-03-01

    Full Text Available Dihydrodipicolinate synthase (DHDPS catalyzes the first committed step in the diaminopimelate pathway of bacteria, yielding amino acids required for cell wall and protein biosyntheses. The essentiality of the enzyme to bacteria, coupled with its absence in humans, validates DHDPS as an antibacterial drug target. Conventional drug design efforts have thus far been unsuccessful in identifying potent DHDPS inhibitors. Here, we make use of contemporary molecular dynamics simulation and Markov state models to explore the interactions between DHDPS from the human pathogen Staphylococcus aureus and its cognate substrate, pyruvate. Our simulations recover the crystallographic DHDPS-pyruvate complex without a priori knowledge of the final bound structure. The highly conserved residue Arg140 was found to have a pivotal role in coordinating the entry of pyruvate into the active site from bulk solvent, consistent with previous kinetic reports, indicating an indirect role for the residue in DHDPS catalysis. A metastable binding intermediate characterized by multiple points of intermolecular interaction between pyruvate and key DHDPS residue Arg140 was found to be a highly conserved feature of the binding trajectory when comparing alternative binding pathways. By means of umbrella sampling we show that these binding intermediates are thermodynamically metastable, consistent with both the available experimental data and the substrate binding model presented in this study. Our results provide insight into an important enzyme-substrate interaction in atomistic detail that offers the potential to be exploited for the discovery of more effective DHDPS inhibitors and, in a broader sense, dynamic protein-drug interactions.

  8. Transport and uptake effects of marine complex lipid liposomes in small intestinal epithelial cell models.

    Science.gov (United States)

    Du, Lei; Yang, Yu-Hong; Xu, Jie; Wang, Yu-Ming; Xue, Chang-Hu; Kurihara, Hideyuki; Takahashi, Koretaro

    2016-04-20

    Nowadays, marine complex lipids, including starfish phospholipids (SFP) and cerebrosides (SFC) separated from Asterias amurensis as well as sea cucumber phospholipids (SCP) and cerebrosides (SCC) isolated from Cucumaria frondosa, have received much attention because of their potent biological activities. However, little information is known on the transport and uptake of these lipids in liposome forms in small intestinal cells. Therefore, this study was undertaken to investigate the effects of these complex lipid liposomes on transport and uptake in Caco-2 and M cell monolayer models. The results revealed that SFP and SCP contained 42% and 47.9% eicosapentaenoic acid (EPA), respectively. The average particle sizes of liposomes prepared in this study were from 169 to 189 nm. We found that the transport of the liposomes across the M cell monolayer model was much higher than the Caco-2 cell monolayer model. The liposomes consisting of SFP or SCP showed significantly higher transport and uptake than soy phospholipid (soy-PL) liposomes in both Caco-2 and M cell monolayer models. Our results also exhibited that treatment with 1 mM liposomes composed of SFP or SCP for 3 h tended to increase the EPA content in phospholipid fractions of both differentiated Caco-2 and M cells. Moreover, it was also found that the hybrid liposomes consisting of SFP/SFC/cholesterol (Chol) revealed higher transport and uptake across the M cell monolayer in comparison with other liposomes. Furthermore, treatment with SFP/SFC/Chol liposomes could notably decrease the trans-epithelial electrical resistance (TEER) values of Caco-2 and M cell monolayers. The present data also showed that the cell viability of differentiated Caco-2 and M cells was not affected after the treatment with marine complex lipids or soy-PL liposomes. Based on the data in this study, it was suggested that marine complex lipid liposomes exhibit prominent transport and uptake in small intestinal epithelial cell models. PMID

  9. The Nature of Genetic Variation for Complex Traits Revealed by GWAS and Regional Heritability Mapping Analyses.

    Science.gov (United States)

    Caballero, Armando; Tenesa, Albert; Keightley, Peter D

    2015-12-01

    We use computer simulations to investigate the amount of genetic variation for complex traits that can be revealed by single-SNP genome-wide association studies (GWAS) or regional heritability mapping (RHM) analyses based on full genome sequence data or SNP chips. We model a large population subject to mutation, recombination, selection, and drift, assuming a pleiotropic model of mutations sampled from a bivariate distribution of effects of mutations on a quantitative trait and fitness. The pleiotropic model investigated, in contrast to previous models, implies that common mutations of large effect are responsible for most of the genetic variation for quantitative traits, except when the trait is fitness itself. We show that GWAS applied to the full sequence increases the number of QTL detected by as much as 50% compared to the number found with SNP chips but only modestly increases the amount of additive genetic variance explained. Even with full sequence data, the total amount of additive variance explained is generally below 50%. Using RHM on the full sequence data, a slightly larger number of QTL are detected than by GWAS if the same probability threshold is assumed, but these QTL explain a slightly smaller amount of genetic variance. Our results also suggest that most of the missing heritability is due to the inability to detect variants of moderate effect (∼0.03-0.3 phenotypic SDs) segregating at substantial frequencies. Very rare variants, which are more difficult to detect by GWAS, are expected to contribute little genetic variation, so their eventual detection is less relevant for resolving the missing heritability problem. PMID:26482794

  10. Genome evolution in the eremothecium clade of the Saccharomyces complex revealed by comparative genomics.

    Science.gov (United States)

    Wendland, Jürgen; Walther, Andrea

    2011-12-01

    We used comparative genomics to elucidate the genome evolution within the pre-whole-genome duplication genus Eremothecium. To this end, we sequenced and assembled the complete genome of Eremothecium cymbalariae, a filamentous ascomycete representing the Eremothecium type strain. Genome annotation indicated 4712 gene models and 143 tRNAs. We compared the E. cymbalariae genome with that of its relative, the riboflavin overproducer Ashbya (Eremothecium) gossypii, and the reconstructed yeast ancestor. Decisive changes in the Eremothecium lineage leading to the evolution of the A. gossypii genome include the reduction from eight to seven chromosomes, the downsizing of the genome by removal of 10% or 900 kb of DNA, mostly in intergenic regions, the loss of a TY3-Gypsy-type transposable element, the re-arrangement of mating-type loci, and a massive increase of its GC content. Key species-specific events are the loss of MNN1-family of mannosyltransferases required to add the terminal fourth and fifth α-1,3-linked mannose residue to O-linked glycans and genes of the Ehrlich pathway in E. cymbalariae and the loss of ZMM-family of meiosis-specific proteins and acquisition of riboflavin overproduction in A. gossypii. This reveals that within the Saccharomyces complex genome, evolution is not only based on genome duplication with subsequent gene deletions and chromosomal rearrangements but also on fungi associated with specific environments (e.g. involving fungal-insect interactions as in Eremothecium), which have encountered challenges that may be reflected both in genome streamlining and their biosynthetic potential. PMID:22384365

  11. Polymorphism of DNA–anionic liposome complexes reveals hierarchy of ion-mediated interactions

    OpenAIRE

    Liang, Hongjun; Harries, Daniel; Gerard C L Wong

    2005-01-01

    Self-assembled DNA delivery systems based on anionic lipids (ALs) complexed with DNA mediated by divalent cations have been recently introduced as an alternative to cationic lipid–DNA complexes because of their low cytotoxicity. We investigate AL–DNA complexes induced by different cations by using synchrotron small angle x-ray scattering and confocal microscopy to show how different ion-mediated interactions are expressed in the self-assembled structures and phase behavior of AL–DNA complexes...

  12. Radiosensitization of EMT6 cells by four platinum complexes

    Energy Technology Data Exchange (ETDEWEB)

    Teicher, B.A.; Rockwell, S.; Lee, J.B.

    1985-05-01

    The compounds described here are dichloro complexes of bivalent platinum with one or two potentially radiosensitizing ligands. The radiosensitization of oxygenated and hypoxic exponentially growing EMT6 cells in vitro was measured. The dose modifying factors obtained with 200 ..mu..M and 400 ..mu..M trans-bis(2-nitroimidazole)dichloroplatinum II (NIPt) in hypoxic cells were 1.5 and 2.1, respectively. For trans-bis(2-amino-5-nitrothiazole)dichloroplatinum II (Plant) under the same conditions, the dose modifying factor was 1.5 at 200 ..mu..M and 1.8 at 400 ..mu..M. Neither compound sensitized oxygenated cells when tested similar protocols. Unlike the trans complexes (1,2-diamino-4-nitrobenzene)dichloroplatinum II (Plato) was cytotoxic toward the hypoxic cells in the absence of X rays. The time course of cytotoxicity for 100 ..mu..M Plato in exponentially growing cells showed rapid killing of hypoxic cells, and much less toxicity toward oxygenated cells. In radiosensitization studies, dose modifying factors of 1.6 and 2.0 were found with 200 ..mu..M and 400 ..mu..M Plato in hypoxic cells. The compound did not sensitize aerobic cells. The well-known platinum complex cis-dipyridinedichloroplatinum II (PyPt) represents a cis-platinum heterocyclic aromatic complex that does not have a nitro-functionality. The dose modifying factor obtained with 400 ..mu..M PyPt in hypoxic cells was 1.7. On a molar basis, the nitro-functional platinum complexes appear to be more effective as hypoxic cell radiosensitizers than the corresponding free ligands.

  13. Radiosensitization of EMT6 cells by four platinum complexes

    International Nuclear Information System (INIS)

    The compounds described here are dichloro complexes of bivalent platinum with one or two potentially radiosensitizing ligands. The radiosensitization of oxygenated and hypoxic exponentially growing EMT6 cells in vitro was measured. The dose modifying factors obtained with 200 μM and 400 μM trans-bis(2-nitroimidazole)dichloroplatinum II (NIPt) in hypoxic cells were 1.5 and 2.1, respectively. For trans-bis(2-amino-5-nitrothiazole)dichloroplatinum II (Plant) under the same conditions, the dose modifying factor was 1.5 at 200 μM and 1.8 at 400 μM. Neither compound sensitized oxygenated cells when tested similar protocols. Unlike the trans complexes (1,2-diamino-4-nitrobenzene)dichloroplatinum II (Plato) was cytotoxic toward the hypoxic cells in the absence of X rays. The time course of cytotoxicity for 100 μM Plato in exponentially growing cells showed rapid killing of hypoxic cells, and much less toxicity toward oxygenated cells. In radiosensitization studies, dose modifying factors of 1.6 and 2.0 were found with 200 μM and 400 μM Plato in hypoxic cells. The compound did not sensitize aerobic cells. The well-known platinum complex cis-dipyridinedichloroplatinum II (PyPt) represents a cis-platinum heterocyclic aromatic complex that does not have a nitro-functionality. The dose modifying factor obtained with 400 μM PyPt in hypoxic cells was 1.7. On a molar basis, the nitro-functional platinum complexes appear to be more effective as hypoxic cell radiosensitizers than the corresponding free ligands

  14. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  15. Power-cancellation of CW-complexes with few cells

    OpenAIRE

    Llerena, Irene

    1992-01-01

    In this paper, we use the fact that the rings of integer matrices have the power-substitution property in order to obtain a powercancellation property for homotopy types of CW-complexes with one cell in dimensions 0 and 4n and a finite number of cells in dimension 2n.

  16. Nanoparticle-Based Immunocytochemistry Reveals Microarchitecture of the Cell Nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Kyoto : International Federation of Societies for Histochemistry and Cytochemistry, Japan Society of Histochemistry, 2012. [14th International Congress of Histochemistry and Cytochemistry. Kyoto (JP), 26.08.2012-29.08.2012] R&D Projects: GA ČR GAP305/11/2232; GA MŠk LC545; GA MŠk(CZ) LC06063; GA MPO(CZ) FRTI3588 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : PIP2 * NMI * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  17. Nanoparticle-based immunocytochemistry reveals microarchitecture of the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Mérida : CIASEM, AMM, 2011. ---. [Inter-American Congress on Microscopy /11./. 25.09.2011-29.09.2011, Mérida] R&D Projects: GA ČR GAP305/11/2232; GA ČR(CZ) GD204/09/H084; GA MŠk LC545; GA MŠk(CZ) LC06063; GA AV ČR KAN200520704 Institutional research plan: CEZ:AV0Z50520514 Keywords : nanoparticles * immunogold detection * histochemistry * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  18. Nanoparticle-based immunocytochemistry reveals microarchitecture of the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Hozák, Pavel

    Istanbul: Turkish Society for Electron Microscopy, 2011. ---. [National Electron Microscopy Congress /20./. 25.10.2011-28.10.2011, Kemer] R&D Projects: GA ČR GAP305/11/2232; GA ČR(CZ) GD204/09/H084; GA MŠk LC545; GA MŠk(CZ) LC06063; GA MŠk 2B06063; GA AV ČR KAN200520704 Institutional research plan: CEZ:AV0Z50520514 Keywords : nanoparticles * immunogold detection * histochemistry * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology

  19. Paramagnetic Europium Salen Complex and Sickle-Cell Anemia

    Science.gov (United States)

    Wynter, Clive I.; Ryan, D. H.; May, Leopold; Oliver, F. W.; Brown, Eugene; Hoffman, Eugene J.; Bernstein, David

    2005-04-01

    A new europium salen complex, Eu(salen)2NH4, was synthesized, and its composition was confirmed by chemical analysis and infrared spectroscopy. Further characterization was carried out by 151 Eu Mössbauer spectroscopy and magnetic susceptibility measurements. Mössbauer spectroscopic measurements were made at varying temperatures between 9 K and room temperature and a value of Debye temperature of 133 ±5 K was computed. Both Mössbauer and magnetic susceptibility measurements confirmed the paramagnetic behavior of this complex and the trivalent state of the europium ion. In view of the fact that the "odd" paramagnetic molecule NO has been shown to reverse sickling of red blood cells in sickle cell anemia, the interaction between the paramagnetic europium salen complex and sickle cells was examined after incubation with this europium complex and shown to have similar effects.

  20. Anticancer Activity Studies of Ruthenium(II) Complex Toward Human Osteosarcoma HOS Cells.

    Science.gov (United States)

    Zhu, Jian-Wei; Liu, Si-Hong; Zhang, Gui-Qiang; Xu, Hui-Hua; Wang, Yu-Xuan; Wu, Yong; Liu, Ya-Min; Wang, Yan; Liang, Jun-Bo; Guo, Qi-Feng

    2016-08-01

    A new Ru(II) complex [Ru(dmp)2(NMIP)](ClO4)2 (dmp = 2,9-dimethyl-1,10-phenanthroline, NMIP = 2'-(2″-nitro-3″,4″-methylenedioxyphenyl)imidazo[4',5'-f][1,10]-phenanthroline) was synthesized and characterized by elemental analysis, ESI-MS and (1)H NMR. The cytotoxic activity of the complex against MG-63, U2OS, HOS, and MC3T3-e1 cell lines was investigated by MTT method. The complex shows moderate cytotoxicity toward HOS (IC50 = 35.6 ± 2.6 µM) and MC3T3-e1 (IC50 = 41.6 ± 2.8 µM) cell lines. The morphological studies show that the complex can induce apoptosis in HOS cells and cause an increase of reactive oxygen species levels and a decrease in the mitochondrial membrane potential. The cell cycle distribution demonstrates that the complex inhibits the cell growth at S phase. Additionally, the antitumor activity in vivo reveals that the complex can induce a decrease in tumor weight. PMID:27007877

  1. Single cell activity reveals direct electron transfer in methanotrophic consortia

    Science.gov (United States)

    McGlynn, Shawn E.; Chadwick, Grayson L.; Kempes, Christopher P.; Orphan, Victoria J.

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  2. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    Science.gov (United States)

    Stingaciu, Laura-Roxana; O'Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  3. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    Directory of Open Access Journals (Sweden)

    Yevgeniy A Grigoryev

    Full Text Available A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+CD62L(- effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  4. Naturally death-resistant precursor cells revealed as the origin of retinoblastoma

    DEFF Research Database (Denmark)

    Trinh, Emmanuelle; Lazzerini Denchi, Eros; Helin, Kristian

    2004-01-01

    The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retin...

  5. Comparative genomic analysis reveals 2-oxoacid dehydrogenase complex lipoylation correlation with aerobiosis in archaea.

    Directory of Open Access Journals (Sweden)

    Kirill Borziak

    Full Text Available Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multienzyme complexes (OADHCs, is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA, which in archaea is typically encoded by two genes (LplA-N and LplA-C, or by a lipoyl(octanoyl transferase (LipB or LipM plus a lipoic acid synthetase (LipA. Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across

  6. Whole-Genome Sequencing Reveals Diverse Models of Structural Variations in Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Cheng, Caixia; Zhou, Yong; Li, Hongyi; Xiong, Teng; Li, Shuaicheng; Bi, Yanghui; Kong, Pengzhou; Wang, Fang; Cui, Heyang; Li, Yaoping; Fang, Xiaodong; Yan, Ting; Li, Yike; Wang, Juan; Yang, Bin; Zhang, Ling; Jia, Zhiwu; Song, Bin; Hu, Xiaoling; Yang, Jie; Qiu, Haile; Zhang, Gehong; Liu, Jing; Xu, Enwei; Shi, Ruyi; Zhang, Yanyan; Liu, Haiyan; He, Chanting; Zhao, Zhenxiang; Qian, Yu; Rong, Ruizhou; Han, Zhiwei; Zhang, Yanlin; Luo, Wen; Wang, Jiaqian; Peng, Shaoliang; Yang, Xukui; Li, Xiangchun; Li, Lin; Fang, Hu; Liu, Xingmin; Ma, Li; Chen, Yunqing; Guo, Shiping; Chen, Xing; Xi, Yanfeng; Li, Guodong; Liang, Jianfang; Yang, Xiaofeng; Guo, Jiansheng; Jia, JunMei; Li, Qingshan; Cheng, Xiaolong; Zhan, Qimin; Cui, Yongping

    2016-02-01

    Comprehensive identification of somatic structural variations (SVs) and understanding their mutational mechanisms in cancer might contribute to understanding biological differences and help to identify new therapeutic targets. Unfortunately, characterization of complex SVs across the whole genome and the mutational mechanisms underlying esophageal squamous cell carcinoma (ESCC) is largely unclear. To define a comprehensive catalog of somatic SVs, affected target genes, and their underlying mechanisms in ESCC, we re-analyzed whole-genome sequencing (WGS) data from 31 ESCCs using Meerkat algorithm to predict somatic SVs and Patchwork to determine copy-number changes. We found deletions and translocations with NHEJ and alt-EJ signature as the dominant SV types, and 16% of deletions were complex deletions. SVs frequently led to disruption of cancer-associated genes (e.g., CDKN2A and NOTCH1) with different mutational mechanisms. Moreover, chromothripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally mis-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromothripsis-derived double-minute chromosome formation (e.g., FGFR1 and LETM2) or BFB-affected chromosomes (e.g., CCND1, EGFR, ERBB2, MMPs, and MYC), with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification. Furthermore, analyses of copy-number alterations reveal high frequency of whole-genome duplication (WGD) and recurrent focal amplification of CDCA7 that might act as a potential oncogene in ESCC. Our findings reveal molecular defects such as chromothripsis and BFB in malignant transformation of ESCCs and demonstrate diverse models of SVs-derived target genes in ESCCs. These genome-wide SV profiles and their underlying mechanisms provide preventive, diagnostic, and therapeutic implications for ESCCs. PMID:26833333

  7. Quantitative trait loci mapping reveals candidate pathways regulating cell cycle duration in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Siwo Geoffrey

    2010-10-01

    Full Text Available Abstract Background Elevated parasite biomass in the human red blood cells can lead to increased malaria morbidity. The genes and mechanisms regulating growth and development of Plasmodium falciparum through its erythrocytic cycle are not well understood. We previously showed that strains HB3 and Dd2 diverge in their proliferation rates, and here use quantitative trait loci mapping in 34 progeny from a cross between these parent clones along with integrative bioinformatics to identify genetic loci and candidate genes that control divergences in cell cycle duration. Results Genetic mapping of cell cycle duration revealed a four-locus genetic model, including a major genetic effect on chromosome 12, which accounts for 75% of the inherited phenotype variation. These QTL span 165 genes, the majority of which have no predicted function based on homology. We present a method to systematically prioritize candidate genes using the extensive sequence and transcriptional information available for the parent lines. Putative functions were assigned to the prioritized genes based on protein interaction networks and expression eQTL from our earlier study. DNA metabolism or antigenic variation functional categories were enriched among our prioritized candidate genes. Genes were then analyzed to determine if they interact with cyclins or other proteins known to be involved in the regulation of cell cycle. Conclusions We show that the divergent proliferation rate between a drug resistant and drug sensitive parent clone is under genetic regulation and is segregating as a complex trait in 34 progeny. We map a major locus along with additional secondary effects, and use the wealth of genome data to identify key candidate genes. Of particular interest are a nucleosome assembly protein (PFL0185c, a Zinc finger transcription factor (PFL0465c both on chromosome 12 and a ribosomal protein L7Ae-related on chromosome 4 (PFD0960c.

  8. Cobalt substituted thiosemicarbazone metal complex induced apoptosis in cancer cells via activation of mitochondrial pathway

    International Nuclear Information System (INIS)

    Thiosemicarbazone (TSC) and their transition metal complexes are broad class of biologically active small molecules and present a great variety of biological activity ranging from antitumour, fungicide, bacteriocide, anti inflammatory and antiviral activities. These characteristics render the whole class of compounds very interesting. In the present study, we sought to examine cytotoxic activity of thiosemicarbazone cobalt complex on various cancer cell lines along with the possible mechanism through which the compound induce apoptosis in these cell lines. Cytotoxicity of TSC cobalt complex was studied by performing standard MTT drug sensitivity assay and determining its IC50 value on various leukemic and solid cancer cell lines like HL-60, MOLT-4, K-562 and COLO-205. Cellular damage upon the treatment of test molecule was analyzed by conducting LDH release assay. DNA fragmentation and morphology of apoptotic cells were assessed respectively by performing ladder assay and acridine orange/ethidium bromide staining. Role of mitochondria in the induction of cell death was studied by measuring mitochondrial membrane potential (ΔΨm) using JC-1 probe. The cytotoxicity studies confirms that TSC cobalt complex is having potent anticancer activity on HL-60, K-562, MOLT-4 and COLO-205 cell lines with the IC50 value in the range 0.225-29.00 μM. Dose dependent increase in the LDH release into the surrounding media depicts the cell membrane disintegrity. DNA fragmentation on treated cells revealed the cell death, which is commonly associated with apoptosis. Fluorescence microscopic imaging of treated cells confirms that the mode of cell death was through apoptosis. Loss of the ΔΨm in treated cells explicates the involvement of mitochondria in the cell death induction. Further increase in caspase-3 activity upon treatment corroborates that molecule induces apoptosis. Taken together, this exploratory study revealed that TSC cobalt complex possesses potent cytotoxic and

  9. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells.

    Science.gov (United States)

    Fang, Yi; Zhang, Qian; Wang, Xin; Yang, Xue; Wang, Xiangyu; Huang, Zhen; Jiao, Yuchen; Wang, Jing

    2016-03-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  10. Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation

    OpenAIRE

    Sugiyama, Takuya; Benitez, Cecil M.; Ghodasara, Amar; Liu, Lucy; McLean, Graeme W.; Lee, Jonghyeob; Blauwkamp, Timothy A; Nusse, Roeland; Wright, Christopher V.E.; Gu, Guoqiang; Kim, Seung K.

    2013-01-01

    Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A...

  11. Asymmetric collapse in biomimetic complex coacervates revealed by local polymer and water dynamics

    OpenAIRE

    Ortony, Julia H.; Hwang, Dong Soo; Franck, John M.; Waite, J. Herbert; Han, Songi

    2013-01-01

    Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micron-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materi...

  12. Mechanistic modeling confronts the complexity of molecular cell biology

    OpenAIRE

    Phair, Robert D.

    2014-01-01

    Mechanistic modeling has the potential to transform how cell biologists contend with the inescapable complexity of modern biology. I am a physiologist–electrical engineer–systems biologist who has been working at the level of cell biology for the past 24 years. This perspective aims 1) to convey why we build models, 2) to enumerate the major approaches to modeling and their philosophical differences, 3) to address some recurrent concerns raised by experimentalists, and then 4) to imagine a fu...

  13. Contextual interactions in a generalized energy model of complex cells

    OpenAIRE

    Dellen, Babette; Clark, John W.; Wessel, Ralf

    2009-01-01

    We propose a generalized energy model of complex cells to describe modulatory contextual influences on the responses of neurons in the primary visual cortex (V1). Many orientationselective cells in V1 respond to contrast of orientation and motion of stimuli exciting the classical receptive field (CRF) and the non-CRF, or surround. In the proposed model, a central spatiotemporal filter, defining the CRF, is nonlinearly combined with a spatiotemporal filter extending into the non- ...

  14. Tutorial: Electroporation of cells in complex materials and tissue

    Science.gov (United States)

    Rems, L.; Miklavčič, D.

    2016-05-01

    Electroporation is being successfully used in biology, medicine, food processing, and biotechnology, and in some environmental applications. Recent applications also include in addition to classical electroporation, where cells are exposed to micro- or milliseconds long pulses, exposures to extremely short nanosecond pulses, i.e., high-frequency electroporation. Electric pulses are applied to cells in different structural configurations ranging from suspended cells to cells in tissues. Understanding electroporation of cells in tissues and other complex environments is a key to its successful use and optimization in various applications. Thus, explanation will be provided theoretically/numerically with relation to experimental observations by scaling our understanding of electroporation from the molecular level of the cell membrane up to the tissue level.

  15. Rapid binding of plasminogen to streptokinase in a catalytic complex reveals a three-step mechanism.

    Science.gov (United States)

    Verhamme, Ingrid M; Bock, Paul E

    2014-10-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys(414) deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-D-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK ·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys(414) deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys(414) to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK · Pg complex compared with SK · Pm is characterized by a ∼ 25-fold weaker encounter complex and ∼ 40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys(414) engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK · Pg complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared with SK · Pm. PMID:25138220

  16. Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism*

    Science.gov (United States)

    Verhamme, Ingrid M.; Bock, Paul E.

    2014-01-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys414 deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-d-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys414 deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys414 to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared with SK·Pm is characterized by a ∼25-fold weaker encounter complex and ∼40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys414 engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex and the expression of a weaker “pro”-exosite for binding of a second Pg in the substrate mode compared with SK·Pm. PMID:25138220

  17. Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba δ-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation

    International Nuclear Information System (INIS)

    The insecticidal nature of Cry δ-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17 A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore

  18. A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression.

    Science.gov (United States)

    Marmorstein, L Y; Kinev, A V; Chan, G K; Bochar, D A; Beniya, H; Epstein, J A; Yen, T J; Shiekhattar, R

    2001-01-26

    Germline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BRCA2-Associated Factor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression. PMID:11207365

  19. Topological similarity of random cell complexes and applications

    Science.gov (United States)

    Schweinhart, B.; Mason, J. K.; MacPherson, R. D.

    2016-06-01

    Although random cell complexes occur throughout the physical sciences, there does not appear to be a standard way to quantify their statistical similarities and differences. The various proposals in the literature are usually motivated by the analysis of particular physical systems and do not necessarily apply to general situations. The central concepts in this paper—the swatch and the cloth—provide a description of the local topology of a cell complex that is general (any physical system that can be represented as a cell complex is admissible) and complete (any statistical question about the local topology can be answered from the cloth). Furthermore, this approach allows a distance to be defined that measures the similarity of the local topology of two cell complexes. The distance is used to identify a steady state of a model grain boundary network, quantify the approach to this steady state, and show that the steady state is independent of the initial conditions. The same distance is then employed to show that the long-term properties in simulations of a specific model of a dislocation network do not depend on the implementation of dislocation intersections.

  20. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

    Science.gov (United States)

    Beringer, Dennis X; Kleijwegt, Fleur S; Wiede, Florian; van der Slik, Arno R; Loh, Khai Lee; Petersen, Jan; Dudek, Nadine L; Duinkerken, Gaby; Laban, Sandra; Joosten, Antoinette; Vivian, Julian P; Chen, Zhenjun; Uldrich, Adam P; Godfrey, Dale I; McCluskey, James; Price, David A; Radford, Kristen J; Purcell, Anthony W; Nikolic, Tatjana; Reid, Hugh H; Tiganis, Tony; Roep, Bart O; Rossjohn, Jamie

    2015-11-01

    Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition. PMID:26437244

  1. Peeling Back the Layers of Policy and School Reform: Revealing the Structural and Social Complexities within

    Science.gov (United States)

    Woodside-Jiron, Haley; Gehsmann, Kristin M.

    2009-01-01

    This article explores the complex process of school change over a six-year period in one high-poverty, urban elementary school in a northeastern city of the United States. The school included in this instrumental case study was identified by its State Department of Education as "being in need of improvement" in March 2000. Findings suggest that…

  2. Behaviour of Zinc Complexes and Zinc Sulphide Nanoparticles Revealed by Using Screen Printed Electrodes and Spectrometry

    Directory of Open Access Journals (Sweden)

    Lukas Nejdl

    2013-10-01

    Full Text Available In this study, we focused on microfluidic electrochemical analysis of zinc complexes (Zn(phen(hisCl2, Zn(hisCl2 and ZnS quantum dots (QDs using printed electrodes. This method was chosen due to the simple (easy to use instrumentation and variable setting of flows. Reduction signals of zinc under the strictly defined and controlled conditions (pH, temperature, flow rate, accumulation time and applied potential were studied. We showed that the increasing concentration of the complexes (Zn(phen(hisCl2, Zn(hisCl2 led to a decrease in the electrochemical signal and a significant shift of the potential to more positive values. The most likely explanation of this result is that zinc is strongly bound in the complex and its distribution on the electrode is very limited. Changing the pH from 3.5 to 5.5 resulted in a significant intensification of the Zn(II reduction signal. The complexes were also characterized by UV/VIS spectrophotometry, chromatography, and ESI-QTOF mass spectrometry.

  3. Genetic and genomic analysis of a fat mass trait with complex inheritance reveals marked sex specificity.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available The integration of expression profiling with linkage analysis has increasingly been used to identify genes underlying complex phenotypes. The effects of gender on the regulation of many physiological traits are well documented; however, "genetical genomic" analyses have not yet addressed the degree to which their conclusions are affected by sex. We constructed and densely genotyped a large F2 intercross derived from the inbred mouse strains C57BL/6J and C3H/HeJ on an apolipoprotein E null (ApoE-/- background. This BXH.ApoE-/- population recapitulates several "metabolic syndrome" phenotypes. The cross consists of 334 animals of both sexes, allowing us to specifically test for the dependence of linkage on sex. We detected several thousand liver gene expression quantitative trait loci, a significant proportion of which are sex-biased. We used these analyses to dissect the genetics of gonadal fat mass, a complex trait with sex-specific regulation. We present evidence for a remarkably high degree of sex-dependence on both the cis and trans regulation of gene expression. We demonstrate how these analyses can be applied to the study of the genetics underlying gonadal fat mass, a complex trait showing significantly female-biased heritability. These data have implications on the potential effects of sex on the genetic regulation of other complex traits.

  4. Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

    DEFF Research Database (Denmark)

    Laursen, Nick Stub; Andersen, Kasper Røjkjær; Braren, Ingke; Spilllner, Edzard; Sottrup-Jensen, Lars; Andersen, Gregers Rom

    2011-01-01

    Complement acts as a danger-sensing system in the innate immune system, and its activation initiates a strong inflammatory response and cleavage of the proteins C3 and C5 by proteolytic enzymes, the convertases. These contain a non-catalytic substrate contacting subunit (C3b or C4b) in complex with...

  5. Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses.

    Science.gov (United States)

    Amundson, S A; Bittner, M; Chen, Y; Trent, J; Meltzer, P; Fornace, A J

    1999-06-17

    The fate of cells exposed to ionizing radiation (IR) may depend greatly on changes in gene expression, so that an improved view of gene induction profiles is important for understanding mechanisms of checkpoint control, repair and cell death following such exposures. We have used a quantitative fluorescent cDNA microarray hybridization approach to identify genes regulated in response to 7-irradiation in the p53 wild-type ML-1 human myeloid cell line. Hybridization of the array to fluorescently-labeled RNA from treated and untreated cells was followed by computer analysis to derive relative changes in expression levels of the genes present in the array, which agreed well with actual quantitative changes in expression. Forty-eight sequences, 30 not previously identified as IR-responsive, were significantly regulated by IR. Induction by IR and other stresses of a subset of these genes, including the previously characterized CIP1/ WAF1, MDM2 and BAX genes, as well as nine genes not previously reported to be IR-responsive, was examined in a panel of 12 human cell lines. Responses varied widely in cell lines with different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses. Two of the newly identified IR-responsive genes, FRA-1 and ATF3, showed a p53-associated component to their IR-induction, and this was confirmed both in isogenic human cell lines and in mouse thymus. The majority of the IR-responsive genes, however, showed no indication of p53-dependent regulation, representing a potentially important class of stress-responsive genes in leukemic cells. PMID:10380890

  6. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis.

    Science.gov (United States)

    De Souza, Amanda P; Alvim Kamei, Claire L; Torres, Andres F; Pattathil, Sivakumar; Hahn, Michael G; Trindade, Luisa M; Buckeridge, Marcos S

    2015-07-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis. PMID:25908240

  7. Asymmetric collapse in biomimetic complex coacervates revealed by local polymer and water dynamics.

    Science.gov (United States)

    Ortony, Julia H; Hwang, Dong Soo; Franck, John M; Waite, J Herbert; Han, Songi

    2013-05-13

    Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micrometer-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications; however, their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties, capabilities not offered by conventional techniques such as X-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) line shape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization. PMID:23540713

  8. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin

    Science.gov (United States)

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G.

    2009-01-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland. PMID:19074636

  9. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin.

    Science.gov (United States)

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G

    2009-02-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland. PMID:19074636

  10. Ruthenium(II) multi carboxylic acid complexes: chemistry and application in dye sensitized solar cells.

    Science.gov (United States)

    Shahroosvand, Hashem; Nasouti, Fahimeh; Sousaraei, Ahmad

    2014-04-01

    Novel ruthenium multi carboxylic complexes (RMCCs) have been synthesized by using ruthenium nitrosyl nitrate, 1,2,4,5-benzenetetracarboxylic acid (H4btec) and 4,7-diphenyl-1,10-phenanthroline (BPhen) as photosensitizers for titanium dioxide semiconductor solar cells. The complexes were characterized by (1)H-NMR, FT-IR, UV-Vis, ICP and CHN analyses. The reaction details and features were then described. SEM analysis revealed that the penetration of dyes into the pores of the nanocrystalline TiO2 surface was improved by increasing the number of btec units. The solar energy to electricity conversion efficiency of complexes shows that the number of attached carboxylates on a dye has an influence on the photoelectrochemical properties of the dye-sensitized electrode. An incident photon-to-current conversion efficiency (IPCE) of 13% at 510 nm was obtained for ruthenium complexes with three btec units. PMID:24500312

  11. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity.

    Science.gov (United States)

    Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4(+)SNS-Cre/TdTomato(+), 2) IB4(-)SNS-Cre/TdTomato(+), and 3) Parv-Cre/TdTomato(+) cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. PMID:25525749

  12. Pushing structural limits to reveal fundamental mechanisms of organic solar cell operation

    Science.gov (United States)

    Rand, Barry

    2015-03-01

    Organic-based solar cells are beginning to emerge with the potential to compete with other thin film photovoltaic technologies, with efficiencies of 12% recently demonstrated. Unique to the function of organic photovoltaics are the creation of tightly bound excitons that can only be efficiently separated at a donor/acceptor (D/A) interface capable of providing the necessary energetic driving force for dissociation. The consequences of this are the need for long exciton diffusion lengths and the presence of charge transfer (CT) states, ground state complexes that exist at the D/A interface. We have found that charge transfer states are more easily separated into free charge if they are delocalized; an aspect that becomes most feasible for highly ordered systems. I will discuss our recent efforts to template and control film morphology and molecular orientation. These studies allow us to understand the importance of molecular orientation, crystallite size, and crystal phase. We will show templated devices utilizing neat films as well as bulk heterojunctions, with crystallite dimensions spanning from the more standard nano-sized grains to those with highly ordered micron-sized crystalline domains revealing unprecedented thin film exciton diffusion lengths of 100s of nm. In these highly ordered films, owing to significant delocalization, we are able to directly measure photocurrent from multiple CT states, an aspect which has important consequences for the design of more efficient photocurrent generation. We acknowledge support from DOE BES Grant #11493344.

  13. A gallbladder tumor revealing metastatic clear cell renal carcinoma: report of case and review of literature

    OpenAIRE

    Ghaouti Merieme; Znati Kaoutar; Jahid Ahmed; Zouaidia Fouad; Bernoussi Zakiya; Fakir Youssef El; Mahassini Najat

    2013-01-01

    Abstract Metastatic renal cell carcinoma in the gallbladder is extremely rare, with reported frequencies of less than 0.6% in large autopsy reviews. Only 40 cases were reported in the literature. We report a first case of gallbladder polypoid tumor revealing metastatic clear cell renal cell carcinoma, which demonstrates the importance of radiological tests, histology and immunohistochemistry when making a definitive diagnosis. These examinations also allow differentiating metastatic clear cel...

  14. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    Directory of Open Access Journals (Sweden)

    Lukas Aeberhard

    2015-06-01

    Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  15. Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

    Energy Technology Data Exchange (ETDEWEB)

    Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander; Wei, Anzhi; Emanuel, Stuart L.; Gupta, Ruchira Das; Janjua, Ahsen; Cheng, Lin; Murdock, Melissa; Abramczyk, Bozena; Cohen, Daniel; Lin, Zheng; Morin, Paul; Davis, Jonathan H.; Dabritz, Michael; McLaughlin, Douglas C.; Russo, Katie A.; Chao, Ginger; Wright, Martin C.; Jenny, Victoria A.; Engle, Linda J.; Furfine, Eric; Sheriff, Steven (BMS)

    2014-10-02

    Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.

  16. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes.

    Science.gov (United States)

    Li, Y; Zakharov, D; Zhao, S; Tappero, R; Jung, U; Elsen, A; Baumann, Ph; Nuzzo, R G; Stach, E A; Frenkel, A I

    2015-01-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction-ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes. PMID:26119246

  17. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    Science.gov (United States)

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R. G.; Stach, E. A.; Frenkel, A. I.

    2015-06-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction--ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  18. Meditation effects within the hippocampal complex revealed by voxel-based morphometry and cytoarchitectonic probabilistic mapping

    OpenAIRE

    Luders, Eileen; Kurth, Florian; Toga, Arthur W.; Narr, Katherine L.; Gaser, Christian

    2013-01-01

    Scientific studies addressing anatomical variations in meditators' brains have emerged rapidly over the last few years, where significant links are most frequently reported with respect to gray matter (GM). To advance prior work, this study examined GM characteristics in a large sample of 100 subjects (50 meditators, 50 controls), where meditators have been practicing close to 20 years, on average. A standard, whole-brain voxel-based morphometry approach was applied and revealed significant m...

  19. Meditation effects within the hippocampal complex revealed by voxel-based morphometry and cytoarchitectonic probabilistic mapping

    OpenAIRE

    Eileen eLuders; Florian eKurth; Toga, Arthur W.; Narr, Katherine L.; Christian eGaser

    2013-01-01

    Scientific studies addressing anatomical variations in meditators’ brains have emerged rapidly over the last few years, where significant links are most frequently reported with respect to gray matter (GM). To advance prior work, this study examined GM characteristics in a large sample of 100 subjects (50 meditators, 50 controls), where meditators have been practicing close to twenty years, on average. A standard, whole-brain voxel-based morphometry approach was applied and revealed significa...

  20. Test of colonisation scenarios reveals complex invasion history of the red tomato spider mite Tetranychus evansi.

    Directory of Open Access Journals (Sweden)

    Angham Boubou

    Full Text Available The spider mite Tetranychus evansi is an emerging pest of solanaceous crops worldwide. Like many other emerging pests, its small size, confusing taxonomy, complex history of associations with humans, and propensity to start new populations from small inocula, make the study of its invasion biology difficult. Here, we use recent developments in Approximate Bayesian Computation (ABC and variation in multi-locus genetic markers to reconstruct the complex historical demography of this cryptic invasive pest. By distinguishing among multiple pathways and timing of introductions, we find evidence for the "bridgehead effect", in which one invasion serves as source for subsequent invasions. Tetranychus evansi populations in Europe and Africa resulted from at least three independent introductions from South America and involved mites from two distinct sources in Brazil, corresponding to highly divergent mitochondrial DNA lineages. Mites from southwest Brazil (BR-SW colonized the African continent, and from there Europe through two pathways in a "bridgehead" type pattern. One pathway resulted in a widespread invasion, not only to Europe, but also to other regions in Africa, southern Europe and eastern Asia. The second pathway involved the mixture with a second introduction from BR-SW leading to an admixed population in southern Spain. Admixture was also detected between invasive populations in Portugal. A third introduction from the Brazilian Atlantic region resulted in only a limited invasion in Europe. This study illustrates that ABC methods can provide insights into, and distinguish among, complex invasion scenarios. These processes are critical not only in understanding the biology of invasions, but also in refining management strategies for invasive species. For example, while reported observations of the mite and outbreaks in the invaded areas were largely consistent with estimates of geographical expansion from the ABC approach, historical

  1. Susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves

    OpenAIRE

    Neibergs, Holly L.; Seabury, Christopher M.; Wojtowicz, Andrzej J.; Wang, Zeping; Scraggs, Erik; Kiser, Jennifer N; Neupane, Mahesh; Womack, James E.; Van Eenennaam, Alison; Hagevoort, Gerald Robert; Lehenbauer, Terry W.; Aly, Sharif; Davis, Jessica; Taylor, Jeremy F; ,

    2014-01-01

    Background Bovine respiratory disease complex (BRDC) is an infectious disease of cattle that is caused by a combination of viral and/or bacterial pathogens. Selection for cattle with reduced susceptibility to respiratory disease would provide a permanent tool for reducing the prevalence of BRDC. The objective of this study was to identify BRDC susceptibility loci in pre-weaned Holstein calves as a prerequisite to using genetic improvement as a tool for decreasing the prevalence of BRDC. High ...

  2. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    OpenAIRE

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U. (Udo); Elsen, A.; Baumann, Ph.; Nuzzo, R.G.; Stach, E A; Frenkel, A. I.

    2015-01-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—e...

  3. Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

    OpenAIRE

    Masedunskas, Andrius; Sramkova, Monika; Parente, Laura; Sales, Katiuchia Uzzun; Amornphimoltham, Panomwat; Thomas H Bugge; Weigert, Roberto

    2011-01-01

    The regulation and the dynamics of membrane trafficking events have been studied primarily in in vitro models that often do not fully reflect the functional complexity found in a living multicellular organism. Here we used intravital microscopy in the salivary glands of live rodents to investigate regulated exocytosis, a fundamental process in all of the secretory organs. We found that β-adrenergic stimulation elicits exocytosis of large secretory granules, which gradually collapse with the a...

  4. Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ

    OpenAIRE

    Pertsinidis, Alexandros; Mukherjee, Konark; Sharma, Manu; Pang, Zhiping P.; Park, Sang Ryul; Zhang, Yunxiang; Brunger, Axel T.; Südhof, Thomas C.; Chu, Steven

    2013-01-01

    Synaptic vesicle fusion is catalyzed by multiprotein complexes that bring two lipid bilayers into close opposition. Several assembly mechanisms have been proposed for the synaptic vesicle fusion machinery, but exactly how these proteins interact in vivo remains unclear. We developed two-color fluorescence nanoscopy to directly visualize molecular interactions in situ and discovered that syntaxin-1, SNAP-25, and Munc18-1 (mammalian uncoordinated-18), three essential components for neurotransmi...

  5. CrypticspeciescompositionandgeneticdiversitywithinBemisiatabaci complex in soybean in India revealed by mtCOI DNA sequence

    Institute of Scientific and Technical Information of China (English)

    Prasanna H C; Kanakala S; Archana K; Jyothsna P; Varma R K; Malathi V G

    2015-01-01

    Bemisia tabaci is a cryptic species complex, causing signiifcant loss on many agricultural y important crops worldwide. Knowledge on species composition and diversity within B. tabaci complex is critical for evolving sustainable pest management strategies. Here we investigate the whitelfy species complex in soybean in major soybean growing states of India. The mitochondrial cytochrome oxidase gene subunit-1 (mtCOI) based phylogenetic relationships established using Bayesian methods indicated the existence of three cryptic species namely Asia I, Asia II 1, and Asia II 7. Al the haplotypes detected in the study could be assigned to these three cryptic species fol owing the species demarcation criteria of 3.5%divergence threshold. Of these, Asia II 1 was found to be predominant with wide spread distribution across the surveyed regions from cool temperate zones to hot and humid tropical plains. On the contrary, cryptic species Asia II 7 showed localized distribu-tion. The Asia II 1 exhibited the highest haplotype diversity and Asia I showed high level of nucleotide diversity. There was a signiifcantly high genetic differentiation among these three cryptic species. The MEAM 1, a dreadful invasive species was not detected in the specimens tested in the current study. The diversity and distribution of three cryptic species is discussed in the light of current knowledge on distribution of whitelfy species in India and yel ow mosaic disease observed during sampling survey.

  6. Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex.

    Science.gov (United States)

    Obado, Samson O; Brillantes, Marc; Uryu, Kunihiro; Zhang, Wenzhu; Ketaren, Natalia E; Chait, Brian T; Field, Mark C; Rout, Michael P

    2016-02-01

    The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes. PMID:26891179

  7. Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I.

    Science.gov (United States)

    Steimle, Stefan; Schnick, Christian; Burger, Eva-Maria; Nuber, Franziska; Krämer, Dorothée; Dawitz, Hannah; Brander, Sofia; Matlosz, Bartlomiej; Schäfer, Jacob; Maurer, Katharina; Glessner, Udo; Friedrich, Thorsten

    2015-10-01

    The NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 Å unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston-like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine-scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spin-probe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement. PMID:26115017

  8. Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

    Science.gov (United States)

    Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

    2016-01-01

    Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

  9. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  10. Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants.

    OpenAIRE

    Fobert, P R; Coen, E S; Murphy, G. J.; Doonan, J H

    1994-01-01

    Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstainin...

  11. Transcriptome analysis of a cnidarian – dinoflagellate mutualism reveals complex modulation of host gene expression

    Directory of Open Access Journals (Sweden)

    Phillips Wendy S

    2006-02-01

    Full Text Available Abstract Background Cnidarian – dinoflagellate intracellular symbioses are one of the most important mutualisms in the marine environment. They form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species. Despite the prevalence of these symbioses, we still know very little about the molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian – algal symbiosis. Results We detected statistically significant differences in host gene expression profiles between sea anemones (Anthopleura elegantissima in a symbiotic and non-symbiotic state. The group of genes, whose expression is altered, is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. In the context of cnidarian – dinoflagellate symbioses, we discuss pivotal host gene expression changes involved in lipid metabolism, cell adhesion, cell proliferation, apoptosis, and oxidative stress. Conclusion Our data do not support the existence of symbiosis-specific genes involved in controlling and regulating the symbiosis. Instead, it appears that the symbiosis is maintained by altering expression of existing genes involved in vital cellular processes. Specifically, the finding of key genes involved in cell cycle progression and apoptosis have led us to hypothesize that a suppression of apoptosis, together with a deregulation of the host cell cycle, create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. This first comprehensive molecular examination of the cnidarian – dinoflagellate associations provides critical insights into the maintenance and regulation of the

  12. Live cell linear dichroism imaging reveals extensive membrane ruffling within the docking structure of natural killer cell immune synapses

    DEFF Research Database (Denmark)

    Benninger, Richard K P; Vanherberghen, Bruno; Young, Stephen;

    2009-01-01

    We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into...... absent from the center of the mature synapse. Understanding the role of such extensive membrane ruffling in the assembly of cytolytic synapses is an intriguing new goal....

  13. Trafficking of the IKs -Complex in MDCK Cells

    DEFF Research Database (Denmark)

    David, Jens-Peter; Andersen, Martin N; Olesen, Søren-Peter; Rasmussen, Hanne B; Schmitt, Nicole

    2013-01-01

    has not been unequivocally resolved yet. We employed trafficking-deficient K 7.1 and KCNE1 mutants to investigate I trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes...... place in a post-endoplasmic reticulum compartment. We demonstrate that K 7.1 targets the I -complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical K 7.1 basolateral targeting signals. Our data provide a possible explanation to the...

  14. The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition

    OpenAIRE

    Kaan, Hung Yi Kristal; Hackney, David D.; Kozielski, Frank

    2011-01-01

    When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 Å and compare it with a structure of the motor domain alone at 2.7 Å. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second po...

  15. Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association

    Science.gov (United States)

    Cenens, William; Makumi, Angela; Govers, Sander K.; Lavigne, Rob; Aertsen, Abram

    2015-01-01

    Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host. PMID:26720743

  16. Metagenomic signatures of a tropical mining-impacted stream reveal complex microbial and metabolic networks.

    Science.gov (United States)

    Reis, Mariana P; Dias, Marcela F; Costa, Patrícia S; Ávila, Marcelo P; Leite, Laura R; de Araújo, Flávio M G; Salim, Anna C M; Bucciarelli-Rodriguez, Mônica; Oliveira, Guilherme; Chartone-Souza, Edmar; Nascimento, Andréa M A

    2016-10-01

    Bacteria from aquatic ecosystems significantly contribute to biogeochemical cycles, but details of their community structure in tropical mining-impacted environments remain unexplored. In this study, we analyzed a bacterial community from circumneutral-pH tropical stream sediment by 16S rRNA and shotgun deep sequencing. Carrapatos stream sediment, which has been exposed to metal stress due to gold and iron mining (21 [g Fe]/kg), revealed a diverse community, with predominance of Proteobacteria (39.4%), Bacteroidetes (12.2%), and Parcubacteria (11.4%). Among Proteobacteria, the most abundant reads were assigned to neutrophilic iron-oxidizing taxa, such as Gallionella, Sideroxydans, and Mariprofundus, which are involved in Fe cycling and harbor several metal resistance genes. Functional analysis revealed a large number of genes participating in nitrogen and methane metabolic pathways despite the low concentrations of inorganic nitrogen in the Carrapatos stream. Our findings provide important insights into bacterial community interactions in a mining-impacted environment. PMID:27441985

  17. The cometary composition of a protoplanetary disk as revealed by complex cyanides

    CERN Document Server

    Oberg, Karin I; Furuya, Kenji; Qi, Chunhua; Aikawa, Yuri; Andrews, Sean M; Loomis, Ryan; Wilner, David J

    2015-01-01

    Observations of comets and asteroids show that the Solar Nebula that spawned our planetary system was rich in water and organic molecules. Bombardment brought these organics to the young Earth's surface, seeding its early chemistry. Unlike asteroids, comets preserve a nearly pristine record of the Solar Nebula composition. The presence of cyanides in comets, including 0.01% of methyl cyanide (CH3CN) with respect to water, is of special interest because of the importance of C-N bonds for abiotic amino acid synthesis. Comet-like compositions of simple and complex volatiles are found in protostars, and can be readily explained by a combination of gas-phase chemistry to form e.g. HCN and an active ice-phase chemistry on grain surfaces that advances complexity[3]. Simple volatiles, including water and HCN, have been detected previously in Solar Nebula analogues - protoplanetary disks around young stars - indicating that they survive disk formation or are reformed in situ. It has been hitherto unclear whether the s...

  18. Multivariate and multiscale dependence in the global climate system revealed through complex networks

    Energy Technology Data Exchange (ETDEWEB)

    Steinhaeuser, Karsten [Oak Ridge National Laboratory, Geographic Information Science and Technology Group, Computational Sciences and Engineering Division, Oak Ridge, TN (United States); University of Notre Dame, Department of Computer Science and Engineering and Interdisciplinary Center for Network Science and Applications, Notre Dame, IN (United States); Ganguly, Auroop R. [Oak Ridge National Laboratory, Geographic Information Science and Technology Group, Computational Sciences and Engineering Division, Oak Ridge, TN (United States); University of Tennessee at Knoxville, Department of Civil and Environmental Engineering, Knoxville, TN (United States); Chawla, Nitesh V. [University of Notre Dame, Department of Computer Science and Engineering and Interdisciplinary Center for Network Science and Applications, Notre Dame, IN (United States)

    2012-08-15

    A systematic characterization of multivariate dependence at multiple spatio-temporal scales is critical to understanding climate system dynamics and improving predictive ability from models and data. However, dependence structures in climate are complex due to nonlinear dynamical generating processes, long-range spatial and long-memory temporal relationships, as well as low-frequency variability. Here we utilize complex networks to explore dependence in climate data. Specifically, networks constructed from reanalysis-based atmospheric variables over oceans and partitioned with community detection methods demonstrate the potential to capture regional and global dependence structures within and among climate variables. Proximity-based dependence as well as long-range spatial relationships are examined along with their evolution over time, yielding new insights on ocean meteorology. The tools are implicitly validated by confirming conceptual understanding about aggregate correlations and teleconnections. Our results also suggest a close similarity of observed dependence patterns in relative humidity and horizontal wind speed over oceans. In addition, updraft velocity, which relates to convective activity over the oceans, exhibits short spatiotemporal decorrelation scales but long-range dependence over time. The multivariate and multi-scale dependence patterns broadly persist over multiple time windows. Our findings motivate further investigations of dependence structures among observations, reanalysis and model-simulated data to enhance process understanding, assess model reliability and improve regional climate predictions. (orig.)

  19. DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes.

    Science.gov (United States)

    Iftikhar, Romana; Ashfaq, Muhammad; Rasool, Akhtar; Hebert, Paul D N

    2016-01-01

    Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5' (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips. PMID:26741134

  20. DNA Barcode Analysis of Thrips (Thysanoptera Diversity in Pakistan Reveals Cryptic Species Complexes.

    Directory of Open Access Journals (Sweden)

    Romana Iftikhar

    Full Text Available Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5' (DNA barcode region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27% at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%. BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci, and one predatory thrips (Aeolothrips intermedius showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.

  1. Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance.

    Science.gov (United States)

    Brooun, Alexei; Gajiwala, Ketan S; Deng, Ya-Li; Liu, Wei; Bolaños, Ben; Bingham, Patrick; He, You-Ai; Diehl, Wade; Grable, Nicole; Kung, Pei-Pei; Sutton, Scott; Maegley, Karen A; Yu, Xiu; Stewart, Al E

    2016-01-01

    Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform. PMID:27122193

  2. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Directory of Open Access Journals (Sweden)

    Riffat I Munir

    Full Text Available Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes, sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199 and carbohydrate binding modules (95 were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  3. Distinct configurations of protein complexes and biochemical pathways revealed by epistatic interaction network motifs

    LENUS (Irish Health Repository)

    Casey, Fergal

    2011-08-22

    Abstract Background Gene and protein interactions are commonly represented as networks, with the genes or proteins comprising the nodes and the relationship between them as edges. Motifs, or small local configurations of edges and nodes that arise repeatedly, can be used to simplify the interpretation of networks. Results We examined triplet motifs in a network of quantitative epistatic genetic relationships, and found a non-random distribution of particular motif classes. Individual motif classes were found to be associated with different functional properties, suggestive of an underlying biological significance. These associations were apparent not only for motif classes, but for individual positions within the motifs. As expected, NNN (all negative) motifs were strongly associated with previously reported genetic (i.e. synthetic lethal) interactions, while PPP (all positive) motifs were associated with protein complexes. The two other motif classes (NNP: a positive interaction spanned by two negative interactions, and NPP: a negative spanned by two positives) showed very distinct functional associations, with physical interactions dominating for the former but alternative enrichments, typical of biochemical pathways, dominating for the latter. Conclusion We present a model showing how NNP motifs can be used to recognize supportive relationships between protein complexes, while NPP motifs often identify opposing or regulatory behaviour between a gene and an associated pathway. The ability to use motifs to point toward underlying biological organizational themes is likely to be increasingly important as more extensive epistasis mapping projects in higher organisms begin.

  4. Virtual Mutagenesis of the Yeast Cyclins Genetic Network Reveals Complex Dynamics of Transcriptional Control Networks

    Czech Academy of Sciences Publication Activity Database

    Vohradská, E.; Vohradský, Jiří

    2011-01-01

    Roč. 6, č. 4 (2011), s. 1-9. E-ISSN 1932-6203 R&D Projects: GA ČR GA310/07/1009; GA ČR GAP302/11/0229 Institutional research plan: CEZ:AV0Z50200510 Keywords : REGULATORY NETWORKS * SACCHAROMYCES - CEREVISIAE * CELL-CYCLE Subject RIV: EE - Microbiology, Virology Impact factor: 4.092, year: 2011

  5. Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

    Science.gov (United States)

    Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

    2016-06-01

    Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. PMID:27194709

  6. Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

    Science.gov (United States)

    Inoue, Shinnosuke; Lee, Hyun-Boo; Becker, Annie L; Weigel, Kris M; Kim, Jong-Hoon; Lee, Kyong-Hoon; Cangelosi, Gerard A; Chung, Jae-Hyun

    2015-10-01

    Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells. PMID:26231690

  7. Chemical Complexity in the Shocked Outflow L1157 Revealed by CARMA

    Science.gov (United States)

    Dollhopf, Niklaus M.; McGuire, Brett A.; Carroll, P. Brandon; Remijan, Anthony J.

    2015-01-01

    Amino acids, the complex organic molecules which are the building blocks of life, have been found in meteoritic samples and, most recently, in samples from Comet Wild-2. Yet, no amino acids have been detected in the gas-phase in the interstellar medium, which seeds and enriches these meteorites and comets. Glycine, the simplest amino acid, has been shown to form in the laboratory through the reaction of hydroxylamine (NH2OH) with acetic acid (CH3COOH), a known interstellar molecule. This has prompted a move to search for NH2OH as a proxy of identifying regions where subsequent searches for glycine may prove the most fruitful.A search for NH2OH was conducted in seven diverse, molecule-rich sources and resulted in non-detections for all seven (Pulliam, et al. 2012). Theoretical work suggested the temperature of the sources was perhaps too low for NH2OH to thermally-desorb into the gas phase. Searches in shocked molecular regions, however, may overcome this barrier, as complex molecules are non-thermally liberated into the gas-phase by these shocks.Here, we present results from a targeted search toward the prototypical shocked outflow L1157. L1157-B0, -B1, and -B2 are shocked regions within the outflow from the infrared source L1157-mm. Using observations from the Combined Array for Research in Millimeter-wave Astronomy (CARMA), we have mapped a variety of molecular tracers in the region and conducted an interferometric search for NH2OH with typical spatial resolutions of ~3'. We find that the prototypical complex molecule methanol (CH3OH) peaks in B2, the newer shock. We compare this with the distributions of HCN and HCO+ and discuss the implications for chemical evolution within the region. HCN, used as a density tracer, also peaks in B2 while HCO+ is shown as diffuse throughout B0. We also present the first maps of isocyanic acid (HNCO) in L1157. HNCO is found to peak in B2, cospatial with CH3OH and HCN. Finally, we report a non-detection of three NH2OH transitions

  8. RNA-Seq reveals complex genetic response to deepwater horizon oil release in Fundulus grandis

    Directory of Open Access Journals (Sweden)

    Garcia Tzintzuni I

    2012-09-01

    Full Text Available Abstract Background The release of oil resulting from the blowout of the Deepwater Horizon (DH drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future. Results Our de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set. Conclusion RNA-Seq may be successfully applied to feral and

  9. A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation

    DEFF Research Database (Denmark)

    Klochendler, Agnes; Weinberg-Corem, Noa; Moran, Maya;

    2012-01-01

    biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to......Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive...... reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of...

  10. Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

    Science.gov (United States)

    Yu, Tao; Wang, Yongtao; Zhang, Huizhen; Johnson, Caroline H; Jiang, Yiming; Li, Xiangjun; Wu, Zeming; Liu, Tian; Krausz, Kristopher W; Yu, Aiming; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2016-06-01

    Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells. PMID:27074779

  11. [Complex metabolic disorders revealing a gastric ulcer of the bulb. A case report].

    Science.gov (United States)

    Neffati, F; Hellara, I; Jelizi, M A; Bahri, J; Douki, W; Amor, A Ben; Najjar, M F

    2009-01-01

    We report the case of a 54-year-old man, without particular pathological antecedents admitted to the emergency of the university hospital of Monastir, for right renal colic. Radiography of the urinary tract without preparation and renal echography showed bilateral renal lithiasis and a right ureteral lithiasis. The interrogation revealed concept of vomiting after which the patient felt relieved. The biological assessment objectified an hypochloremic metabolic alcalosis, an increase in the anion gap, a severe impaired renal function of obstructive origin and an hypokaliemia. The presence of the lithiasis did not explain on its own the metabolic disorders of this patient. The other investigations showed that initial pathology was an evolutionary bulb ulcer into pre-stenosis justifying treatment by omeprazole and explaining the biological disorders. PMID:19654086

  12. Computer simulations reveal complex distribution of haemodynamic forces in a mouse retina model of angiogenesis

    CERN Document Server

    Bernabeu, Miguel O; Jones, Martin; Nielsen, Jens H; Krüger, Timm; Nash, Rupert W; Groen, Derek; Hetherington, James; Gerhardt, Holger; Coveney, Peter V

    2013-01-01

    There is currently limited understanding of the role played by haemodynamic forces on the processes governing vascular development. One of many obstacles to be overcome is being able to measure those forces, at the required resolution level, on vessels only a few micrometres thick. In the current paper, we present an in silico method for the computation of the haemodynamic forces experienced by murine retinal vasculature (a widely used vascular development animal model) beyond what is measurable experimentally. Our results show that it is possible to reconstruct high-resolution three-dimensional geometrical models directly from samples of retinal vasculature and that the lattice-Boltzmann algorithm can be used to obtain accurate estimates of the haemodynamics in these domains. Our findings show that the flow patterns recovered are complex, that branches of predominant flow exist from early development stages, and that the pruning process tends to make the wall shear stress experienced by the capillaries incre...

  13. Dengue in China: Comprehensive Phylogenetic Evaluation Reveals Evidence of Endemicity and Complex Genetic Diversity.

    Science.gov (United States)

    Chen, Rubing; Han, Guan-Zhu

    2016-01-01

    Despite the increasing threat of dengue outbreaks in China, it is still considered as an imported disease and its introduction and/or circulation patterns remain obscure. On the basis of the most extensive phylogenetic analysis to date, we showed highly complex genetic diversity of dengue viruses (DENVs) in south China with up to 20 different clades/lineages from multiple serotypes co-circulating in the same year. Despite that most of these clades/lineages were resulted from imported cases, evidence of local persistence of DENV serotype 1 (DENV-1) was observed, indicating its potential endemicity in Guangdong province. This study, therefore, provided an overview of DENV genetic diversity and evolutionary dynamics in China, which will be useful for developing policies to prevent and control future dengue outbreaks in China. PMID:26458780

  14. Shadows of complexity: what biological networks reveal about epistasis and pleiotropy.

    Science.gov (United States)

    Tyler, Anna L; Asselbergs, Folkert W; Williams, Scott M; Moore, Jason H

    2009-02-01

    Pleiotropy, in which one mutation causes multiple phenotypes, has traditionally been seen as a deviation from the conventional observation in which one gene affects one phenotype. Epistasis, or gene-gene interaction, has also been treated as an exception to the Mendelian one gene-one phenotype paradigm. This simplified perspective belies the pervasive complexity of biology and hinders progress toward a deeper understanding of biological systems. We assert that epistasis and pleiotropy are not isolated occurrences, but ubiquitous and inherent properties of biomolecular networks. These phenomena should not be treated as exceptions, but rather as fundamental components of genetic analyses. A systems level understanding of epistasis and pleiotropy is, therefore, critical to furthering our understanding of human genetics and its contribution to common human disease. Finally, graph theory offers an intuitive and powerful set of tools with which to study the network bases of these important genetic phenomena. PMID:19204994

  15. Collaborative play in young children as a complex dynamic system: revealing gender related differences.

    Science.gov (United States)

    Steenbeek, Henderien; van der Aalsvoort, Diny; van Geert, Paul

    2014-07-01

    This study was focused on the role of gender-related differences in collaborative play, by examining properties of play as a complex system, and by using micro-genetic analysis techniques. A complex dynamic systems model of dyadic play was used to make predictions with regard to duration and number of contact-episodes during play of same-sex dyads, both on the micro- (i.e., per individual session), meso- (i.e., in smoothed data), and macro time scale (i.e., the change over six consecutive play sessions). The empirical data came from a study that examined the collaborative play skills of children who experienced six twenty minute play sessions within a three week period of time. Monte Carlo permutation analyses were used to compare model predictions and empirical data. The findings point to strongly asymmetric distributions in the duration and number of contact episodes in all dyads over the six sessions, as a direct consequence of the underlying dynamics of the play system. The model prediction that girls-dyads would show longer contact episodes than boys-dyads was confirmed, but the prediction regarding the difference in number of peaks was not confirmed. In addition, the majority of the model predictions regarding changes over the course of six sessions were consistent with the data. That is, the average duration and the maximum duration of contact-episodes increases both in boys-dyads and girls-dyads, but differences occur in the strength of the increase. Contrary to expectation, the number of contact-episodes decreases both in boys-dyads and in girls-dyads. PMID:24894265

  16. Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Aube, Michel, E-mail: 4aubem@videotron.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Larochelle, Christian, E-mail: christian.larochelle@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Ayotte, Pierre, E-mail: pierre.ayotte@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Laboratoire de Toxicologie, Institut national de sante publique du Quebec, 945 avenue Wolfe, Quebec, QC, Canada G1V 5B3 (Canada)

    2011-04-15

    Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cells and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100x10{sup 3} and 50x10{sup 3} dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10x10{sup 3} and 5x10{sup 3} dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p<0.05 at the 50x10{sup 3} dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, {beta}-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The

  17. Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines

    International Nuclear Information System (INIS)

    Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cells and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100x103 and 50x103 dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10x103 and 5x103 dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p3 dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, β-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The proliferative effect of the mixture on CAMA-1 cells in

  18. Structural Identification of the Vps18 β-Propeller Reveals a Critical Role in the HOPS Complex Stability and Function*

    Science.gov (United States)

    Behrmann, Heide; Lürick, Anna; Kuhlee, Anne; Balderhaar, Henning Kleine; Bröcker, Cornelia; Kümmel, Daniel; Engelbrecht-Vandré, Siegfried; Gohlke, Ulrich; Raunser, Stefan; Heinemann, Udo; Ungermann, Christian

    2014-01-01

    Membrane fusion at the vacuole, the lysosome equivalent in yeast, requires the HOPS tethering complex, which is recruited by the Rab7 GTPase Ypt7. HOPS provides a template for the assembly of SNAREs and thus likely confers fusion at a distinct position on vacuoles. Five of the six subunits in HOPS have a similar domain prediction with strong similarity to COPII subunits and nuclear porins. Here, we show that Vps18 indeed has a seven-bladed β-propeller as its N-terminal domain by revealing its structure at 2.14 Å. The Vps18 N-terminal domain can interact with the N-terminal part of Vps11 and also binds to lipids. Although deletion of the Vps18 N-terminal domain does not preclude HOPS assembly, as revealed by negative stain electron microscopy, the complex is instable and cannot support membrane fusion in vitro. We thus conclude that the β-propeller of Vps18 is required for HOPS stability and function and that it can serve as a starting point for further structural analyses of the HOPS tethering complex. PMID:25324549

  19. Bioinformatic analysis of the neprilysin (M13 family of peptidases reveals complex evolutionary and functional relationships

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    Pinney John W

    2008-01-01

    Full Text Available Abstract Background The neprilysin (M13 family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2, which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles. Results The phylogenetic analysis successfully resolved vertebrate M13 peptidases into seven classes, one of which appears to be specific to mammals, and insect genes into five functional classes and a series of expansions, which may include inactive peptidases. Nematode genes primarily resolved into groups containing no other taxa, bar the two nematode genes associated with Drosophila DmeNEP1 and DmeNEP4. This analysis reconstructed only one relationship between chordate and invertebrate clusters, that of the ECE sub-group and the DmeNEP3 related genes. Analysis of amino acid utilisation in the active site of M13 peptidases reveals a basis for their biochemical properties. A relatively invariant S1' subsite gives the majority of M13 peptidases their strong preference for hydrophobic residues in P1' position. The greater variation in the S2' subsite may be instrumental in determining the specificity of M13 peptidases for their substrates

  20. Latent physiological factors of complex human diseases revealed by independent component analysis of clinarrays

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    Chen David P

    2010-10-01

    Full Text Available Abstract Background Diagnosis and treatment of patients in the clinical setting is often driven by known symptomatic factors that distinguish one particular condition from another. Treatment based on noticeable symptoms, however, is limited to the types of clinical biomarkers collected, and is prone to overlooking dysfunctions in physiological factors not easily evident to medical practitioners. We used a vector-based representation of patient clinical biomarkers, or clinarrays, to search for latent physiological factors that underlie human diseases directly from clinical laboratory data. Knowledge of these factors could be used to improve assessment of disease severity and help to refine strategies for diagnosis and monitoring disease progression. Results Applying Independent Component Analysis on clinarrays built from patient laboratory measurements revealed both known and novel concomitant physiological factors for asthma, types 1 and 2 diabetes, cystic fibrosis, and Duchenne muscular dystrophy. Serum sodium was found to be the most significant factor for both type 1 and type 2 diabetes, and was also significant in asthma. TSH3, a measure of thyroid function, and blood urea nitrogen, indicative of kidney function, were factors unique to type 1 diabetes respective to type 2 diabetes. Platelet count was significant across all the diseases analyzed. Conclusions The results demonstrate that large-scale analyses of clinical biomarkers using unsupervised methods can offer novel insights into the pathophysiological basis of human disease, and suggest novel clinical utility of established laboratory measurements.

  1. Understanding the complexities of Salmonella-host crosstalk as revealed by in vivo model organisms.

    Science.gov (United States)

    Verma, Smriti; Srikanth, Chittur V

    2015-07-01

    Foodborne infections caused by non-typhoidal Salmonellae, such as Salmonella enterica serovar Typhimurium (ST), pose a major challenge in the developed and developing world. With constant rise of drug-resistant strains, understanding the epidemiology, microbiology, pathogenesis and host-pathogen interactions biology is a mandatory requirement to enable health systems to be ready to combat these illnesses. Patient data from hospitals, at least from some parts of the world, have aided in epidemiological understanding of ST-mediated disease. Most of the other aspects connected to Salmonella-host crosstalk have come from model systems that offer convenience, genetic tractability and low maintenance costs that make them extremely valuable tools. Complex model systems such as the bovine model have helped in understanding key virulence factors needed for infection. Simple systems such as fruit flies and Caenorhabditis elegans have aided in identification of novel virulence factors, host pathways and mechanistic details of interactions. Some of the path-breaking concepts of the field have come from mice model of ST colitis, which allows genetic manipulations as well as high degree of similarity to human counterpart. Together, they are invaluable for correlating in vitro findings of ST-induced disease progression in vivo. The current review is a compilation of various advances of ST-host interactions at cellular and molecular levels that has come from investigations involving model organisms. PMID:26179888

  2. Enrichment of SNPs in Functional Categories Reveals Genes Affecting Complex Traits.

    Science.gov (United States)

    Zhao, Huiying; Fan, Dongsheng; Nyholt, Dale R; Yang, Yuedong

    2016-08-01

    Genome-wide association studies (GWAS) have indicated potential to identify heritability of common complex phenotypes, but traditional approaches have limited ability to detect hiding signals because single SNP has weak effect size accounting for only a small fraction of overall phenotypic variations. To improve the power of GWAS, methods have been developed to identify truly associated genes by jointly testing effects of all SNPs. However, equally considering all SNPs within a gene might dilute strong signals of SNPs in real functional categories. Here, we observed a consistent pattern on enrichment of significant SNPs in eight functional categories across six phenotypes, with the highest enrichment in coding and both UTR regions while the lowest enrichment in the intron. Based on the pattern of SNP enrichment in functional categories, we developed a new approach for detecting gene associations on traits (DGAT) by selecting the most significant functional category and then using SNPs within it to assess gene associations. The method was found to be robust in type I error rate on simulated data, and to have mostly higher power in detecting associated genes for three different diseases than other methods. Further analysis indicated ability of the DGAT to detect novel genes. The DGAT is available by http://sparks-lab.org/server/DGAT. PMID:27113629

  3. Protein dynamics revealed in the excitonic spectra of single LH2 complexes

    International Nuclear Information System (INIS)

    The fluorescence emission spectrum of single peripheral light-harvesting (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila exhibits remarkable dynamics on a time scale of several minutes. Often the spectral properties are quasi-stable; sometimes large spectral jumps to the blue or to the red are observed. To explain the dynamics, every pigment is proposed to be in two conformational substates with different excitation energies, which originate from the conformational state of the protein as a result of pigment-protein interaction. Due to the excitonic coupling in the ring of 18 pigments, the two-state assumption generates a substantial amount of distinct spectroscopic states, which reflect part of the inhomogeneous distributed spectral properties of LH2. To describe the observed dynamics, spontaneous and light-induced transitions are introduced between the two states. For each 'realization of the disorder', the spectral properties are calculated using a disordered exciton model combined with the modified Redfield theory to obtain realistic spectral line shapes. The single-molecule fluorescence peak (FLP) distribution, the distribution dependence on the excitation intensity, and the FLP time traces are well described within the framework of this model

  4. Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins.

    Science.gov (United States)

    Lavy, Meirav; Prigge, Michael J; Tao, Sibo; Shain, Stephanie; Kuo, April; Kirchsteiger, Kerstin; Estelle, Mark

    2016-01-01

    The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs. PMID:27247276

  5. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility.

    Science.gov (United States)

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A-H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A-H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A-H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  6. Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins

    Science.gov (United States)

    Lavy, Meirav; Prigge, Michael J; Tao, Sibo; Shain, Stephanie; Kuo, April; Kirchsteiger, Kerstin; Estelle, Mark

    2016-01-01

    The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs. DOI: http://dx.doi.org/10.7554/eLife.13325.001 PMID:27247276

  7. Enlightening intracellular complexity of living cells with quantitative phase microscopy

    Science.gov (United States)

    Martinez Torres, C.; Laperrousaz, B.; Berguiga, L.; Boyer Provera, E.; Elezgaray, J.; Nicolini, F. E.; Maguer-Satta, V.; Arneodo, A.; Argoul, F.

    2016-03-01

    The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

  8. Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes.

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2015-07-01

    Full Text Available The RNA polymerase II (Pol II is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the "secondary channel" is the only route for NTP to reach the active site of the enzyme or if the "main channel" could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.

  9. Global terrestrial water storage connectivity revealed using complex climate network analyses

    Directory of Open Access Journals (Sweden)

    A. Y. Sun

    2015-04-01

    Full Text Available Terrestrial water storage (TWS exerts a key control in global water, energy, and biogeochemical cycles. Although certain causal relationships exist between precipitation and TWS, the latter also reflects impacts of anthropogenic activities. Thus, quantification of the spatial patterns of TWS will not only help to understand feedbacks between climate dynamics and hydrologic cycle, but also provide new model calibration constraints for improving the current land surface models. In this work, the connectivity of TWS is quantified using the climate network theory, which has received broad attention in the climate modeling community in recent years. Complex networks of TWS anomalies are built using two global TWS datasets, a remote-sensing product that is obtained from the Gravity Recovery and Climate Experiment (GRACE satellite mission, and a model-generated dataset from the global land data assimilation system's NOAH model (GLDAS-NOAH. Both datasets have 1 ° × 1 ° resolutions and cover most global land areas except for permafrost regions. TWS networks are built by first quantifying pairwise correlation among all valid TWS anomaly time series, and then applying a statistical cutoff threshold to retain only the most important features in the network. Basinwise network connectivity maps are used to illuminate connectivity of individual river basins with other regions. The constructed network degree centrality maps show TWS hotspots around the globe and the patterns are consistent with recent GRACE studies. Parallel analyses of networks constructed using the two datasets indicate that the GLDAS-NOAH model captures many of the spatial patterns shown by GRACE, although significant discrepancies exist in some regions. Thus, our results provide important insights for constraining land surface models, especially in data sparse regions.

  10. NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation.

    Science.gov (United States)

    Murayama, Emi; Sarris, Milka; Redd, Michael; Le Guyader, Dorothée; Vivier, Catherine; Horsley, Wyatt; Trede, Nikolaus; Herbomel, Philippe

    2015-01-01

    The ontogeny of haematopoietic niches in vertebrates is essentially unknown. Here we show that the stromal cells of the caudal haematopoietic tissue (CHT), the first niche where definitive haematopoietic stem/progenitor cells (HSPCs) home in zebrafish development, derive from the caudal somites through an epithelial-mesenchymal transition (EMT). The resulting stromal cell progenitors accompany the formation of the caudal vein sinusoids, the other main component of the CHT niche, and mature into reticular cells lining and interconnecting sinusoids. We characterize a zebrafish mutant defective in definitive haematopoiesis due to a deficiency in the nascent polypeptide-associated complex alpha subunit (NACA). We demonstrate that the defect resides not in HSPCs but in the CHT niche. NACA-deficient stromal cell progenitors initially develop normally together with the sinusoids, and HSPCs home to the resulting niche, but stromal cell maturation is compromised, leading to a niche that is unable to support HSPC maintenance, expansion and differentiation. PMID:26411530

  11. Autonomy and Non-autonomy of Angiogenic Cell Movements Revealed by Experiment-Driven Mathematical Modeling

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    Kei Sugihara

    2015-12-01

    Full Text Available Angiogenesis is a multicellular phenomenon driven by morphogenetic cell movements. We recently reported morphogenetic vascular endothelial cell (EC behaviors to be dynamic and complex. However, the principal mechanisms orchestrating individual EC movements in angiogenic morphogenesis remain largely unknown. Here we present an experiment-driven mathematical model that enables us to systematically dissect cellular mechanisms in branch elongation. We found that cell-autonomous and coordinated actions governed these multicellular behaviors, and a cell-autonomous process sufficiently illustrated essential features of the morphogenetic EC dynamics at both the single-cell and cell-population levels. Through refining our model and experimental verification, we further identified a coordinated mode of tip EC behaviors regulated via a spatial relationship between tip and follower ECs, which facilitates the forward motility of tip ECs. These findings provide insights that enhance our mechanistic understanding of not only angiogenic morphogenesis, but also other types of multicellular phenomenon.

  12. Structural Studies of Complex Carbohydrates of Plant Cell Walls

    Energy Technology Data Exchange (ETDEWEB)

    Darvill, Alan [Univ. of Georgia, Athens, GA (United States); Hahn, Michael G. [Univ. of Georgia, Athens, GA (United States); O' Neill, Malcolm A. [Univ. of Georgia, Athens, GA (United States); York, William S. [Univ. of Georgia, Athens, GA (United States)

    2015-02-17

    Most of the solar energy captured by land plants is converted into the polysaccharides (cellulose, hemicellulose, and pectin) that are the predominant components of the cell wall. These walls, which account for the bulk of plant biomass, have numerous roles in the growth and development of plants. Moreover, these walls have a major impact on human life as they are a renewable source of biomass, a source of diverse commercially useful polymers, a major component of wood, and a source of nutrition for humans and livestock. Thus, understanding the molecular mechanisms that lead to wall assembly and how cell walls and their component polysaccharides contribute to plant growth and development is essential to improve and extend the productivity and value of plant materials. The proposed research will develop and apply advanced analytical and immunological techniques to study specific changes in the structures and interactions of the hemicellulosic and pectic polysaccharides that occur during differentiation and in response to genetic modification and chemical treatments that affect wall biosynthesis. These new techniques will make it possible to accurately characterize minute amounts of cell wall polysaccharides so that subtle changes in structure that occur in individual cell types can be identified and correlated to the physiological or developmental state of the plant. Successful implementation of this research will reveal fundamental relationships between polysaccharide structure, cell wall architecture, and cell wall functions.

  13. Cdt1 revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells

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    Fujita Masatoshi

    2006-10-01

    Full Text Available Abstract In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1 functional and SCFSkp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2 replication-coupled proteolysis mediated by the Cullin4-DDB1Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.

  14. Conditional deletion of Sox17 reveals complex effects on uterine adenogenesis and function.

    Science.gov (United States)

    Guimarães-Young, Amy; Neff, Traci; Dupuy, Adam J; Goodheart, Michael J

    2016-06-15

    The importance of canonical Wnt signaling to murine uterine development is well established. Mouse models in which uterine-specific Wnt ligands, β-catenin, or Lef1 are disrupted result in failure of postnatal endometrial gland development. Sox17 is a transcription factor characterized in numerous tissues as an antagonist of Wnt signaling. Thus, we hypothesized that conditional ablation of Sox17 would lead to hyperproliferation of endometrial glands in mice. Contrary to our prediction, disruption of Sox17 in epithelial and stromal compartments led to inhibition of endometrial adenogenesis and a loss of reproductive capacity. Epithelium-specific Sox17 disruption resulted in normal adenogenesis although reproductive capacity remained impaired. These findings suggest that non-epithelial, Sox17-positive cells are necessary for adenogenesis and that glands require Sox17 to properly function. To our knowledge, these findings are the first to implicate Sox17 in endometrial gland formation and reproductive success. The data presented herein underscore the importance of studying Sox17 in uterine homeostasis and function. PMID:27102016

  15. Kinetic modeling reveals a common death niche for newly formed and mature B cells.

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    Gitit Shahaf

    Full Text Available BACKGROUND: B lymphocytes are subject to elimination following strong BCR ligation in the absence of appropriate second signals, and this mechanism mediates substantial cell losses during late differentiation steps in the bone marrow and periphery. Mature B cells may also be eliminated through this mechanism as well as through normal turnover, but the population containing mature cells destined for elimination has not been identified. Herein, we asked whether the transitional 3 (T3 subset, which contains most newly formed cells undergoing anergic death, could also include mature B cells destined for elimination. METHODOLOGY/PRINCIPAL FINDINGS: To interrogate this hypothesis and its implications, we applied mathematical models to previously generated in vivo labeling data. Our analyses reveal that the death rate of T3 B cells is far higher than the death rates of all other splenic B cell subpopulations. Further, the model, in which the T3 pool includes both newly formed and mature primary B cells destined for apoptotic death, shows that this cell loss may account for nearly all mature B cell turnover. CONCLUSIONS/SIGNIFICANCE: This finding has implications for the mechanism of normal mature B cell turnover.

  16. Polycomb repressive complex 2 component Suz12 is required for hematopoietic stem cell function and lymphopoiesis.

    Science.gov (United States)

    Lee, Stanley C W; Miller, Sarah; Hyland, Craig; Kauppi, Maria; Lebois, Marion; Di Rago, Ladina; Metcalf, Donald; Kinkel, Sarah A; Josefsson, Emma C; Blewitt, Marnie E; Majewski, Ian J; Alexander, Warren S

    2015-07-01

    Polycomb repressive complex 2 (PRC2) is a chromatin modifier that regulates stem cells in embryonic and adult tissues. Loss-of-function studies of PRC2 components have been complicated by early embryonic dependence on PRC2 activity and the partial functional redundancy of enhancer of zeste homolog 1 (Ezh1) and enhancer of zeste homolog 2 (Ezh2), which encode the enzymatic component of PRC2. Here, we investigated the role of PRC2 in hematopoiesis by conditional deletion of suppressor of zeste 12 protein homolog (Suz12), a core component of PRC2. Complete loss of Suz12 resulted in failure of hematopoiesis, both in the embryo and the adult, with a loss of maintenance of hematopoietic stem cells (HSCs). In contrast, partial loss of PRC2 enhanced HSC self-renewal. Although Suz12 was required for lymphoid development, deletion in individual blood cell lineages revealed that it was dispensable for the development of granulocytic, monocytic, and megakaryocytic cells. Collectively, these data reveal the multifaceted role of PRC2 in hematopoiesis, with divergent dose-dependent effects in HSC and distinct roles in maturing blood cells. Because PRC2 is a potential target for cancer therapy, the significant consequences of modest changes in PRC2 activity, as well as the cell and developmental stage-specific effects, will need to be carefully considered in any therapeutic context. PMID:26036803

  17. Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

    Science.gov (United States)

    Nakayama, Naomi; Kato, Hiroaki; Sakashita, Gyosuke; Nariai, Yuko; Nakayama, Kentaro; Kyo, Satoru; Urano, Takeshi

    2016-09-15

    Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells. PMID:27424155

  18. Multiple mating reveals complex patterns of assortative mating by personality and body size.

    Science.gov (United States)

    Montiglio, Pierre-Olivier; Wey, Tina W; Chang, Ann T; Fogarty, Sean; Sih, Andrew

    2016-01-01

    Understanding patterns of non-random mating is central to predicting the consequences of sexual selection. Most studies quantifying assortative mating focus on testing for correlations among partners' phenotypes in mated pairs. Few studies have distinguished between assortative mating arising from preferences for similar partners (expressed by all or a subset of the population) vs. from phenotypic segregation in the environment. Also, few studies have assessed the robustness of assortative mating against temporal changes in social conditions. We tracked multiple matings by stream water striders (Aquarius remigis) across variable social conditions to investigate mating patterns by both body size and behavioural type (personality). We documented temporal changes in partner availability and used a mixed model approach to analyse individual behaviours and changes in mating status recorded on an hourly basis. We assessed whether all or only a subset of individuals in the population expressed a tendency to mate with similar phenotypes. Our analyses took into account variation in the level of competition and in the phenotypes of available partners. Males and females exhibited significant assortative mating by body size: the largest males and females, and the smallest males and females mated together more often than random. However, individuals of intermediate size were equally likely to mate with small, intermediate or large partners. Individuals also displayed two contrasting patterns of assortative mating by personality (activity level). Individuals generally mated preferentially with partners of similar activity level. However, beyond that general trend, individuals with more extreme personalities tended to exhibit disassortative mating: the most active males mated disproportionately with less active females and the least active males tended to mate with more active females. Our analyses thus revealed multiple, distinct patterns of nonrandom mating. These mating

  19. Complexity of genome evolution by segmental rearrangement in Brassica rapa revealed by sequence-level analysis

    Directory of Open Access Journals (Sweden)

    Paterson Andrew H

    2009-11-01

    Full Text Available Abstract Background The Brassica species, related to Arabidopsis thaliana, include an important group of crops and represent an excellent system for studying the evolutionary consequences of polyploidy. Previous studies have led to a proposed structure for an ancestral karyotype and models for the evolution of the B. rapa genome by triplication and segmental rearrangement, but these have not been validated at the sequence level. Results We developed computational tools to analyse the public collection of B. rapa BAC end sequence, in order to identify candidates for representing collinearity discontinuities between the genomes of B. rapa and A. thaliana. For each putative discontinuity, one of the BACs was sequenced and analysed for collinearity with the genome of A. thaliana. Additional BAC clones were identified and sequenced as part of ongoing efforts to sequence four chromosomes of B. rapa. Strikingly few of the 19 inter-chromosomal rearrangements corresponded to the set of collinearity discontinuities anticipated on the basis of previous studies. Our analyses revealed numerous instances of newly detected collinearity blocks. For B. rapa linkage group A8, we were able to develop a model for the derivation of the chromosome from the ancestral karyotype. We were also able to identify a rearrangement event in the ancestor of B. rapa that was not shared with the ancestor of A. thaliana, and is represented in triplicate in the B. rapa genome. In addition to inter-chromosomal rearrangements, we identified and analysed 32 BACs containing the end points of segmental inversion events. Conclusion Our results show that previous studies of segmental collinearity between the A. thaliana, Brassica and ancestral karyotype genomes, although very useful, represent over-simplifications of their true relationships. The presence of numerous cryptic collinear genome segments and the frequent occurrence of segmental inversions mean that inference of the positions

  20. Complex Crustal Structure Beneath Western Turkey Revealed by 3D Seismic Full Waveform Inversion (FWI)

    Science.gov (United States)

    Cubuk-Sabuncu, Yesim; Taymaz, Tuncay; Fichtner, Andreas

    2016-04-01

    We present a 3D radially anisotropic velocity model of the crust and uppermost mantle structure beneath the Sea of Marmara and surroundings based on the full waveform inversion method. The intense seismic activity and crustal deformation are observed in the Northwest Turkey due to transition tectonics between the strike-slip North Anatolian Fault (NAF) and the extensional Aegean region. We have selected and simulated complete waveforms of 62 earthquakes (Mw > 4.0) occurred during 2007-2015, and recorded at (Δ Unified Seismic Network (HUSN, Greece) and Earthquake Research Center of Turkey (AFAD-DAD). The spectral-element solver of the wave equation, SES3D algorithm, is used to simulate seismic wave propagation in 3D spherical coordinates (Fichtner, 2009). The Large Scale Seismic Inversion Framework (LASIF) workflow tool is also used to perform full seismic waveform inversion (Krischer et al., 2015). The initial 3D Earth model is implemented from the multi-scale seismic tomography study of Fichtner et al. (2013). Discrepancies between the observed and simulated synthetic waveforms are determined using the time-frequency misfits which allows a separation between phase and amplitude information (Fichtner et al., 2008). The conjugate gradient optimization method is used to iteratively update the initial Earth model when minimizing the misfit. The inversion is terminated after 19 iterations since no further advances are observed in updated models. Our analysis revealed shear wave velocity variations of the shallow and deeper crustal structure beneath western Turkey down to depths of ~35-40 km. Low shear wave velocity anomalies are observed in the upper and mid crustal depths beneath major fault zones located in the study region. Low velocity zones also tend to mark the outline of young volcanic areas. Our final 3D Earth model is tested using forward wave simulations of earthquakes (M ≥ 3.7) that were not used during the inversion process. The comparison of observed

  1. Complex frictional behaviour of clay-bearing carbonate faults revealed by integrated field and experimental investigation

    Science.gov (United States)

    Bullock, R. J.; De Paola, N.; Holdsworth, R.

    2013-12-01

    Seismicity in the Northern Apennines of Italy nucleates in and propagates through a complex carbonate multilayer sequence, comprising limestones with regular marl interbeds. Observations from the Gubbio fault (1984, Ms = 5.2) indicate that earthquake displacement is localized within narrow principal slip zones, characterized by cataclasites, gouges and calcite veins, and containing up to 50% phyllosilicate. Due to their predominantly velocity-strengthening nature, phyllosilicates are often considered to promote aseismic behaviour during the inter-seismic and post-seismic (afterslip) periods, and may act as barriers to rupture propagation in the upper crust. To assess the effect of clay content on the frictional behaviour and microstructural evolution of carbonate faults during earthquake propagation, we performed high-velocity friction (HVF) experiments, using a rotary-shear apparatus, on end-member gouges of calcite (Cal), montmorillonite (Mont) and mixed-layer illite-smectite (I/S), and on 50:50 and 80:20 mixtures of Cal+Mont and Cal+I/S. Experiments were conducted at room temperature, 1.3 m/s slip rate and 9 MPa normal load. Each sample was run under dry and water-saturated conditions, and experiments terminated both at peak friction (μp) and steady-state friction (μss) for comparison of microstructures at contrasting strength stages. Results for the dry gouges are in-line with previous HVF experiments: all samples attain a peak in friction at the onset of slip, ranging from 0.59 (Mont) to 0.72 (Cal), followed by a dramatic decrease in strength within the first ~0.5 m, after which friction maintains a constant μss value, ranging from 0.15 (Cal) to 0.22 (Mont). Frictional behaviour of the wet gouges is very different: clay-bearing samples typically do not exhibit a peak in friction; steady-state behaviour is attained immediately at the onset of sliding with friction coefficient values as low as 0.05. In summary, when dry, calcite controls the strength of the

  2. Temporal trends in mammal responses to fire reveals the complex effects of fire regime attributes.

    Science.gov (United States)

    Lindenmayer, David B; Blanchard, Wade; MacGregor, Christopher; Barton, Philip; Banks, Sam C; Crane, Mason; Michael, Damian; Okada, Sachiko; Berry, Laurence; Florance, Daniel; Gill, Malcolm

    2016-03-01

    guide management of when and where (prescribed) fire or, conversely, long-unburned vegetation is needed. The complexity of observed responses highlights the need for large reserves in which patterns of heterogeneity in fire regimes can be sustained in space and over time. PMID:27209795

  3. Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation

    OpenAIRE

    Alvarez-Dominguez, Juan R.; Hu, Wenqian; Yuan, Bingbing; Shi, Jiahai; Park, Staphany S.; Gromatzky, Austin A.; van Oudenaarden, Alexander; Lodish, Harvey F.

    2014-01-01

    Global lncRNA discovery reveals novel erythroid-specific lncRNAs that are dynamically expressed and targeted by GATA1, TAL1, and KLF1.Multiple types of lncRNAs promote red cell maturation by regulating neighboring loci, including DLEU2 and a novel Band 3 enhancer lncRNA.

  4. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    OpenAIRE

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M.

    2012-01-01

    Rubin and colleagues describe the first structures of full-length and phosphorylated Retinoblastoma (Rb) protein. These structures reveal the mechanism of Rb inactivation and provide valuable insight into this critical tumor suppressor protein's allosteric inhibition via multisite Cdk phosphorylation and its E2F and cell cycle regulation.

  5. Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells

    International Nuclear Information System (INIS)

    Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 deg. C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells

  6. Dynamics inside the cancer cell attractor reveal cell heterogeneity, limits of stability, and escape.

    Science.gov (United States)

    Li, Qin; Wennborg, Anders; Aurell, Erik; Dekel, Erez; Zou, Jie-Zhi; Xu, Yuting; Huang, Sui; Ernberg, Ingemar

    2016-03-01

    The observed intercellular heterogeneity within a clonal cell population can be mapped as dynamical states clustered around an attractor point in gene expression space, owing to a balance between homeostatic forces and stochastic fluctuations. These dynamics have led to the cancer cell attractor conceptual model, with implications for both carcinogenesis and new therapeutic concepts. Immortalized and malignant EBV-carrying B-cell lines were used to explore this model and characterize the detailed structure of cell attractors. Any subpopulation selected from a population of cells repopulated the whole original basin of attraction within days to weeks. Cells at the basin edges were unstable and prone to apoptosis. Cells continuously changed states within their own attractor, thus driving the repopulation, as shown by fluorescent dye tracing. Perturbations of key regulatory genes induced a jump to a nearby attractor. Using the Fokker-Planck equation, this cell population behavior could be described as two virtual, opposing influences on the cells: one attracting toward the center and the other promoting diffusion in state space (noise). Transcriptome analysis suggests that these forces result from high-dimensional dynamics of the gene regulatory network. We propose that they can be generalized to all cancer cell populations and represent intrinsic behaviors of tumors, offering a previously unidentified characteristic for studying cancer. PMID:26929366

  7. Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells

    DEFF Research Database (Denmark)

    Ding, Li; Paszkowski-Rogacz, Maciej; Winzi, Maria;

    2015-01-01

    Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell...... identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a...

  8. Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency.

    Science.gov (United States)

    Ehinger, Johannes K; Piel, Sarah; Ford, Rhonan; Karlsson, Michael; Sjövall, Fredrik; Frostner, Eleonor Åsander; Morota, Saori; Taylor, Robert W; Turnbull, Doug M; Cornell, Clive; Moss, Steven J; Metzsch, Carsten; Hansson, Magnus J; Fliri, Hans; Elmér, Eskil

    2016-01-01

    Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [(13)C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction. PMID:27502960

  9. Some technetium complexes for labelling red blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Emery, M.F.

    1988-01-01

    A new approach to produce technetium labelled red blood cells, used routinely in diagnostic nuclear medicine, is reported. The enzyme Carbonic Anhydrase (CA), present in erythrocytes, is strongly inhibited by primary aromatic sulphonamides, which bind at the enzyme active site. Three types of ligand able to coordinate to technetium and suitable for modification to include a primary aromatic sulphonamide group were studied; bis(thiosemicarbazones), Schiff bases and some propylene amine oximes. The experimental conditions needed to label the ligands were determined. Both the thiosemicarbazone and propyleneamine oxime derivatives were labelled, but under no conditions attempted were the Schiff bases complexed by Technetium. The two major isozymes of Human Carbonic Anhydrase, HCA I and HCA II, were isolated from blood. The strength of binding of the free ligands SET, PN130 and PN135 with each of the isozymes was measured and expressed as the Dissociation Constant K{sub d}. The rate of uptake of the technetium complexes into washed RBCs and whole blood was measured and found to be much slower in whole blood. The biodistribution of both TcPN130 and TcPN135 in rats was determined and scintigraphic images for the TcPN130 complex were recorded. Attempts to synthesise the Tc-99 analogues on the milligram scale to allow chemical characterisation of these complexes were unsuccessful. (author).

  10. Some technetium complexes for labelling red blood cells

    International Nuclear Information System (INIS)

    A new approach to produce technetium labelled red blood cells, used routinely in diagnostic nuclear medicine, is reported. The enzyme Carbonic Anhydrase (CA), present in erythrocytes, is strongly inhibited by primary aromatic sulphonamides, which bind at the enzyme active site. Three types of ligand able to coordinate to technetium and suitable for modification to include a primary aromatic sulphonamide group were studied; bis(thiosemicarbazones), Schiff bases and some propylene amine oximes. The experimental conditions needed to label the ligands were determined. Both the thiosemicarbazone and propyleneamine oxime derivatives were labelled, but under no conditions attempted were the Schiff bases complexed by Technetium. The two major isozymes of Human Carbonic Anhydrase, HCA I and HCA II, were isolated from blood. The strength of binding of the free ligands SET, PN130 and PN135 with each of the isozymes was measured and expressed as the Dissociation Constant Kd. The rate of uptake of the technetium complexes into washed RBCs and whole blood was measured and found to be much slower in whole blood. The biodistribution of both TcPN130 and TcPN135 in rats was determined and scintigraphic images for the TcPN130 complex were recorded. Attempts to synthesise the Tc-99 analogues on the milligram scale to allow chemical characterisation of these complexes were unsuccessful. (author)

  11. Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq.

    Science.gov (United States)

    Chan, Sunny Sun-Kin; Chan, Howe H W; Kyba, Michael

    2016-06-01

    Mesp1 is a transcription factor that promotes differentiation of pluripotent cells into different mesoderm lineages including hematopoietic, cardiac and skeletal myogenic. This occurs via at least two transient cell populations: a common hematopoietic/cardiac progenitor population and a common cardiac/skeletal myogenic progenitor population. It is not established whether Mesp1-induced mesoderm cells are intrinsically heterogeneous, or are simply capable of multiple lineage decisions. In the current study, we applied single-cell RNA-seq to analyze Mesp1+ mesoderm. Initial whole transcriptome analysis showed a surprising homogeneity among Mesp1-induced mesoderm cells. However, this apparent global homogeneity masked an intrinsic heterogeneity revealed by interrogating a panel of early mesoderm patterning factors. This approach enabled discovery of subpopulations primed for hematopoietic or cardiac development. These studies demonstrate the heterogeneic nature of Mesp1+ mesoderm. PMID:27131741

  12. Genomic instability of micronucleated cells revealed by single-cell comparative genomic hybridization.

    NARCIS (Netherlands)

    Imle, A.; Polzer, B.; Alexander, S.; Klein, C.A.; Friedl, P.H.A.

    2009-01-01

    Nuclear variation in size and shape and genomic instability are hallmarks of dedifferentiated cancer cells. Although micronuclei are a typical long-term consequence of DNA damage, their contribution to chromosomal instability and clonal diversity in cancer disease is unclear. We isolated cancer cell

  13. Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

    Science.gov (United States)

    Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M

    2015-11-15

    The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis. PMID:26213183

  14. C3b complexation diversifies naturally processed T cell epitopes.

    OpenAIRE

    Cretin, François,; Serra, Vincent,; Villiers, Marie-Bernadette; Laharie, Anne-Marie; Marche, Patrice; Gabert, Françoise,

    2007-01-01

    International audience In addition to its well-established role in innate immunity, the complement component C3 is of critical importance in modulating the humoral response. In this study, we examined the effect of C3b linkage to tetanus toxin (TeNT) in the production of antigenic peptides inside human APC. We purified HLA-DR associated peptides isolated either from TeNT or TeNT-C3b pulsed cells. This study revealed that TeNT-C3b derived antigenic peptides are different and more numerous t...

  15. Complex trait subtypes identification using transcriptome profiling reveals an interaction between two QTL affecting adiposity in chicken

    Directory of Open Access Journals (Sweden)

    Blum Yuna

    2011-11-01

    Full Text Available Abstract Background Integrative genomics approaches that combine genotyping and transcriptome profiling in segregating populations have been developed to dissect complex traits. The most common approach is to identify genes whose eQTL colocalize with QTL of interest, providing new functional hypothesis about the causative mutation. Another approach includes defining subtypes for a complex trait using transcriptome profiles and then performing QTL mapping using some of these subtypes. This approach can refine some QTL and reveal new ones. In this paper we introduce Factor Analysis for Multiple Testing (FAMT to define subtypes more accurately and reveal interaction between QTL affecting the same trait. The data used concern hepatic transcriptome profiles for 45 half sib male chicken of a sire known to be heterozygous for a QTL affecting abdominal fatness (AF on chromosome 5 distal region around 168 cM. Results Using this methodology which accounts for hidden dependence structure among phenotypes, we identified 688 genes that are significantly correlated to the AF trait and we distinguished 5 subtypes for AF trait, which are not observed with gene lists obtained by classical approaches. After exclusion of one of the two lean bird subtypes, linkage analysis revealed a previously undetected QTL on chromosome 5 around 100 cM. Interestingly, the animals of this subtype presented the same q paternal haplotype at the 168 cM QTL. This result strongly suggests that the two QTL are in interaction. In other words, the "q configuration" at the 168 cM QTL could hide the QTL existence in the proximal region at 100 cM. We further show that the proximal QTL interacts with the previous one detected on the chromosome 5 distal region. Conclusion Our results demonstrate that stratifying genetic population by molecular phenotypes followed by QTL analysis on various subtypes can lead to identification of novel and interacting QTL.

  16. Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates

    Science.gov (United States)

    Yuan, Bo; Liu, Pengfei; Gupta, Aditya; Beck, Christine R.; Tejomurtula, Anusha; Campbell, Ian M.; Gambin, Tomasz; Simmons, Alexandra D.; Withers, Marjorie A.; Harris, R. Alan; Rogers, Jeffrey; Schwartz, David C.; Lupski, James R.

    2015-01-01

    Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases—about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual’s susceptibility to acquiring disease-associated alleles. PMID:26641089

  17. Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates.

    Directory of Open Access Journals (Sweden)

    Bo Yuan

    2015-12-01

    Full Text Available Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100 is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases-about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual's susceptibility to acquiring disease-associated alleles.

  18. Early evolution of large micro-organisms with cytological complexity revealed by microanalyses of 3.4 Ga organic-walled microfossils.

    Science.gov (United States)

    Sugitani, K; Mimura, K; Takeuchi, M; Lepot, K; Ito, S; Javaux, E J

    2015-11-01

    The Strelley Pool Formation (SPF) is widely distributed in the East Pilbara Terrane (EPT) of the Pilbara Craton, Western Australia, and represents a Paleoarchean shallow-water to subaerial environment. It was deposited ~3.4 billion years ago and displays well-documented carbonate stromatolites. Diverse putative microfossils (SPF microfossils) were recently reported from several localities in the East Strelley, Panorama, Warralong, and Goldsworthy greenstone belts. Thus, the SPF provides unparalleled opportunities to gain insights into a shallow-water to subaerial ecosystem on the early Earth. Our new micro- to nanoscale ultrastructural and microchemical studies of the SPF microfossils show that large (20-70 μm) lenticular organic-walled flanged microfossils retain their structural integrity, morphology, and chain-like arrangements after acid (HF-HCl) extraction (palynology). Scanning and transmitted electron microscopy of extracted microfossils revealed that the central lenticular body is either alveolar or hollow, and the wall is continuous with the surrounding smooth to reticulated discoidal flange. These features demonstrate the evolution of large micro-organisms able to form an acid-resistant recalcitrant envelope or cell wall with complex morphology and to form colonial chains in the Paleoarchean era. This study provides evidence of the evolution of very early and remarkable biological innovations, well before the presumed late emergence of complex cells. PMID:26073280

  19. The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

    Science.gov (United States)

    Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

    2014-02-01

    Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

  20. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2014-02-01

    Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

  1. Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

    Directory of Open Access Journals (Sweden)

    Dmitri Papatsenko

    2015-08-01

    Full Text Available Analyses of gene expression in single mouse embryonic stem cells (mESCs cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM. In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.

  2. Nanotube action between human mesothelial cells reveals novel aspects of inflammatory responses.

    Directory of Open Access Journals (Sweden)

    Julia Ranzinger

    Full Text Available A well-known role of human peritoneal mesothelial cells (HPMCs, the resident cells of the peritoneal cavity, is the generation of an immune response during peritonitis by activation of T-cells via antigen presentation. Recent findings have shown that intercellular nanotubes (NTs mediate functional connectivity between various cell types including immune cells - such as T-cells, natural killer (NK cells or macrophages - by facilitating a spectrum of long range cell-cell interactions. Although of medical interest, the relevance of NT-related findings for human medical conditions and treatment, e.g. in relation to inflammatory processes, remains elusive, particularly due to a lack of appropriate in vivo data. Here, we show for the first time that primary cultures of patient derived HPMCs are functionally connected via membranous nanotubes. NT formation appears to be actin cytoskeleton dependent, mediated by the action of filopodia. Importantly, significant variances in NT numbers between different donors as a consequence of pathophysiological alterations were observable. Furthermore, we show that TNF-α induces nanotube formation and demonstrate a strong correlation of NT connectivity in accordance with the cellular cholesterol level and distribution, pointing to a complex involvement of NTs in inflammatory processes with potential impact for clinical treatment.

  3. Investigating complex I deficiency in Purkinje cells and synapses in patients with mitochondrial disease

    Science.gov (United States)

    Chrysostomou, Alexia; Grady, John P.; Laude, Alex; Taylor, Robert W.; Turnbull, Doug M.

    2015-01-01

    Aims Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus. Methods We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age‐matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses. Results Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses. Conclusions This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia. PMID:26337858

  4. Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification

    Science.gov (United States)

    Boube, Muriel; Faucher, Christian; Joulia, Laurent; Cribbs, David L.; Bourbon, Henri-Marc

    2000-01-01

    The origins of specificity in gene expression are a central concern in understanding developmental control. Mediator protein complexes regulate transcriptional initiation, acting as modular adaptors linking specific transcription factors to core RNA polymerase II. Here, we identified the Drosophila homologs of 23 human mediator genes and mutations of two, dTRAP240 and of dTRAP80 (the putative fly homolog of yeast SRB4). Clonal analysis indicates a general role for dTRAP80 necessary for cell viability. The dTRAP240 gene is also essential, but cells lacking its function are viable and proliferate normally. Clones reveal localized developmental activities including a sex comb cell identity function. This contrasts with the ubiquitous nuclear accumulation of dTRAP240 protein in imaginal discs. Synergistic genetic interactions support shared developmental cell and segment identity functions of dTRAP240 and dTRAP80, potentially within a common complex. Further, they identify the homeotic Sex combs reduced product, required for the same cell/tissue identities, as a functional partner of these mediator proteins. PMID:11090137

  5. Discovery of a dual-targeting organometallic ruthenium complex with high activity inducing early stage apoptosis of cancer cells.

    Science.gov (United States)

    Du, Jun; Zhang, Erlong; Zhao, Yao; Zheng, Wei; Zhang, Yang; Lin, Yu; Wang, Zhaoying; Luo, Qun; Wu, Kui; Wang, Fuyi

    2015-12-01

    Ruthenium based complexes are promising antitumour candidates due to their lower toxicity and better water-solubility compared to the platinum antitumour complexes. An epidermal growth factor receptor (EGFR) has been found to be overexpressed in a large set of tumour cells. In this work, a series of organoruthenium complexes containing EGFR-inhibiting 4-anilinoquinazoline pharmacophores were synthesised and characterised. These complexes exhibited excellent inhibitory activity against EGFR and high affinity to interact with DNA via minor groove binding, featuring dual-targeting properties. In vitro screening demonstrated that the as-prepared ruthenium complexes are anti-proliferating towards a series of cancer cell lines, in particular the non-small-cell lung cancer cell line A549. Fluorescence-activated cell sorting analysis and fluorescence microscopy revealed that the most active complex 3 induced much more early-stage cell apoptosis than its cytotoxic arene ruthenium analogue and the EGFR-inhibiting 4-anilinoquinazolines, verifying the synergetic effect of the two mono-functional pharmacophores. PMID:26446567

  6. Cell division control by the Chromosomal Passenger Complex

    Energy Technology Data Exchange (ETDEWEB)

    Waal, Maike S. van der; Hengeveld, Rutger C.C.; Horst, Armando van der; Lens, Susanne M.A., E-mail: s.m.a.lens@umcutrecht.nl

    2012-07-15

    The Chromosomal Passenger Complex (CPC) consisting of Aurora B kinase, INCENP, Survivin and Borealin, is essential for genomic stability by controlling multiple processes during both nuclear and cytoplasmic division. In mitosis it ensures accurate segregation of the duplicated chromosomes by regulating the mitotic checkpoint, destabilizing incorrectly attached spindle microtubules and by promoting the axial shortening of chromosomal arms in anaphase. During cytokinesis the CPC most likely prevents chromosome damage by imposing an abscission delay when a chromosome bridge connects the two daughter cells. Moreover, by controlling proper cytoplasmic division, the CPC averts tetraploidization. This review describes recent insights on how the CPC is capable of conducting its various functions in the dividing cell to ensure chromosomal stability.

  7. An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

    Directory of Open Access Journals (Sweden)

    Cecil M Benitez

    2014-10-01

    Full Text Available The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

  8. Pyramidal cells in prefrontal cortex: comparative observations reveal unparalleled specializations in neuronal structure among primate species.

    Directory of Open Access Journals (Sweden)

    Guy eElston

    2011-02-01

    Full Text Available The most ubiquitous neuron in the cerebral cortex, the pyramidal cell, is characterised by markedly different dendritic structure among different cortical areas. The complex pyramidal cell phenotype in granular prefrontal cortex (gPFC of higher primates endows specific biophysical properties and patterns of connectivity, which differ to those in other cortical regions. However, within the gPFC, data have been sampled from only a select few cortical areas. The gPFC of species such as human and macaque monkey includes more than 10 cortical areas. It remains unknown as to what degree pyramidal cell structure may vary among these cortical areas. Here we undertook a survey of pyramidal cells in the dorsolateral, medial and orbital gPFC of cercopethicid primates. We found marked heterogeneity in pyramidal cell structure within and between these regions. Moreover, trends for gradients in neuronal complexity varied among species. As neuron structure determines it’s computational abilities and memory storage capacity and connectivity, we propose that these specializations in the pyramidal cell phenotype are an important determinant of species specific executive cortical functions in primates.

  9. DNA damaging, cell cytotoxicity and serum albumin binding efficacy of the rutin-Cu(ii) complex.

    Science.gov (United States)

    Roy, Atanu Singha; Tripathy, Debi Ranjan; Samanta, Sintu; Ghosh, Sudip K; Dasgupta, Swagata

    2016-04-26

    Flavonoids are widely used as anti-oxidants, anti-cancer agents and possess metal ion chelation properties. In this report we have investigated the DNA binding (and damaging), cell cytotoxicity and serum albumin (SA) binding efficacy of the rutin-Cu(ii) complex using differential spectroscopic methods. The rutin-Cu(ii) complex was able to intercalate into calf thymus DNA (ct-DNA) at lower concentrations and its DNA damaging properties were also confirmed from the agarose gel based assay, fluorescence and UV-vis studies. The copper complex was found to be effective against the growth of HeLa cells in vivo. The binding constants (Kb) of the rutin-Cu(ii) complex towards HSA and BSA were found to be (0.98 ± 0.03) and (1.05 ± 0.02) × 10(5) M(-1), respectively, at 299 K and observed to increase with the increase in temperature. Site selectivity studies revealed that the rutin-Cu(ii) complex binds near site 1 (subdomain IIA) of SAs. Thermodynamic parameters indicated that the mode of interaction of rutin and its copper complex with SAs are different from each other. Both ΔH° and ΔS° were observed to be positive for the interaction of the rutin-Cu(ii) complex with SAs, indicating the presence of hydrophobic association in binding. The values of ΔH° were estimated to be negative (-42.07 ± 2.92 and -23.29 ± 2.33 kJ mol(-1) for HSA and BSA respectively) in the binding of rutin with SAs. It implies that after chelation with Cu(ii) ion, rutin alters its binding mode which could have varying applications to its other physicochemical activities. PMID:27035097

  10. Complex Systems Analysis of Arrested Neural Cell Differentiation during Development and Analogous Cell Cycling Models in Carcinogenesis

    OpenAIRE

    Baianu, Professor I.C.; Prisecaru, M.S. V

    2004-01-01

    A new approach to the modular, complex systems analysis of nonlinear dynamics of arrested neural cell Differentiation--induced cell proliferation during organismic development and the analogous cell cycling network transformations involved in carcinogenesis is proposed. Neural tissue arrested differentiation that induces cell proliferation during perturbed development and Carcinogenesis are complex processes that involve dynamically inter-connected biomolecules in the intercellular, membrane...

  11. Genetically Induced Cell Death in Bulge Stem Cells Reveals Their Redundancy for Hair and Epidermal Regeneration

    OpenAIRE

    Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

    2014-01-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Set...

  12. Of macrophages and red blood cells; a complex love story.

    Directory of Open Access Journals (Sweden)

    RobinVan Bruggen

    2014-01-01

    Full Text Available Macrophages tightly control the production and clearance of red blood cells (RBC. During steady state haematopoiesis, approximately 1010 red blood cells are produced per hour within erythroblastic islands in humans. In these erythroblastic islands, resident bone marrow macrophages provide erythroblasts with interactions that are essential for erythroid development. New evidence suggests that not only under homeostasis but also under stress conditions, macrophages play an important role in promoting erythropoiesis. Once RBC have matured, these cells remain in circulation for about 120 days. At the end of their life span, RBC are cleared by macrophages residing in the spleen and the liver. Current theories about the removal of senescent RBC and the essential role of macrophages will be discussed as well as the role of macrophages in facilitating the removal of damaged cellular content from the RBC. In this review we will provide an overview on the role of macrophages in the regulation of RBC production, maintenance and clearance. In addition, we will discuss the interactions between these two cell types during transfer of immune complexes and pathogens from RBC to macrophages.

  13. In vivo fluorescence imaging reveals the promotion of mammary tumorigenesis by mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Chien-Chih Ke

    Full Text Available Mesenchymal stromal cells (MSCs are multipotent adult stem cells which are recruited to the tumor microenvironment (TME and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.

  14. Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

    International Nuclear Information System (INIS)

    Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

  15. Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

    Energy Technology Data Exchange (ETDEWEB)

    Rockel, Beate [Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany); Schmaler, Tilo; Huang, Xiaohua [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany); Dubiel, Wolfgang, E-mail: Wolfgang.dubiel@charite.de [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany)

    2014-07-25

    Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

  16. Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human β Cell Function.

    Science.gov (United States)

    Arda, H Efsun; Li, Lingyu; Tsai, Jennifer; Torre, Eduardo A; Rosli, Yenny; Peiris, Heshan; Spitale, Robert C; Dai, Chunhua; Gu, Xueying; Qu, Kun; Wang, Pei; Wang, Jing; Grompe, Markus; Scharfmann, Raphael; Snyder, Michael S; Bottino, Rita; Powers, Alvin C; Chang, Howard Y; Kim, Seung K

    2016-05-10

    Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however, establishing these age-dependent changes in humans has been challenging. Here, we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α cells or β cells and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function. PMID:27133132

  17. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

    Science.gov (United States)

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-03-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction.

  18. Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

    Science.gov (United States)

    Schmidt, Jens C; Zaug, Arthur J; Cech, Thomas R

    2016-08-25

    Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end. PMID:27523609

  19. Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

    Science.gov (United States)

    Vanderlinden, Willem; Lipfert, Jan; Demeulemeester, Jonas; Debyser, Zeger; de Feyter, Steven

    2014-04-01

    LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease.LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease. Electronic supplementary information (ESI) available: SFM topographs of phage lambda DNA in situ, in the absence and presence of LEDGF/p75; model-independent tests for DNA chain equilibration in 2D; SFM topographs of

  20. Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy.

    Science.gov (United States)

    Caldwell, Brittany; Ustione, Alessandro; Piston, David W

    2016-08-01

    Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic β-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in β-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 β-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gβ and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gβ and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gβγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gβγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gβγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in β-cells occurs at the level of G-protein release. PMID:27508444

  1. Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Xuewei Wang

    Full Text Available The regulation of complex cellular activities in palmitate treated HepG2 cells, and the ensuing cytotoxic phenotype, involves cooperative interactions between genes. While previous approaches have largely focused on identifying individual target genes, elucidating interacting genes has thus far remained elusive. We applied the concept of information synergy to reconstruct a "gene-cooperativity" network for palmititate-induced cytotoxicity in liver cells. Our approach integrated gene expression data with metabolic profiles to select a subset of genes for network reconstruction. Subsequent analysis of the network revealed insulin signaling as the most significantly enriched pathway, and desmoplakin (DSP as its top neighbor. We determined that palmitate significantly reduces DSP expression, and treatment with insulin restores the lost expression of DSP. Insulin resistance is a common pathological feature of fatty liver and related ailments, whereas loss of DSP has been noted in liver carcinoma. Reduced DSP expression can lead to loss of cell-cell adhesion via desmosomes, and disrupt the keratin intermediate filament network. Our findings suggest that DSP expression may be perturbed by palmitate and, along with insulin resistance, may play a role in palmitate induced cytotoxicity, and serve as potential targets for further studies on non-alcoholic fatty liver disease (NAFLD.

  2. Effect of different agents onto multidrug resistant cells revealed by fluorescence correlation spectroscopy

    Science.gov (United States)

    Boutin, C.; Roche, Y.; Jaffiol, R.; Millot, J.-M.; Millot, C.; Plain, J.; Deturche, R.; Jeannesson, P.; Manfait, M.; Royer, P.

    Fluorescence correlation spectroscopy (FCS), which is a sensitive and non invasive technique, has been used to characterize the plasma membrane fluidity and heterogeneity of multidrug resistant living cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been analyzed. Firstly, we reveal a modification of plasma membrane microviscosity according to the addition of a fluidity modulator, benzyl alcohol. In the other hand, revertant such as verapamil and cyclosporin-A appears to act more specifically on the slow diffusion sites as microdomains.

  3. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    Science.gov (United States)

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  4. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity.

    Science.gov (United States)

    Noutsi, Pakiza; Gratton, Enrico; Chaieb, Sahraoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines. PMID:27362860

  5. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    KAUST Repository

    Noutsi, Pakiza

    2016-06-30

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  6. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

    International Nuclear Information System (INIS)

    The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

  7. Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism

    OpenAIRE

    Meierhofer, D.; Mayr, J. A.; Fink, K.; Schmeller, N.; Kofler, B; Sperl, W.

    2006-01-01

    Previously, renal cell carcinoma tissues were reported to display a marked reduction of components of the respiratory chain. To elucidate a possible relationship between tumourigenesis and alterations of oxidative phosphorylation, we screened for mutations of the mitochondrial DNA (mtDNA) in renal carcinoma tissues and patient-matched normal kidney cortex. Seven of the 15 samples investigated revealed at least one somatic heteroplasmic mutation as determined by denaturating HPLC analysis (DHP...

  8. Early events leading to erythroid differentiation in mouse Friend cells revealed by cell fusion experiments

    International Nuclear Information System (INIS)

    Cell fusion with two genetically marked Friend (murine erythroleukemia) cells has made it possible to characterize the very early events leading to erythroid differentiation, particularly the nature of reactions initiated by inducers such as dimethyl sulfoxide. We have found that brief exposure of Friend cells to dimethyl sulfoxide (as well as butyric acid or hexamethylene-bisacetamide) induces an early cellular activity required for erythroid differentiation which is detected only by fusion with ultraviolet-irradiated cells. The induction process of this activity consists of at least two distinct stages. In the first stage, the reaction proceeds without supply of metabolites from the medium and exhibits sensitivity to tumor promoters. The second stage is tightly coupled to cellular metabolic activity, notably protein synthesis. Under normal conditions, the induced activity is short-lived, suggesting turnover of the molecules responsible for this activity. There appears to be a signal produced following dimethyl sulfoxide pulse which acts as an inducer for this activity. The signal remains active for as long as 40 hr when protein synthesis is blocked

  9. Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay.

    Directory of Open Access Journals (Sweden)

    Kaoru Miyazaki

    Full Text Available BACKGROUND: Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP, but not endometrial main population cells (EMP, exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. METHODOLOGY/PRINCIPAL FINDINGS: ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom, a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. CONCLUSIONS/SIGNIFICANCE: We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo

  10. Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

    Science.gov (United States)

    Petropoulos, S; Panula, S P; Schell, J P; Lanner, F

    2016-09-01

    Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line. PMID:27046137

  11. The Paleoproterozoic Singo granite in south-central Uganda revealed as a nested igneous ring complex using geophysical data

    Science.gov (United States)

    Abdelsalam, Mohamed G.; Katumwehe, Andrew B.; Atekwana, Estella A.; Le Pera, Alan K.; Achang, Mercy

    2016-04-01

    We used high-resolution airborne magnetic and radiometric data and satellite gravity data to investigate the form of occurrence of the Paleoproterozoic Singo granite in west-central Uganda. This granitic body covers an area of ∼700 km2, intrudes Paleoproterozoic crystalline rocks and overlain by Paleoproterozoic-Mesoproterozoic sedimentary rocks, both of which belong to the Rwenzori terrane, and it is host to hydrothermally-formed economic minerals such as gold and tungsten. Our analysis provided unprecedented geometrical details of the granitic body and revealed the following: (1) the margins of the Singo granite are characterized by a higher magnetic signature compared to the interior of the granitic body as well as the surroundings. These anomalies are apparent in both the total magnetic field and horizontal derivative images and define eight overlapping ring features. (2) the depth continuation of these magnetic anomalies define outward but steeply-dipping features as indicated by the tilt images extracted from the airborne magnetic data. This is further supported by forward modeling of the magnetic and gravity data. (3) the Singo granite is characterized by relatively high and evenly-distributed equivalent concentration of Uranium (eU) and Thorium (eTh) compared to the surroundings and this is apparent in the Potassium (K)-eTh-eU radiometric ternary image. (4) the granitic body is defined by a gravity low anomaly that persisted to a depth of three km as shown by the Bouguer anomaly image and its five km upward continuation. We used these observations to identify this granitic body as a nested igneous ring complex and we refer to it as the Singo Igneous Ring Complex (SIRC). We further interpreted the eight ring structures as individual igneous ring complexes aligned in an E-W and NE-SW direction and these were developed due to repeated calderas collapse. Additionally, we interpreted the ring-shaped magnetic anomalies as due to hydrothermally-altered margins

  12. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

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    Xiaoyun Wu

    Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

  13. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

    Science.gov (United States)

    Wu, Xiaoyun; Shi, Zhen; Cui, Mingxue; Han, Min; Ruvkun, Gary

    2012-01-01

    The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene. PMID:22412383

  14. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  15. Genome-wide association analyses reveal complex genetic architecture underlying natural variation for flowering time in canola.

    Science.gov (United States)

    Raman, H; Raman, R; Coombes, N; Song, J; Prangnell, R; Bandaranayake, C; Tahira, R; Sundaramoorthi, V; Killian, A; Meng, J; Dennis, E S; Balasubramanian, S

    2016-06-01

    Optimum flowering time is the key to maximize canola production in order to meet global demand of vegetable oil, biodiesel and canola-meal. We reveal extensive variation in flowering time across diverse genotypes of canola under field, glasshouse and controlled environmental conditions. We conduct a genome-wide association study and identify 69 single nucleotide polymorphism (SNP) markers associated with flowering time, which are repeatedly detected across experiments. Several associated SNPs occur in clusters across the canola genome; seven of them were detected within 20 Kb regions of a priori candidate genes; FLOWERING LOCUS T, FRUITFUL, FLOWERING LOCUS C, CONSTANS, FRIGIDA, PHYTOCHROME B and an additional five SNPs were localized within 14 Kb of a previously identified quantitative trait loci for flowering time. Expression analyses showed that among FLC paralogs, BnFLC.A2 accounts for ~23% of natural variation in diverse accessions. Genome-wide association analysis for FLC expression levels mapped not only BnFLC.C2 but also other loci that contribute to variation in FLC expression. In addition to revealing the complex genetic architecture of flowering time variation, we demonstrate that the identified SNPs can be modelled to predict flowering time in diverse canola germplasm accurately and hence are suitable for genomic selection of adaptative traits in canola improvement programmes. PMID:26428711

  16. The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, T.B.; Lerch, T.F.; Cook, R.W.; Woodruff, T.K.; Jardetzky, T.S. (NWU)

    2010-03-08

    TGF-{beta} ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-{beta} ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.

  17. Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

    Science.gov (United States)

    Yamaguchi, Masaya; VanderLinden, Ryan; Weissmann, Florian; Qiao, Renping; Dube, Prakash; Brown, Nicholas G; Haselbach, David; Zhang, Wei; Sidhu, Sachdev S; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-08-18

    The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation. PMID:27522463

  18. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.

    Science.gov (United States)

    Lechtenberg, Bernhard C; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K; Ware, Carl F; Mace, Peter D; Riedl, Stefan J

    2016-01-28

    Ubiquitination is a central process affecting all facets of cellular signalling and function. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate. The RING-between-RING (RBR) family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases. The RBR family includes Parkin and HOIP, the central catalytic factor of the LUBAC (linear ubiquitin chain assembly complex). While structural insights into the RBR E3 ligases Parkin and HHARI in their overall auto-inhibited forms are available, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely unknown. Here we present the first structure, to our knowledge, of the fully active human HOIP RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP RBR adopts a conformation markedly different from that of auto-inhibited RBRs. HOIP RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centres ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, three distinct helix-IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~ubiquitin conjugate and, surprisingly, an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  19. A transcriptional regulatory role of the THAP11-HCF-1 complex in colon cancer cell function.

    Science.gov (United States)

    Parker, J Brandon; Palchaudhuri, Santanu; Yin, Hanwei; Wei, Jianjun; Chakravarti, Debabrata

    2012-05-01

    The recently identified Thanatos-associated protein (THAP) domain is an atypical zinc finger motif with sequence-specific DNA-binding activity. Emerging data suggest that THAP proteins may function in chromatin-dependent processes, including transcriptional regulation, but the roles of most THAP proteins in normal and aberrant cellular processes remain largely unknown. In this work, we identify THAP11 as a transcriptional regulator differentially expressed in human colon cancer. Immunohistochemical analysis of human colon cancers revealed increased THAP11 expression in both primary tumors and metastases. Knockdown of THAP11 in SW620 colon cancer cells resulted in a significant decrease in cell proliferation, and profiling of gene expression in these cells identified a novel gene set composed of 80 differentially expressed genes, 70% of which were derepressed by THAP11 knockdown. THAP11 was found to associate physically with the transcriptional coregulator HCF-1 (host cell factor 1) and recruit HCF-1 to target promoters. Importantly, THAP11-mediated gene regulation and its chromatin association require HCF-1, while HCF-1 recruitment at these genes requires THAP11. Collectively, these data provide the first characterization of THAP11-dependent gene expression in human colon cancer cells and suggest that the THAP11-HCF-1 complex may be an important transcriptional and cell growth regulator in human colon cancer. PMID:22371484

  20. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    Directory of Open Access Journals (Sweden)

    Schuren Frank H

    2008-12-01

    Full Text Available Abstract Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability.

  1. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells.

    Science.gov (United States)

    Xu, Bo; Chen, Minjian; Ji, Xiaoli; Yao, Mengmeng; Mao, Zhilei; Zhou, Kun; Xia, Yankai; Han, Xiao; Tang, Wei

    2015-10-01

    Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1α, IL-1β, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment. PMID:26165742

  2. A computer-assisted 3D model for analyzing the aggregation of tumorigenic cells reveals specialized behaviors and unique cell types that facilitate aggregate coalescence.

    Directory of Open Access Journals (Sweden)

    Amanda Scherer

    Full Text Available We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity, and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named "facilitators" and "probes." A third cell type, the "dervish", is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.

  3. Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells

    International Nuclear Information System (INIS)

    Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34+ cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML

  4. Transcriptomic changes in human renal proximal tubular cells revealed under hypoxic conditions by RNA sequencing.

    Science.gov (United States)

    Yu, Wenmin; Li, Yiping; Wang, Zhi; Liu, Lei; Liu, Jing; Ding, Fengan; Zhang, Xiaoyi; Cheng, Zhengyuan; Chen, Pingsheng; Dou, Jun

    2016-09-01

    Chronic hypoxia often occurs among patients with chronic kidney disease (CKD). Renal proximal tubular cells may be the primary target of a hypoxic insult. However, the underlying transcriptional mechanisms remain undefined. In this study, we revealed the global changes in gene expression in HK‑2 human renal proximal tubular cells under hypoxic and normoxic conditions. We analyzed the transcriptome of HK‑2 cells exposed to hypoxia for 24 h using RNA sequencing. A total of 279 differentially expressed genes was examined, as these genes could potentially explain the differences in HK‑2 cells between hypoxic and normoxic conditions. Moreover, 17 genes were validated by qPCR, and the results were highly concordant with the RNA seqencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these differentially expressed genes. The upregulated genes appeared to be significantly enriched in the pathyway of extracellular matrix (ECM)-receptor interaction, and in paticular, the pathway of renal cell carcinoma was upregulated under hypoxic conditions. The downregulated genes were enriched in the signaling pathway related to antigen processing and presentation; however, the pathway of glutathione metabolism was downregulated. Our analysis revealed numerous novel transcripts and alternative splicing events. Simultaneously, we also identified a large number of single nucleotide polymorphisms, which will be a rich resource for future marker development. On the whole, our data indicate that transcriptome analysis provides valuable information for a more in depth understanding of the molecular mechanisms in CKD and renal cell carcinoma. PMID:27432315

  5. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics.

    Science.gov (United States)

    Hsu, Shang-Te D; Breukink, Eefjan; Tischenko, Eugene; Lutters, Mandy A G; de Kruijff, Ben; Kaptein, Robert; Bonvin, Alexandre M J J; van Nuland, Nico A J

    2004-10-01

    The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics. PMID:15361862

  6. Distinctive Structure of the EphA3/Ephrin-A5 Complex Reveals a Dual Mode of Eph Receptor Interaction for Ephrin-A5.

    Directory of Open Access Journals (Sweden)

    Garry Jason Forse

    Full Text Available The Eph receptor tyrosine kinase/ephrin ligand system regulates a wide spectrum of physiological processes, while its dysregulation has been implicated in cancer progression. The human EphA3 receptor is widely upregulated in the tumor microenvironment and is highly expressed in some types of cancer cells. Furthermore, EphA3 is among the most highly mutated genes in lung cancer and it is also frequently mutated in other cancers. We report the structure of the ligand-binding domain of the EphA3 receptor in complex with its preferred ligand, ephrin-A5. The structure of the complex reveals a pronounced tilt of the ephrin-A5 ligand compared to its orientation when bound to the EphA2 and EphB2 receptors and similar to its orientation when bound to EphA4. This tilt brings an additional area of ephrin-A5 into contact with regions of EphA3 outside the ephrin-binding pocket thereby enlarging the size of the interface, which is consistent with the high binding affinity of ephrin-A5 for EphA3. This large variation in the tilt of ephrin-A5 bound to different Eph receptors has not been previously observed for other ephrins.

  7. The FasFADD death domain complex structure reveals the basis of DISC assembly and disease mutations

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Liwei [Weill Cornell Medical College, New York, NY (United States); Yang, Jin Kuk [Weill Cornell Medical College, New York, NY (United States); Soongsil Univ., Seoul (Korea); Kabaleeswaran, Venkataraman [Weill Cornell Medical College, New York, NY (United States); Rice, Amanda J. [Harvard Medical School, Boston, MA (United States); Cruz, Anthony C. [National Inst. of Health (NIH), Bethesda, MD (United States); Park, Ah Young [Univ. of Oxford (United Kingdom); Yin, Qian [Weill Cornell Medical College, New York, NY (United States); Damko, Ermelinda [Weill Cornell Medical College, New York, NY (United States); Jang, Se Bok [Weill Cornell Medical College, New York, NY (United States); Pusan National Univ., Busan (Korea, Republic of); Raunser, Stefan [Harvard Medical School, Boston, MA (United States); Robinson, Carol V. [Univ. of Oxford (United Kingdom); Siegel, Richard M. [National Inst. of Health (NIH), Bethesda, MD (United States); Walz, Thomas [Harvard Medical School, Boston, MA (United States); Wu, Hao [Weill Cornell Medical College, New York, NY (United States)

    2010-10-10

    The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

  8. m(6)A-LAIC-seq reveals the census and complexity of the m(6)A epitranscriptome.

    Science.gov (United States)

    Molinie, Benoit; Wang, Jinkai; Lim, Kok Seong; Hillebrand, Roman; Lu, Zhi-Xiang; Van Wittenberghe, Nicholas; Howard, Benjamin D; Daneshvar, Kaveh; Mullen, Alan C; Dedon, Peter; Xing, Yi; Giallourakis, Cosmas C

    2016-08-01

    N(6)-Methyladenosine (m(6)A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m(6)A modified ('m(6)A levels') or the relationship of m(6)A modification(s) to alternative RNA isoforms. To deconvolute the m(6)A epitranscriptome, we developed m(6)A-level and isoform-characterization sequencing (m(6)A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m(6)A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3' untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m(6)A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m(6)A modifications. PMID:27376769

  9. Complex patterns of divergence among green-sensitive (RH2a African cichlid opsins revealed by Clade model analyses

    Directory of Open Access Journals (Sweden)

    Weadick Cameron J

    2012-10-01

    Full Text Available Abstract Background Gene duplications play an important role in the evolution of functional protein diversity. Some models of duplicate gene evolution predict complex forms of paralog divergence; orthologous proteins may diverge as well, further complicating patterns of divergence among and within gene families. Consequently, studying the link between protein sequence evolution and duplication requires the use of flexible substitution models that can accommodate multiple shifts in selection across a phylogeny. Here, we employed a variety of codon substitution models, primarily Clade models, to explore how selective constraint evolved following the duplication of a green-sensitive (RH2a visual pigment protein (opsin in African cichlids. Past studies have linked opsin divergence to ecological and sexual divergence within the African cichlid adaptive radiation. Furthermore, biochemical and regulatory differences between the RH2aα and RH2aβ paralogs have been documented. It thus seems likely that selection varies in complex ways throughout this gene family. Results Clade model analysis of African cichlid RH2a opsins revealed a large increase in the nonsynonymous-to-synonymous substitution rate ratio (ω following the duplication, as well as an even larger increase, one consistent with positive selection, for Lake Tanganyikan cichlid RH2aβ opsins. Analysis using the popular Branch-site models, by contrast, revealed no such alteration of constraint. Several amino acid sites known to influence spectral and non-spectral aspects of opsin biochemistry were found to be evolving divergently, suggesting that orthologous RH2a opsins may vary in terms of spectral sensitivity and response kinetics. Divergence appears to be occurring despite intronic gene conversion among the tandemly-arranged duplicates. Conclusions Our findings indicate that variation in selective constraint is associated with both gene duplication and divergence among orthologs in African

  10. Spindle cell carcinoma of the breast as complex cystic lesion:a case report

    Institute of Scientific and Technical Information of China (English)

    Masahiro Kitada; Satoshi Hayashi; Yoshinari Matsuda; Kei Ishibashi; Keisuke Oikawa; Naoyuki Miyokawa

    2014-01-01

    Spindle cell carcinoma of the breast is a rare tumor. hTis tumor can proliferate rapidly and cause cystic changes because of internal tissue necrosis. We evaluated a 54-year-old woman with right breast lump. Mammography showed a category four mass with a diameter of 2.5 cm. Ultrasonography (US) revealed a complex cystic lesion, and ifne-needle aspiration (FNA) cytology demonstrated bloody fluid and malignant cells. Partial breast resection and sentinel lymph node biopsy were performed. Immunohistology revealed spindle cells with positive results for cytokeratin (AE1/AE3) and vimentin, partially positive results for s-100, and negative results for desmin and α-actin. The pathological stage was IIA, and biochemical characterization showed that the tumor was triple negative. Six courses of FEC-100 chemotherapy (5-fluorouracil 500 mg/m2, epirubicin 100 mg/m2, and cyclophosphamide 500 mg/m2) were administered. Radiotherapy was performed. hTis case is discussed with reference to the literature.

  11. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    Energy Technology Data Exchange (ETDEWEB)

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  12. Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis.

    Directory of Open Access Journals (Sweden)

    Guoying Dong

    Full Text Available H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A-G. Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.

  13. A comparison of the rest complex binding patterns in embryonic stem cells and epiblast stem cells.

    Directory of Open Access Journals (Sweden)

    Masahide Seki

    Full Text Available We detected and characterized the binding sites of the representative Rest complex components Rest, Sin3A, and Lsd1. We compared their binding patterns in mouse embryonic stem (ES cells and epiblast stem (EpiS cells. We found few Rest sites unique to the EpiS cells. The ES-unique site features were distinct from those of the common sites, namely, the signal intensities were weaker, and the characteristic gene function categories differed. Our analyses showed that the Rest binding sites do not always overlap with the Sin3A and Lsd1 binding sites. The Sin3A binding pattern differed remarkably between the ES and EpiS cells and was accompanied by significant changes in acetylated-histone patterns in the surrounding regions. A series of transcriptome analyses in the same cell types unexpectedly showed that the putative target gene transcript levels were not dramatically different despite dynamic changes in the Rest complex binding patterns and chromatin statuses, which suggests that Rest is not the sole determinant of repression at its targets. Nevertheless, we identified putative Rest targets with explicitly enhanced transcription upon Rest knock-down in 143 and 60 common and ES-unique Rest target genes, respectively. Among such sites, several genes are involved in ES cell proliferation. In addition, we also found that long, intergenic non-coding RNAs were apparent Rest targets and shared similar features with the protein-coding target genes. Interestingly, such non-coding target genes showed less conservation through evolution than protein-coding targets. As a result of differences in the components and targets of the Rest complex, its functional roles may differ in ES and EpiS cells.

  14. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation.

    Directory of Open Access Journals (Sweden)

    Peter Rellos

    Full Text Available UNLABELLED: Long-term potentiation (LTP, a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM-dependent kinase II (CaMKII. CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.

  15. Effect of Toxoplasma gondii infection on the junctional complex of retinal pigment epithelial cells.

    Science.gov (United States)

    Nogueira, Alanderson R; Leve, Fernanda; Morgado-Diaz, José; Tedesco, Roberto Carlos; Pereira, Mirian Claudia S

    2016-04-01

    Ocular toxoplasmosis is the most frequent cause of uveitis, leading to partial or total loss of vision, with the retina the main affected structure. The cells of the retinal pigment epithelium (RPE) play an important role in the physiology of the retina and formation of the blood-retinal barrier. Several pathogens induce barrier dysfunction by altering tight junction (TJ) integrity. Here, we analysed the effect of infection by Toxoplasma gondii on TJ integrity in ARPE-19 cells. Loss of TJ integrity was demonstrated in T. gondii-infected ARPE-19 cells, causing increase in paracellular permeability and disturbance of the barrier function of the RPE. Confocal microscopy also revealed alteration in the TJ protein occludin induced by T. gondii infection. Disruption of junctional complex was also evidenced by scanning and transmission electron microscopy. Cell-cell contact loss was noticed in the early stages of infection by T. gondii with the visualization of small to moderate intercellular spaces. Large gaps were mostly observed with the progression of the infection. Thus, our data suggest that the alterations induced by T. gondii in the structural organization of the RPE may contribute to retinal injury evidenced by ocular toxoplasmosis. PMID:26928468

  16. [Acute intestinal obstruction revealing enteropathy associated t-cell lymphoma, about a case].

    Science.gov (United States)

    Garba, Abdoul Aziz; Adamou, Harissou; Magagi, Ibrahim Amadou; Brah, Souleymane; Habou, Oumarou

    2016-01-01

    Enteropathy associated T-cell lymphoma (EATL) is a rare complication of celiac disease (CD). We report a case of EATL associated with CD revealed by acute intestinal obstruction. A North African woman of 38 years old with a history of infertility and chronic abdominal pain was admitted in emergency with acute intestinal obstruction. During the surgery, we found a tumor on the small intestine with mesenteric lymphadenopathy. Histology and immunohistochemistry of the specimen objectified a digestive T lymphoma CD3+ and immunological assessment of celiac disease was positive. The diagnosis of EATL was thus retained. Chemotherapy (CHOEP protocol) was established as well as gluten-free diet with a complete response to treatment. The EATL is a rare complication of CD that can be revealed by intestinal obstruction. The prognosis can be improved by early treatment involving surgery and chemotherapy. Its prevention requires early diagnosis of celiac and gluten-free diets. PMID:27217874

  17. The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation.

    Science.gov (United States)

    Rodor, Julie; Pan, Qun; Blencowe, Benjamin J; Eyras, Eduardo; Cáceres, Javier F

    2016-09-01

    Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC. PMID:27365209

  18. Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization.

    Science.gov (United States)

    Huebinger, Jan; Han, Hong-Mei; Hofnagel, Oliver; Vetter, Ingrid R; Bastiaens, Philippe I H; Grabenbauer, Markus

    2016-02-23

    Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth. PMID:26541066

  19. Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response.

    OpenAIRE

    Owens, Kelly,; Cunningham, Dale,; Macdonald, Glen; Rubel, Edwin,; Raible, David,; Pujol, Remy

    2007-01-01

    Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycos...

  20. An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas

    DEFF Research Database (Denmark)

    Cirilo, Priscila Daniele Ramos; Marchi, Fábio Albuquerque; Barros Filho, Mateus de Camargo;

    2013-01-01

    integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell...... transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs....

  1. Neurotransmitters and synaptic components in the Merkel cell-neurite complex, a gentle touch receptor

    OpenAIRE

    Maksimovic, Srdjan; Baba, Yoshichika; Lumpkin, Ellen A.

    2013-01-01

    Merkel cells are an enigmatic group of rare cells found in the skin of vertebrates. Most make contacts with somatosensory afferents to form Merkel cell-neurite complexes, which are gentle-touch receptors that initiate slowly adapting type I responses. The function of Merkel cells within the complex remains debated despite decades of research. Numerous anatomical studies demonstrate that Merkel cells form synaptic-like contacts with sensory afferent terminals. Moreover, recent molecular analys...

  2. An integrated RNA-Seq and network study reveals a complex regulation process of rice embryo during seed germination.

    Science.gov (United States)

    Wei, Ting; He, Zilong; Tan, XinYu; Liu, Xue; Yuan, Xiao; Luo, Yingfeng; Hu, Songnian

    2015-08-14

    Seed germination is a crucial stage for plant development and agricultural production. To investigate its complex regulation process, the RNA-Seq study of rice embryo was conducted at three time points of 0, 12 and 48 h post imbibition (HPI). Dynamic transcriptional alterations were observed, especially in the early stage (0-12 HPI). Seed related genes, especially those encoding desiccation inducible proteins and storage reserves in embryo, decreased drastically after imbibition. The expression profiles of phytohormone related genes indicated distinct roles of abscisic acid (ABA), gibberellin (GA) and brassinosteroid (BR) in germination. Moreover, network analysis revealed the importance of protein phosphorylation in phytohormone interactions. Network and gene ontology (GO) analyses suggested that transcription factors (TFs) played a regulatory role in functional transitions during germination, and the enriched TF families at 0 HPI implied a regulation of epigenetic modification in dry seeds. In addition, 35 germination-specific TF genes in embryo were identified and seven genes were verified by qRT-PCR. Besides, enriched TF binding sites (TFBSs) supported physiological changes in germination. Overall, this study expands our comprehensive knowledge of multiple regulation factors underlying rice seed germination. PMID:26116530

  3. Genome re-sequencing of semi-wild soybean reveals a complex Soja population structure and deep introgression.

    Directory of Open Access Journals (Sweden)

    Jie Qiu

    Full Text Available Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou and a wild line (Lanxi 1 collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1 no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2 besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3 high heterozygous rates (0.19-0.49 were observed in several semi-wild lines; and (4 over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure.

  4. Seabird and louse coevolution: complex histories revealed by 12S rRNA sequences and reconciliation analyses.

    Science.gov (United States)

    Paterson, A M; Wallis, G P; Wallis, L J; Gray, R D

    2000-09-01

    We investigated the coevolutionary history of seabirds (orders Procellariiformes and Sphenisciformes) and their lice (order Phthiraptera). Independent trees were produced for the seabirds (tree derived from 12S ribosomal RNA, isoenzyme, and behavioral data) and their lice (trees derived from 12S rRNA data). Brook's parsimony analysis (BPA) supported a general history of cospeciation (consistency index = 0.84, retention index = 0.81). We inferred that the homoplasy in the BPA was caused by one intrahost speciation, one potential host-switching, and eight or nine sorting events. Using reconciliation analysis, we quantified the cost of fitting the louse tree onto the seabird tree. The reconciled trees postulated one host-switching, nine cospeciation, three or four intrahost speciation, and 11 to 14 sorting events. The number of cospeciation events was significantly more than would be expected from chance alone (P seabirds and lice. Neither data set displayed significant rate heterogeneity. An examination of the codivergent nodes revealed that seabirds and lice have cospeciated synchronously and that lice have evolved at approximately 5.5 times the rate of seabirds. The degree of sequence divergence supported some of the postulated intrahost speciation events (e.g., Halipeurus predated the evolution of their present hosts). The sequence data also supported some of the postulated host-switching events. These results demonstrate the value of sequence data and reconciliation analyses in unraveling complex histories between hosts and their parasites. PMID:12116418

  5. Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis Flowers[W][OPEN

    Science.gov (United States)

    Ginglinger, Jean-François; Boachon, Benoit; Höfer, René; Paetz, Christian; Köllner, Tobias G.; Miesch, Laurence; Lugan, Raphael; Baltenweck, Raymonde; Mutterer, Jérôme; Ullmann, Pascaline; Beran, Franziska; Claudel, Patricia; Verstappen, Francel; Fischer, Marc J.C.; Karst, Francis; Bouwmeester, Harro; Miesch, Michel; Schneider, Bernd; Gershenzon, Jonathan; Ehlting, Jürgen; Werck-Reichhart, Danièle

    2013-01-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (−)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined. PMID:24285789

  6. High-density PhyloChip profiling of stimulated aquifer microbial communities reveals a complex response to acetate amendment

    Energy Technology Data Exchange (ETDEWEB)

    Handley, Kim M. [Univ. of California, Berkeley, CA (United States); Wrighton, Kelly C. [Univ. of California, Berkeley, CA (United States); Piceno, Yvette M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Andersen, Gary L. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); DeSantis, Todd Z. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Williams, Kenneth H. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Wilkins, Michael J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); N' Guessan, A. Lucie [Univ. of Massachusetts, Amherst, MA (United States); Peacock, Aaron [Haley & Aldrich, Oak Ridge, TN (United States); Bargar, John [SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL); Long, Philip E. [Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99353, USA; Banfield, Jillian F.

    2012-04-13

    There is increasing interest in harnessing the functional capacities of indigenous microbial communities to transform and remediate a wide range of environmental contaminants. Information about which community members respond to stimulation can guide the interpretation and development of remediation approaches. To comprehensively determine community membership and abundance patterns among a suite of samples associated with uranium bioremediation experiments we employed a high-density microarray (PhyloChip). Samples were unstimulated, naturally reducing, or collected during Fe(III) (early) and sulfate reduction (late biostimulation) from an acetate re-amended/amended aquifer in Rifle, Colorado, and from laboratory experiments using field-collected materials. Deep community sampling with PhyloChip identified hundreds-to-thousands of operational taxonomic units (OTUs) present during amendment, and revealed close similarity among highly enriched taxa from drill-core and groundwater well-deployed column sediment. Overall, phylogenetic data suggested stimulated community membership was most affected by a carryover effect between annual stimulation events. Nevertheless, OTUs within the Fe(III)- and sulfate-reducing lineages, Desulfuromonadales and Desulfobacterales, were repeatedly stimulated. Less consistent, co-enriched taxa represented additional lineages associated with Fe(III) and sulfate reduction (for example, Desulfovibrionales; Syntrophobacterales; Peptococcaceae) and autotrophic sulfur oxidation (Sulfurovum; Campylobacterales). These data imply complex membership among highly stimulated taxa, and by inference biogeochemical responses to acetate, a non-fermentable substrate.

  7. Tetrapositive plutonium, neptunium, uranium, and thorium coordination complexes: chemistry revealed by electron transfer and collision induced dissociation.

    Science.gov (United States)

    Gong, Yu; Tian, Guoxin; Rao, Linfeng; Gibson, John K

    2014-04-17

    The Pu(4+), Np(4+), and U(4+) ions, which have large electron affinities of ∼34.6, ∼33.6, and ∼32.6 eV, respectively, were stabilized from solution to the gas phase upon coordination by three neutral tetramethyl-3-oxa-glutaramide ligands (TMOGA). Both collision induced dissociation (CID) and electron transfer dissociation (ETD) of Pu(TMOGA)3(4+) reveal the propensity for reduction of Pu(IV) to Pu(III), by loss of TMOGA(+) in CID and by simple electron transfer in ETD. The reduction of Pu(IV) is in distinct contrast to retention of Th(IV) in both CID and ETD of Th(TMOGA)3(4+), where only the C-Oether bond cleavage product was observed. U(TMOGA)3(4+) behaves similarly to Th(TMOGA)3(4+) upon CID and ETD, while the fragmentation patterns of Np(TMOGA)3(4+) lie between those of Pu(TMOGA)3(4+) and U(TMOGA)3(4+). It is notable that the gas-phase fragmentation behaviors of these exceptional tetrapositive complexes parallel fundamental differences in condensed phase chemistry within the actinide series, specifically the tendency for reduction from the IV to III oxidation states. PMID:24660979

  8. Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2011-11-01

    Full Text Available As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium and prototrophic (minimal medium culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens and an important target.

  9. Differentiation of Human Parthenogenetic Pluripotent Stem Cells Reveals Multiple Tissue- and Isoform-Specific Imprinted Transcripts

    Directory of Open Access Journals (Sweden)

    Yonatan Stelzer

    2015-04-01

    Full Text Available Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5′ RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes.

  10. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    Energy Technology Data Exchange (ETDEWEB)

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  11. Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging.

    Directory of Open Access Journals (Sweden)

    Margaret Clarke

    Full Text Available BACKGROUND: The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized. METHODOLOGY: To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins. PRINCIPAL FINDINGS: We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved. CONCLUSIONS/SIGNIFICANCE: Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

  12. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

    Science.gov (United States)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  13. Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Liang-Hui Chu

    Full Text Available Angiogenesis involves stimulation of endothelial cells (EC by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the "angiome" could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A. We used the Short Time-series Expression Miner (STEM to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC and human microvascular EC (MEC. The results show that VEGFR1-VEGFR2 levels are more closely coupled than VEGFR1-VEGFR3 or VEGFR2-VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.

  14. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    Science.gov (United States)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  15. Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3.

    Science.gov (United States)

    Kolinko, Sebastian; Jogler, Christian; Katzmann, Emanuel; Wanner, Gerhard; Peplies, Jörg; Schüler, Dirk

    2012-07-01

    Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that orient along magnetic fields using membrane-coated magnetic nanocrystals of magnetite (Fe(3) O(4) ) or greigite (Fe(3) S(4) ), the magnetosomes. Previous phylogenetic analysis of MTB has been limited to few cultivated species and most abundant members of natural populations, which were assigned to Proteobacteria and the Nitrospirae phyla. Here, we describe a single cell-based approach that allowed the targeted phylogenetic and ultrastructural analysis of the magnetotactic bacterium SKK-01, which was low abundant in sediments of Lake Chiemsee. Morphologically conspicuous single cells of SKK-01 were micromanipulated from magnetically collected multi-species MTB populations, which was followed by whole genome amplification and ultrastructural analysis of sorted cells. Besides intracellular sulphur inclusions, the large ovoid cells of SKK-01 harbour ∼175 bullet-shaped magnetosomes arranged in multiple chains that consist of magnetite as revealed by TEM and EDX analysis. Sequence analysis of 16 and 23S rRNA genes from amplified genomic DNA as well as fluorescence in situ hybridization assigned SKK-01 to the candidate division OP3, which so far lacks any cultivated representatives. SKK-01 represents the first morphotype that can be assigned to the OP3 group as well as the first magnetotactic member of the PVC superphylum. PMID:22003954

  16. Combined in silico and in vivo analyses reveal role of Hes1 in taste cell differentiation.

    Directory of Open Access Journals (Sweden)

    Masato S Ota

    2009-04-01

    Full Text Available The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction, the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP, we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1(-/ (- mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds.

  17. CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Yue Hu

    Full Text Available We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC and adenocarcinoma (AC subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.

  18. Surface complexation of neptunium (V) onto whole cells and cell componets of Shewanella alga

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Donald Timothy [Los Alamos National Laboratory; Deo, Randhir P [ASU; Rittmann, Bruce E [ASU; Songkasiri, Warinthorn [UNAFFILIATED

    2008-01-01

    We systematically quantified surface complexation of neptunium(V) onto whole cells of Shewanella alga strain BrY and onto cell wall and extracellular polymeric substances (EPS) of S. alga. We first performed acid and base titrations and used the mathematical model FITEQL with constant-capacitance surface-complexation to determine the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl site associated with amino acids (pK{sub a} {approx} 2.4), a carboxyl group not associated with amino acids (pK{sub a} {approx} 5), a phosphoryl site (pK{sub a} {approx} 7.2), and an amine site (pK{sub a} > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components at different pHs. Results show that solution pH influenced the speciation of Np(V) and each of the surface functional groups. We used the speciation sub-model of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, NpO{sub 2}{sup +} was the dominant form of Np(V), and its log K values for the low-pK{sub a} carboxyl, other carboxyl, and phosphoryl groups were 1.75, 1.75, and 2.5 to 3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH3+, which complexed with NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-}. The log K for NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-} complexed onto the amine groups was 3.1 to 3.6. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results point towards the important role of surface complexation in defining key actinide-microbiological interactions in the subsurface.

  19. Networks of neuroblastoma cells on porous silicon substrates reveal a small world topology

    KAUST Repository

    Marinaro, Giovanni

    2015-01-01

    The human brain is a tightly interweaving network of neural cells where the complexity of the network is given by the large number of its constituents and its architecture. The topological structure of neurons in the brain translates into its increased computational capabilities, low energy consumption, and nondeterministic functions, which differentiate human behavior from artificial computational schemes. In this manuscript, we fabricated porous silicon chips with a small pore size ranging from 8 to 75 nm and large fractal dimensions up to Df ∼ 2.8. In culturing neuroblastoma N2A cells on the described substrates, we found that those cells adhere more firmly to and proliferate on the porous surfaces compared to the conventional nominally flat silicon substrates, which were used as controls. More importantly, we observed that N2A cells on the porous substrates create highly clustered, small world topology patterns. We conjecture that neurons with a similar architecture may elaborate information more efficiently than in random or regular grids. Moreover, we hypothesize that systems of neurons on nano-scale geometry evolve in time to form networks in which the propagation of information is maximized. This journal is

  20. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  1. Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina

    Directory of Open Access Journals (Sweden)

    Harris William A

    2009-07-01

    Full Text Available Abstract Background The condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described. Results Here, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the cap-g locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in cap-g mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes. Conclusion Our findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.

  2. DNA-based digital tension probes reveal integrin forces during early cell adhesion

    Science.gov (United States)

    Zhang, Yun; Ge, Chenghao; Zhu, Cheng; Salaita, Khalid

    2014-10-01

    Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging. Here to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20- to 30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA hairpins with tunable force response thresholds, ligands and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized RGD over linear RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial resolution limitations of traction force microscopy.

  3. Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells.

    Directory of Open Access Journals (Sweden)

    M Kristen Hall

    Full Text Available The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these

  4. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Frazier, M.E.; Samuel, J.E.

    1985-02-01

    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  5. Genome Editing of Lineage Determinants in Human Pluripotent Stem Cells Reveals Mechanisms of Pancreatic Development and Diabetes.

    Science.gov (United States)

    Zhu, Zengrong; Li, Qing V; Lee, Kihyun; Rosen, Bess P; González, Federico; Soh, Chew-Li; Huangfu, Danwei

    2016-06-01

    Directed differentiation of human pluripotent stem cells (hPSCs) into somatic counterparts is a valuable tool for studying disease. However, examination of developmental mechanisms in hPSCs remains challenging given complex multi-factorial actions at different stages. Here, we used TALEN and CRISPR/Cas-mediated gene editing and hPSC-directed differentiation for a systematic analysis of the roles of eight pancreatic transcription factors (PDX1, RFX6, PTF1A, GLIS3, MNX1, NGN3, HES1, and ARX). Our analysis not only verified conserved gene requirements between mice and humans but also revealed a number of previously unsuspected developmental mechanisms with implications for type 2 diabetes. These include a role of RFX6 in regulating the number of pancreatic progenitors, a haploinsufficient requirement for PDX1 in pancreatic β cell differentiation, and a potentially divergent role of NGN3 in humans and mice. Our findings support use of systematic genome editing in hPSCs as a strategy for understanding mechanisms underlying congenital disorders. PMID:27133796

  6. Diversity and Complexity of Ceramide Generation After Exposure of Jurkat Leukemia Cells to Irradiation

    International Nuclear Information System (INIS)

    Purpose: To define which intracellular pools of sphingomyelin and ceramide are involved in the triggering of apoptosis of Jurkat leukemia cells in response to γ-ray exposure. Methods and Materials: We examined the kinetics of ceramide generation at the whole-cell level and in different subcellular compartments (plasma membrane rafts, mitochondria, and endoplasmic reticulum) after irradiation with photons. Ceramide was measured by high-performance liquid chromatography or after pulse labeling experiments, and the presence of sphingomyelinase within mitochondria was assessed by electron microscopy. Results: Irradiation of Jurkat leukemia cells resulted in the sequential triggering of sphingomyelin hydrolysis, followed by de novo synthesis that led to a late ceramide response (from 24 h) correlated with the triggering of apoptosis. At the subcellular level, pulse-label experiments, using [3H]-palmitate as a precursor, strengthened the involvement of the radiation-induced sphingomyelin breakdown and revealed a very early peak (15 min) of ceramide in plasma membrane rafts. A second peak in mitochondria was measured 4 h after irradiation, resulting from an increase of the sphingomyelin content relating to the targeting of acid sphingomyelinase toward this organelle. Conclusion: These data confirm that ceramide is a major determinant in the triggering of radiation-induced apoptosis and highlight the complexity of the sequential compartment-specific ceramide-mediated response of Jurkat leukemia cells to γ-rays

  7. The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

    Energy Technology Data Exchange (ETDEWEB)

    Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui; Wong, Sui-Lam; Ng, Kenneth K. S., E-mail: ngk@ucalgary.ca [University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4 (Canada)

    2013-05-01

    The structure of the SBP-Tag–streptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12–14) and a C-terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

  8. Functional malignant cell heterogeneity in pancreatic neuroendocrine tumors revealed by targeting of PDGF-DD

    Science.gov (United States)

    Cortez, Eliane; Gladh, Hanna; Braun, Sebastian; Bocci, Matteo; Cordero, Eugenia; Björkström, Niklas K.; Miyazaki, Hideki; Michael, Iacovos P.; Eriksson, Ulf; Folestad, Erika; Pietras, Kristian

    2016-01-01

    Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRβ, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRβ. The presence of a subclonal population of tumor cells characterized by PDGFRβ expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRβ axis. PMID:26831065

  9. Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

    Science.gov (United States)

    Kreder, Rémy; Pyrshev, Kyrylo A; Darwich, Zeinab; Kucherak, Oleksandr A; Mély, Yves; Klymchenko, Andrey S

    2015-06-19

    Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research. PMID:25710589

  10. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    International Nuclear Information System (INIS)

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

  11. Live-cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe oryzae.

    Science.gov (United States)

    Mochizuki, Susumu; Minami, Eiichi; Nishizawa, Yoko

    2015-12-01

    The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live-cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane-rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High-resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first-invaded cell. Furthermore, a newly developed, long-term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first-invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection. PMID:26472068

  12. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris; Kiessling, Ann A

    2016-01-15

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  13. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    Energy Technology Data Exchange (ETDEWEB)

    Economou, Nicoleta J.; Zentner, Isaac J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States); Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian [Brookhaven National Laboratory, Upton, NY 11973 (United States); Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States)

    2013-04-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.

  14. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    International Nuclear Information System (INIS)

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance

  15. Live-cell microscopy reveals small molecule inhibitor effects on MAPK pathway dynamics.

    Directory of Open Access Journals (Sweden)

    Daniel J Anderson

    Full Text Available Oncogenic mutations in the mitogen activated protein kinase (MAPK pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2. We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.

  16. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  17. Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

    Directory of Open Access Journals (Sweden)

    Lulu Cao

    Full Text Available The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76 plays a critical scaffolding role in T cell receptor (TCR signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.

  18. Repeatability of Perimacular Ganglion Cell Complex Analysis with Spectral-Domain Optical Coherence Tomography

    OpenAIRE

    Ng, Dorothy S. K.; Preeti Gupta; Yih Chung Tham; Chye Fong Peck; Tien Yin Wong; Mohammad Kamran Ikram; Cheung, Carol Y.

    2015-01-01

    Purpose. To assess the repeatability of spectral-domain optical coherence tomography to measure macular and perimacular ganglion cell complex thicknesses and compare retinal ganglion cell parameters between algorithms. Methods. Ninety-two nonglaucomatous eyes from 92 participants underwent macular and perimacular ganglion cell complex thickness measurement using OCT-HS100 Glaucoma 3D algorithm and these measurements were repeated for 34 subjects. All subjects also had macular ganglion cell-in...

  19. The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

    Science.gov (United States)

    Allen-Worthington, Krystal; Xie, Jianjun; Brown, Jessica L; Edmunson, Alexa M; Dowling, Abigail; Navratil, Amy M; Scavelli, Kurt; Yoon, Hojean; Kim, Do-Geun; Bynoe, Margaret S; Clarke, Iain; Roberson, Mark S

    2016-09-01

    Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion. PMID:27482602

  20. Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

    Directory of Open Access Journals (Sweden)

    Liu Liwen

    2009-09-01

    Full Text Available Abstract Background Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast. Results By integrating genome-wide data from multiple time course cell cycle microarray experiments we reconstructed a gene regulatory network. Based on the network, we discovered in addition to previously known regulatory hubs in M phase, a new putative regulatory hub in the form of the HMG box transcription factor SPBC19G7.04. Further, we inferred periodic activities of several less known transcription factors over the course of the cell cycle, identified over 500 putative regulatory targets and detected many new phase-specific and conserved cis-regulatory motifs. In particular, we show that SPBC19G7.04 has highly significant periodic activity that peaks in early M phase, which is coordinated with the late G2 activity of the forkhead transcription factor fkh2. Finally, using an enhanced Bayesian algorithm to co-cluster the expression data, we obtained 31 clusters of co-regulated genes 1 which constitute regulatory modules from different phases of the cell cycle, 2 whose phase order is coherent across the 10 time course experiments, and 3 which lead to identification of phase-specific control elements at both the transcriptional and post-transcriptional levels in S. pombe. In particular, the ribosome biogenesis clusters expressed in G2 phase reveal new, highly conserved RNA motifs. Conclusion Using a systems-level analysis of the phase-specific nature of the S. pombe cell cycle gene regulation, we have provided new testable evidence for post-transcriptional regulation in the G2 phase of the fission yeast cell cycle

  1. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation

    Czech Academy of Sciences Publication Activity Database

    Klučková, Katarína; Sticha, M.; Černý, Jiří; Mráček, Tomáš; Dong, L.; Drahota, Zdeněk; Gottlieb, E.; Neužil, Jiří; Rohlena, Jakub

    2015-01-01

    Roč. 6, May 2015 (2015), e1749. ISSN 2041-4889 R&D Projects: GA ČR GAP301/12/1851; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GB14-36804G Institutional support: RVO:86652036 ; RVO:67985823 Keywords : ALPHA-TOCOPHERYL SUCCINATE * ELECTRON-TRANSPORT CHAIN * RESPIRATORY-CHAIN Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.014, year: 2014

  2. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry

    OpenAIRE

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F.

    2013-01-01

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the en...

  3. Efficient transformation of mammalian cells using DNA interpolyelectrolyte complexes with carbon chain polycations.

    Science.gov (United States)

    Kabanov, A V; Astafieva, I V; Maksimova, I V; Lukanidin, E M; Georgiev, G P; Kabanov, V A

    1993-01-01

    A new method for mammalian cell transformation is proposed which is based on incorporation of plasmids into interpolyelectrolyte complexes (IPECs) with carbon chain polycations. The method is illustrated by examples of pRSV CAT and p beta-Gal plasmid IPECs with poly(N-ethyl-4-vinylpyridinium bromide) (C2PVP) and poly(N-ethyl-4-vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium+ ++) bromides random copolymer (C16PVP). These IPECs are produced spontaneously due to formation of a cooperative system of interchain electrostatic bonds after mixing DNA and polycation solutions. The interaction of IPEC with normal mouse fibroblasts NIH 3T3, human T-lymphoma "Jurkat", and Mardin Darby canine kidney cells has been studied. The data obtained has revealed that plasmid incorporation into IPECs significantly enhances both DNA adsorption on the plasma membrane and DNA uptake into a cell. The in vitro transformation of NIH 3T3 cells was monitored by a standard cloramphenicol acetyltransferase (CAT) assay (pRSV CAT plasmid) and by detection of beta-galactosidase (beta-Gal) expression using 4-methylumbeliferril beta-D-galactopyranoside as a substrate (p beta-Gal plasmid). In both cases it has been proved that IPEC-incorporated plasmids possess an ability for efficient cell transformation. The transforming activity of IPECs depends on their composition and polycation chemical structure. Under optimal conditions the efficiency of cell transformation with IPECs is several fold higher than that observed during standard calcium phosphate precipitation. The mechanism of the phenomenon observed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8305514

  4. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    International Nuclear Information System (INIS)

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

  5. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  6. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  7. Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes.

    Science.gov (United States)

    Hamaji, Takashi; Mogi, Yuko; Ferris, Patrick J; Mori, Toshiyuki; Miyagishima, Shinya; Kabeya, Yukihiro; Nishimura, Yoshiki; Toyoda, Atsushi; Noguchi, Hideki; Fujiyama, Asao; Olson, Bradley J S C; Marriage, Tara N; Nishii, Ichiro; Umen, James G; Nozaki, Hisayoshi

    2016-01-01

    Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT-, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale-V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs. PMID:26921294

  8. Complexing 7,3',4'-tryoxiflavonol with cell phosphatidylcholine

    OpenAIRE

    Nusratullin V. M.; Fahretdinova E. R.; Nasibullin R. S.; Galeeva R. I.

    2010-01-01

    Aim. To investigate the complex formation of 7,3',4'-try-oxyflavonol of flavonoids group with cellular phosphatidylcholine. Methods. Semi-empirical quantum chemistry, spectroscopy NMR. Results. The changes in conformational status of 7,3',4'-tryoxy- flavonol at complex formation have been shown. Conclusions. The conformational changes in phosphatidylcholine take place under the 7,3',4'-tryoxyflavonol/phosphatidylcholine complex formation.

  9. A simple cell-based assay reveals that diverse neuropsychiatric risk genes converge on primary cilia.

    Directory of Open Access Journals (Sweden)

    Aaron Marley

    Full Text Available Human genetic studies are beginning to identify a large number of genes linked to neuropsychiatric disorders. It is increasingly evident that different genes contribute to risk for similar syndromes and, conversely, the same genes or even the same alleles cross over traditional diagnostic categories. A current challenge is to understand the cellular biology of identified risk genes. However, most genes associated with complex neuropsychiatric phenotypes are not related through a known biochemical pathway, and many have an entirely unknown cellular function. One possibility is that diverse disease-linked genes converge at a higher-level cellular structure. The synapse is already known to be one such convergence, and emerging evidence suggests the primary cilium as another. Because many genes associated with neuropsychiatric illness are expressed also outside the nervous system, as are cilia, we tested the hypothesis that such genes affect conserved features of the primary cilium. Using RNA interference to test 41 broadly expressed candidate genes associated with schizophrenia, bipolar affective disorder, autism spectrum disorder and intellectual disability, we found 20 candidates that reduce ciliation in NIH3T3 cells when knocked down, and three whose manipulation increases cilia length. Three of the candidate genes were previously implicated in cilia formation and, altogether, approximately half of the candidates tested produced a ciliary phenotype. Our results support the hypothesis that primary cilia indeed represent a conserved cellular structure at which the effects of diverse neuropsychiatric risk genes converge. More broadly, they suggest a relatively simple cell-based approach that may be useful for exploring the complex biological underpinnings of neuropsychiatric disease.

  10. Revealing nonergodic dynamics in living cells from a single particle trajectory

    Science.gov (United States)

    Lanoiselée, Yann; Grebenkov, Denis S.

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  11. Formation of Interpolymer Complexes on Polypropylene Textiles via Layer-by-Layer Modification as Revealed by FTIR Method

    Directory of Open Access Journals (Sweden)

    Dawid STAWSKI

    2012-06-01

    Full Text Available Oppositely-charged polyelectrolytes such poly(acrylic acid and poly(allylamine hydrochloride have been deposited on a polypropylene nonwoven by the layer-by-layer technique. The complex formation of this modified material has been studied by FTIR spectroscopy. It has been found that external reflectance FTIR is an efficient technique for the identification of non-complexed or complexed carboxylic groups in the modified polypropylene material.

  12. Electrophoretic behavior of DNA-methyl-CpG-binding domain protein complexes revealed by capillary electrophoreses laser-induced fluorescence.

    Science.gov (United States)

    Zhong, Shangwei; Zou, Dandan; Zhao, Bailin; Zhang, Dapeng; Li, Xiangjun; Wang, Hailin

    2015-12-01

    The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (μ0 /μ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the μ0 and μ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors. PMID:26377303

  13. Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics

    OpenAIRE

    Smits, A. H.; Jansen, P. W. T. C.; Poser, I; Hyman, A. A.; Vermeulen, M.

    2013-01-01

    Many cellular proteins assemble into macromolecular protein complexes. The identification of protein–protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not sui...

  14. Cellular responses induced by Cu(II quinolinonato complexes in human tumor and hepatic cells

    Directory of Open Access Journals (Sweden)

    Trávníček Zdeněk

    2012-12-01

    Full Text Available Abstract Background Inspired by the unprecedented historical success of cisplatin, one of the most important research directions in bioinorganic and medicinal chemistry is dedicated to the development of new anticancer compounds with the potential to surpass it in antitumor activity, while having lower unwanted side-effects. Therefore, a series of copper(II mixed-ligand complexes of the type [Cu(qui(L]Y · xH2O (1–6, where Hqui = 2-phenyl-3-hydroxy-4(1H-quinolinone, Y = NO3 (1, 3, 5 or BF4 (2, 4, 6, and L = 1,10-phenanthroline (phen (1, 2, 5-methyl-1,10-phenanthroline (mphen (3, 4 and bathophenanthroline (bphen (5, 6, was studied for their in vitro cytotoxicity against several human cancer cell lines (A549 lung carcinoma, HeLa cervix epitheloid carcinoma, G361 melanoma cells, A2780 ovarian carcinoma, A2780cis cisplatin-resistant ovarian carcinoma, LNCaP androgen-sensitive prostate adenocarcinoma and THP-1 monocytic leukemia. Results The tested complexes displayed a stronger cytotoxic effect against all the cancer cells as compared to cisplatin. The highest cytotoxicity was found for the complexes 4 (IC50 = 0.36 ± 0.05 μM and 0.56 ± 0.15 μM, 5 (IC50 = 0.66 ± 0.07 μM and 0.73 ± 0.08 μM and 6 (IC50 = 0.57 ± 0.11 μM and 0.70 ± 0.20 μM against A2780, and A2780cis respectively, as compared with the values of 12.0 ± 0.8 μM and 27.0 ± 4.6 μM determined for cisplatin. Moreover, the tested complexes were much less cytotoxic to primary human hepatocytes than to the cancer cells. The complexes 5 and 6 exhibited significantly high ability to modulate secretion of the pro-inflammatory cytokines TNF-α (2873 ± 238 pg/mL and 3284 ± 139 pg/mL for 5, and 6 respectively and IL-1β (1177 ± 128 pg/mL and 1087 ± 101 pg/mL for 5, and 6 respectively tested on the lipopolysaccharide (LPS-stimulated THP-1 cells as compared with the values of 1173

  15. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

    Science.gov (United States)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D.; Otero, Carolina

    2016-02-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  16. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    Science.gov (United States)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes. PMID:26847692

  17. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation.

    Science.gov (United States)

    Chen, Xiaoying; Zhang, Kunshan; Zhou, Liqiang; Gao, Xinpei; Wang, Junbang; Yao, Yinan; He, Fei; Luo, Yuping; Yu, Yongchun; Li, Siguang; Cheng, Liming; Sun, Yi E

    2016-03-01

    The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions. PMID:26883038

  18. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Directory of Open Access Journals (Sweden)

    Shufeng Liu

    Full Text Available Hepatitis C virus (HCV poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP, UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1 and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection.

  19. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Science.gov (United States)

    Liu, Shufeng; Zhao, Ting; Song, BenBen; Zhou, Jianhua; Wang, Tony T

    2016-01-01

    Hepatitis C virus (HCV) poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP), UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1) and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection. PMID:26808496

  20. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    Science.gov (United States)

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-04-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

  1. Variations in cell morphology in the canine cruciate ligament complex.

    Science.gov (United States)

    Smith, K D; Vaughan-Thomas, A; Spiller, D G; Clegg, P D; Innes, J F; Comerford, E J

    2012-08-01

    Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology. PMID:22465617

  2. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    Science.gov (United States)

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production. PMID:26820137

  3. Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation

    Directory of Open Access Journals (Sweden)

    Treuner-Lange Anke

    2010-04-01

    Full Text Available Abstract Background Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities. Results Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate

  4. Synergistic Effects between mTOR Complex 1/2 and Glycolysis Inhibitors in Non-Small-Cell Lung Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Suhua Jiang

    Full Text Available Cancer metabolism has greatly interested researchers. Mammalian target of rapamycin (mTOR is dysregulated in a variety of cancers and considered to be an appealing therapeutic target. It has been proven that growth factor signal, mediated by mTOR complex 1 (mTORC1, drives cancer metabolism by regulating key enzymes in metabolic pathways. However, the role of mTORC2 in cancer metabolism has not been thoroughly investigated. In this study, by employing automated spectrophotometry, we found the level of glucose uptake was decreased in non-small-cell lung carcinoma (NSCLC A549, PC-9 and SK-MES-1 cells treated with rapamycin or siRNA against Raptor, indicating that the inhibition of mTORC1 attenuated glycolytic metabolism in NSCLC cells. Moreover, the inhibition of AKT reduced glucose uptake in the cells as well, suggesting the involvement of AKT pathway in mTORC1 mediated glycolytic metabolism. Furthermore, our results showed a significant decrease in glucose uptake in rictor down-regulated NSCLC cells, implying a critical role of mTORC2 in NSCLC cell glycolysis. In addition, the experiments for MTT, ATP, and clonogenic assays demonstrated a reduction in cell proliferation, cell viability, and colony forming ability in mTOR inhibiting NSCLC cells. Interestingly, the combined application of mTORC1/2 inhibitors and glycolysis inhibitor not only suppressed the cell proliferation and colony formation, but also induced cell apoptosis, and such an effect of the combined application was stronger than that caused by mTORC1/2 inhibitors alone. In conclusion, this study reports a novel effect of mTORC2 on NSCLC cell metabolism, and reveals the synergistic effects between mTOR complex 1/2 and glycolysis inhibitors, suggesting that the combined application of mTORC1/2 and glycolysis inhibitors may be a new promising approach to treat NSCLC.

  5. Cytotoxic Effects of Newly Synthesized Palladium(II Complexes of Diethyldithiocarbamate on Gastrointestinal Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Shahram Hadizadeh

    2014-01-01

    Full Text Available As a part of a drug development program to discover novel therapeutic and more effective palladium (Pd based anticancer drugs, a series of water-soluble Pd complexes have been synthesized by interaction between [Pd (phen(H2O2(NO32] and alkylenebisdithiocarbamate(al-bis-dtc disodium salts. This study was undertaken to examine the possible cytotoxic effect of three novel complexes (0.125–64 µg/mL on human gastric carcinoma (AGS, esophageal squamous cell carcinoma (Kyse-30, and hepatocellular carcinoma (HepG2 cell lines. The cytotoxicity was examined using cell proliferation and acridine orange/ethidium bromide (AO/EB assay. In order to examine the effects of new Pd(II complexes on cell cycle status, we performed cell cycle analysis. The complexes were found to have completely lethal effects on the cell lines, and the half maximal inhibitory concentration (IC50 values obtained for the cell lines were much lower in comparison with cisplatin. We demonstrated that the three new Pd(II complexes are able to induce G2/M phase arrest in AGS and HepG2; in addition, the Pd(II complexes caused an S phase arrest in Kyse-30 cell line. Our results indicate that newly synthesized Pd(II complexes may provide a novel class of chemopreventive compounds for anticancer therapy.

  6. The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

    OpenAIRE

    Gavina, Manuela; Za, Lorena; Molteni, Raffaella; Pardi, Ruggero; Curtis, Ivan de

    2010-01-01

    Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-...

  7. Low Cost Ferritic Stainless Steel in Dye Sensitized Solar Cells with Cobalt Complex Electrolyte

    OpenAIRE

    Miettunen, Kati; Jouttijärvi, Sami; Jiang, Roger; Saukkonen, Tapio; Romu, Jyrki; Halme, Janne; Lund, Peter

    2014-01-01

    Cheap ferritic stainless steel is applied here as the counter electrode substrate in dye sensitized solar cells with cobalt complex electrolyte. A 5.0% efficiency was reached with these type of cells which is more than 2.5 times higher compared to previously reported devices with metal counter electrode and cobalt complex electrolyte. The electrochemical impedance spectra analysis showed that the best cells with the ferritic steel counter electrode had as low charge transfer resistance (3.6 Ω...

  8. Runx-CBFβ complexes control Foxp3 expression in regulatory T cells

    OpenAIRE

    Rudra, Dipayan; Egawa, Takeshi; Chong, Mark M.W.; Treuting, Piper; Dan R. Littman; Rudensky, Alexander Y.

    2009-01-01

    Foxp3 plays an indispensable role in establishing stable transcriptional and functional programs of regulatory T (Treg) cells. Loss of Foxp3 expression in mature Treg cells results in a failure of suppressor function, yet the molecular mechanisms ensuring steady heritable Foxp3 expression in the Treg cell lineage remain unknown. Using Treg cell-specific gene targeting we found that Runx-CBFβ complexes were required for maintenance of Foxp3 mRNA and protein expression in Treg cells. Consequent...

  9. Comparative study of antithrombin III. Protease complex metabolism by fibroblasts and vascular endothelial cells

    International Nuclear Information System (INIS)

    125I-labeled human antithrombin III (125I-AT III).protease complexes are specifically bound to both cultured human skin fibroblast (HSF) cells and adult bovine aortic endothelial (ABAE) cells; however, there is a significant difference in the rate and degree of metabolism of the complexes by these two cell types. HSF cells appear to internalize the complexes at a rate of about 2.5 pmole/1 X 10(6) cells/h and subsequently degrade them at a rate of 0.6 pmole/1 X 10(6) cells/h. ABAE cells internalize and degrade the complexes at rates approximately 100 and 30 times lower, respectively. Neither cell type interacts with free 125I-AT III but only with its combined form with either thrombin or trypsin. These data indicate the major role of HSF cells in the removal of AT III.protease complexes from extravascular spaces in the body, in contrast to the inert vascular surface with regard to AT III.protease complexes provided by the vascular endothelium

  10. Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde

    International Nuclear Information System (INIS)

    Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3σ and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: → 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. → Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. → The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. → The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.

  11. Introgressive hybridization and the evolutionary history of the herring gull complex revealed by mitochondrial and nuclear DNA

    OpenAIRE

    Zhang Jun; Ritz Markus S; Liebers-Helbig Dorit; Sternkopf Viviane; Helbig Andreas J; de Knijff Peter

    2010-01-01

    Abstract Background Based on extensive mitochondrial DNA (mtDNA) sequence data, we previously showed that the model of speciation among species of herring gull (Larus argentatus) complex was not that of a ring species, but most likely due more complex speciation scenario's. We also found that two species, herring gull and glaucous gull (L. hyperboreus) displayed an unexpected biphyletic distribution of their mtDNA haplotypes. It was evident that mtDNA sequence data alone were far from suffici...

  12. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

    NARCIS (Netherlands)

    Souza, De Amanda P.; Lessa Alvim Kamei, Claire; Torres Salvador, Andres Francisco; Pattathil, Sivakumar; Hahn, Michael G.; Trindade, Luisa M.; Buckeridge, Marcos S.

    2015-01-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell wal

  13. The metabolic enzyme ManA reveals a link between cell wall integrity and chromosome morphology.

    OpenAIRE

    Maya Elbaz; Sigal Ben-Yehuda

    2010-01-01

    Author Summary The bacterial cell is resistant to extremes of osmotic pressure and protected against mechanical damages by the existence of a rigid outer shell defined as the cell wall. The strength of the cell wall is achieved by the presence of long glycan strands cross-linked by peptide side bridges. The cell wall is a dynamic structure continuously being synthesized and modified to allow for cell growth and division. Damaging the cell wall leads to abnormal cellular morphologies and cell ...

  14. Complexity

    CERN Document Server

    Gershenson, Carlos

    2011-01-01

    The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.

  15. Role of topology in complex functional networks of beta cells.

    Science.gov (United States)

    Cherubini, Christian; Filippi, Simonetta; Gizzi, Alessio; Loppini, Alessandro

    2015-10-01

    The activity of pancreatic β cells can be described by biological networks of coupled nonlinear oscillators that, via electrochemical synchronization, release insulin in response to augmented glucose levels. In this work, we analyze the emergent behavior of regular and percolated β-cells clusters through a stochastic mathematical model where "functional" networks arise. We show that the emergence and robustness of the synchronized dynamics depend both on intrinsic and extrinsic parameters. In particular, cellular noise level, glucose concentration, network spatial architecture, and cell-to-cell coupling strength are the key factors for the generation of a rhythmic and robust activity. Their role in the functional network topology associated with β-cells clusters is analyzed and discussed. PMID:26565267

  16. Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

    Science.gov (United States)

    Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

    2016-07-01

    Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. PMID:27184390

  17. Introgressive hybridization and the evolutionary history of the herring gull complex revealed by mitochondrial and nuclear DNA

    Directory of Open Access Journals (Sweden)

    Zhang Jun

    2010-11-01

    Full Text Available Abstract Background Based on extensive mitochondrial DNA (mtDNA sequence data, we previously showed that the model of speciation among species of herring gull (Larus argentatus complex was not that of a ring species, but most likely due more complex speciation scenario's. We also found that two species, herring gull and glaucous gull (L. hyperboreus displayed an unexpected biphyletic distribution of their mtDNA haplotypes. It was evident that mtDNA sequence data alone were far from sufficient to obtain a more accurate and detailed insight into the demographic processes that underlie speciation of this complex, and that extensive autosomal genetic analysis was warranted. Results For this reason, the present study focuses on the reconstruction of the phylogeographic history of a limited number of gull species by means of a combined approach of mtDNA sequence data and 230 autosomal amplified fragment length polymorphism (AFLP loci. At the species level, the mtDNA and AFLP genetic data were largely congruent. Not only for argentatus and hyperboreus, but also among a third species, great black-backed gull (L. marinus we observed two distinct groups of mtDNA sequence haplotypes. Based on the AFLP data we were also able to detect distinct genetic subgroups among the various argentatus, hyperboreus, and marinus populations, supporting our initial hypothesis that complex demographic scenario's underlie speciation in the herring gull complex. Conclusions We present evidence that for each of these three biphyletic gull species, extensive mtDNA introgression could have taken place among the various geographically distinct subpopulations, or even among current species. Moreover, based on a large number of autosomal AFLP loci, we found evidence for distinct and complex demographic scenario's for each of the three species we studied. A more refined insight into the exact phylogeographic history within the herring gull complex is still impossible, and requires

  18. Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex

    Directory of Open Access Journals (Sweden)

    Fuchs Pinhas

    2009-09-01

    Full Text Available Abstract Background Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes. This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. Results We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within" or by infection with a high amount of virus particles per cell (fusion "from without". Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Conclusion Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

  19. Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

    Directory of Open Access Journals (Sweden)

    Margret E Berg Miller

    Full Text Available BACKGROUND: Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. METHODOLOGY/PRINCIPAL FINDINGS: The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb, and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs, polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs. Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315 exhibited the highest levels of up-regulation. CONCLUSIONS/SIGNIFICANCE: The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional

  20. Detonation nanodiamond complexes with cancer stem cells inhibitors or paracrine products of mesenchymal stem cells as new potential medications

    Science.gov (United States)

    Konoplyannikov, A. G.; Alekseenskiy, A. E.; Zlotin, S. G.; Smirnov, B. B.; Kalsina, S. Sh.; Lepehina, L. A.; Semenkova, I. V.; Agaeva, E. V.; Baboyan, S. B.; Rjumshina, E. A.; Nosachenko, V. V.; Konoplyannikov, M. A.

    2015-09-01

    Combined use of complexes of the most active chemotherapeutic drugs and detonation nanodiamonds (DND) is a new trend in cancer therapy, which is probably related to selective chemotherapeutic drug delivery by DND to the zone of so-called cancer stem cells (CSC). Stable DND complexes of 4-5 nm size with salinomycin—a strong CSC inhibitor—have been obtained (as a suspension). It has been demonstrated that a complex administration considerably increases the drug antitumor effect on the transplantable tumor of LLC mice. A similar effect has been observed in CSC models in vivo, obtained by exposure of stem cells of normal mice tissues to a carcinogen 1,2-dimethylhydrazine. It has also been found out, that administration of DND complexes with the conditioned medium from mesenchymal stem cells (MSC) cultures to mice results in a considerable stimulation of stem cell pools in normal mice tissues, which can be used in regenerative medicine.

  1. Study on sickle cell disease haplotypes reveals the African origin of Amapá's population

    Directory of Open Access Journals (Sweden)

    Natália de Morais Castelo

    2014-04-01

    Full Text Available Introduction:Sickle cell disease (SCD is a hereditary, hematologic, multifactorial disease, with high prevalence worldwide; its cause is a mutation in the sixth codon of the beta globin gene (βs.Objective:To identify the haplotypes present in people with SCD in Amapá, and relate them to African descent.Methods:We analyzed, by molecular techniques, 46 blood samples from people with SCD in Macapá, the capital of Amapá, with the purpose of obtaining information about haplotype frequency distribution, which helps understand the ethnic background of Amapá's population.Results:Our study revealed that the most frequent haplotype is Bantu (61.2%, followed by Benin (26.6% and Senegal (12.2%. Results showed statistical differences from studies conducted in other regions. A high frequency of the Senegal haplotype stands out, in comparison with some Brazilian studies.Conclusion:Amapá's results exhibit unique characteristics when compared to haplotypes in other regions, with high frequency of Senegal and Benin haplotypes, absence of atypical, Cameroon and Saudi, confirming that Brazil shows ethnic background diversity, as well as different haplotype frequencies.

  2. Single-cell genomics reveal metabolic strategies for microbial growth and survival in an oligotrophic aquifer

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, Michael J.; Kennedy, David W.; Castelle, Cindy; Field, Erin; Stepanauskas, Ramunas; Fredrickson, Jim K.; Konopka, Allan

    2014-02-09

    Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site and have been shown to significantly change in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single cell genomics techniques to shed light on the physiological niche of these microorganisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3­-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide-range of both intra-and extra­-cellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors (TBDRs), which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. CRISPR-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic microorganisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area subsurface and biogeochemical shifts associated with Columbia River water intrusion.

  3. Poly(vinyl alcohol)–LiBOB complexes for lithium–air cells

    International Nuclear Information System (INIS)

    Highlights: • Mobility and diffusivity can control conductivity even if charge carriers increase. • PVA–LiBOB electrolytes can produce high conductivity in wide temperature range. • Suitable packaging of Li–air cell can reduce moisture interaction with Li metal. -- Abstract: Poly(vinyl alcohol) or PVA complexes with lithium bis(oxalato)borate or LiBOB have been prepared by solution casting. X-ray diffraction reveals that the amorphousness of the polymer increased until 40 wt.% LiBOB salt concentration. The increase in amorphousness is accompanied by a decrease in the glass transition temperature, Tg. The diffusion coefficient of lithium ion obtained from cyclic voltammetry (CV) is 1.98 × 10−8 cm2 s−1. From infrared and impedance spectroscopic studies, it is inferred that conductivity is governed by charge carrier density, ionic mobility and diffusion coefficient for PVA incorporated with 10–40 wt.% LiBOB. However, for the sample with equal weight ratio of PVA and LiBOB, ionic mobility and diffusivity have a greater influence over charge carrier density in determining conductivity at room temperature (RT). PVA can solvate a large amount of salt and for the highest conducting composition of 60 wt.% PVA–40 wt.% LiBOB, conductivity extends from 10−4 S cm−1 at RT (25 °C) to 10−3 S cm−1 at 100 °C. This sample was tested as an electrolyte in a Li–air cell

  4. Jarid2 regulates hematopoietic stem cell function by acting with polycomb repressive complex 2.

    Science.gov (United States)

    Kinkel, Sarah A; Galeev, Roman; Flensburg, Christoffer; Keniry, Andrew; Breslin, Kelsey; Gilan, Omer; Lee, Stanley; Liu, Joy; Chen, Kelan; Gearing, Linden J; Moore, Darcy L; Alexander, Warren S; Dawson, Mark; Majewski, Ian J; Oshlack, Alicia; Larsson, Jonas; Blewitt, Marnie E

    2015-03-19

    Polycomb repressive complex 2 (PRC2) plays a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use short hairpin RNA-mediated knockdown to survey the function of PRC2 accessory factors that were defined in embryonic stem cells (ESCs) by testing the competitive reconstitution capacity of transduced murine HSPCs. We find that, similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult mouse HSPCs. Furthermore, we demonstrate that depletion of JARID2 enhances the in vitro expansion and in vivo reconstitution capacity of human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function. PMID:25645357

  5. Jarid2 regulates hematopoietic stem cell function by acting with polycomb repressive complex 2

    Science.gov (United States)

    Kinkel, Sarah A.; Galeev, Roman; Flensburg, Christoffer; Keniry, Andrew; Breslin, Kelsey; Gilan, Omer; Lee, Stanley; Liu, Joy; Chen, Kelan; Gearing, Linden J.; Moore, Darcy L.; Alexander, Warren S.; Dawson, Mark; Majewski, Ian J.; Oshlack, Alicia; Larsson, Jonas

    2015-01-01

    Polycomb repressive complex 2 (PRC2) plays a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use short hairpin RNA–mediated knockdown to survey the function of PRC2 accessory factors that were defined in embryonic stem cells (ESCs) by testing the competitive reconstitution capacity of transduced murine HSPCs. We find that, similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult mouse HSPCs. Furthermore, we demonstrate that depletion of JARID2 enhances the in vitro expansion and in vivo reconstitution capacity of human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function. PMID:25645357

  6. In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes[OPEN

    Science.gov (United States)

    Husbands, Aman Y.; Aggarwal, Vasudha; Ha, Taekjip; Timmermans, Marja C.P.

    2016-01-01

    Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies. PMID:27385814

  7. Characterization and 454 pyrosequencing of Major Histocompatibility Complex class I genes in the great tit reveal complexity in a passerine system

    Directory of Open Access Journals (Sweden)

    Sepil Irem

    2012-05-01

    Full Text Available Abstract Background The critical role of Major Histocompatibility Complex (Mhc genes in disease resistance and their highly polymorphic nature make them exceptional candidates for studies investigating genetic effects on survival, mate choice and conservation. Species that harbor many Mhc loci and high allelic diversity are particularly intriguing as they are potentially under strong selection and studies of such species provide valuable information as to the mechanisms maintaining Mhc diversity. However comprehensive genotyping of complex multilocus systems has been a major challenge to date with the result that little is known about the consequences of this complexity in terms of fitness effects and disease resistance. Results In this study, we genotyped the Mhc class I exon 3 of the great tit (Parus major from two nest-box breeding populations near Oxford, UK that have been monitored for decades. Characterization of Mhc class I exon 3 was adopted and bidirectional sequencing was carried using the 454 sequencing platform. Full analysis of sequences through a stepwise variant validation procedure allowed reliable typing of more than 800 great tits based on 214,357 reads; from duplicates we estimated the repeatability of typing as 0.94. A total of 862 alleles were detected, and the presence of at least 16 functional loci was shown - the highest number characterized in a wild bird species. Finally, the functional alleles were grouped into 17 supertypes based on their antigen binding affinities. Conclusions We found extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. The presence of many functional loci was shown, together with a pseudogene family and putatively non-functional alleles; there was clear evidence that functional alleles were under strong balancing selection. This study is the first step towards an in-depth analysis of this gene complex in this species, which will help

  8. High-resolution mapping of a fruit firmness-related quantitative trait locus in tomato reveals epistatic interactions associated with a complex combinatorial locus.

    Science.gov (United States)

    Chapman, Natalie H; Bonnet, Julien; Grivet, Laurent; Lynn, James; Graham, Neil; Smith, Rebecca; Sun, Guiping; Walley, Peter G; Poole, Mervin; Causse, Mathilde; King, Graham J; Baxter, Charles; Seymour, Graham B

    2012-08-01

    Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Fir(s.p.)QTL2.1 to Fir(s.p.)QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Fir(s.p.)QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Fir(s.p.)QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus. PMID:22685170

  9. Structure of a helicase–helicase loader complex reveals insights into the mechanism of bacterial primosome assembly

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bin; Eliason, William K.; Steitz, Thomas A.

    2013-09-19

    During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the ~570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that the shift from the ‘open-ring’ to the ‘open-spiral’ and then the ‘closed-spiral’ state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release.

  10. Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity.

    Science.gov (United States)

    Markovski, Monica; Bohrhunter, Jessica L; Lupoli, Tania J; Uehara, Tsuyoshi; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2016-04-26

    To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed. PMID:27071112

  11. Identification of Stem Cells in the Secretory Complex of Salivary Glands.

    Science.gov (United States)

    Kwak, M; Alston, N; Ghazizadeh, S

    2016-07-01

    Salivary glands have an essential secretory function for maintaining oral and overall health. The epithelial compartment of the gland is composed of several highly specialized cell types that cooperate to secrete and deliver saliva to the oral cavity. The mouse submandibular gland has been used as a model for major salivary glands in human. The secretory complex in this model is composed of 2 secretory compartments, including acini and granular ducts connected by intercalated ducts. Contractile myoepithelial cells surround the secretory complex to facilitate salivary flow. Whether differentiated cells in the secretory complex are maintained by self-duplication or contribution from stem cells has remained an open question. Here, in analyzing the expression of basal cytokeratin (K) 14 in the secretory complex, we discovered a subset of K14(+) ductal cells in the intercalated ducts of the adult gland. These cells are distinct from the K14-expressing basal/myoepithelial cells, proliferate at a significantly higher rate than any other epithelial cell type in the gland, and reside in a spatially defined domain within the intercalated duct. Using inducible genetic lineage tracing, we show that K14(+) ductal cells represent a long-lived yet cycling population of stem cells that are established during development and contribute to the formation and maintenance of the granular ducts throughout life. Our data provide direct evidence for the existence of stem cells contributing to homeostasis of salivary glands, as well as new insights into glandular pathobiology. PMID:26936214

  12. The genome of Mycobacterium africanum West African 2 reveals a lineage-specific locus and genome erosion common to the M. tuberculosis complex.

    Directory of Open Access Journals (Sweden)

    Stephen D Bentley

    Full Text Available BACKGROUND: M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the "modern" M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle. CONCLUSIONS/SIGNIFICANCE: The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa.

  13. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells

    Energy Technology Data Exchange (ETDEWEB)

    Abou-El-Ardat, Khalil, E-mail: kabouela@sckcen.be [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Monsieurs, Pieter [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Anastasov, Natasa; Atkinson, Mike [Department of Radiation Sciences, Helmholtz Zentrum Muenchen, Munich (Germany); Derradji, Hanane [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); De Meyer, Tim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Department of Applied Mathematics, Biometrics and Process Control, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Bekaert, Sofie [Clinical Research Center, Faculty for Medicine and Health Sciences, Universiteit Gent, 185 De Pintelaan, 9000 Ghent (Belgium); Van Criekinge, Wim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); and others

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4 Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4 Gy in both RET/PTC-positive systems but no common genes at 62.5 mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent.

  14. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells.

    Science.gov (United States)

    Abou-El-Ardat, Khalil; Monsieurs, Pieter; Anastasov, Nataša; Atkinson, Mike; Derradji, Hanane; De Meyer, Tim; Bekaert, Sofie; Van Criekinge, Wim; Baatout, Sarah

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4Gy in both RET/PTC-positive systems but no common genes at 62.5mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent. PMID:22027090

  15. Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target

    OpenAIRE

    Fedeles, Bogdan I.; Zhu, Angela Y.; Young, Kellie S.; Hillier, Shawn M.; Proffitt, Kyle D.; Essigmann, John M.; Croy, Robert G.

    2011-01-01

    The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action...

  16. Side Population Cells from Human Melanoma Tumors Reveal Diverse Mechanisms for Chemoresistance

    OpenAIRE

    Luo, Yuchun; Ellis, Lixia Z; Dallaglio, Katiuscia; Takeda, Moe; Robinson, William A.; Robinson, Steven; Liu, Weimin; Lewis, Karl D.; McCarter, Martin D; Gonzalez, Rene; David A Norris; Roop, Dennis R.; Spritz, Richard A.; Ahn, Natalie G.; Fujita, Mayumi

    2012-01-01

    Side population (SP) is identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a sub...

  17. Analyzing the complex machinery of cell wall biosynthesis

    OpenAIRE

    Timmers, J.F.P.

    2009-01-01

    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a highly interesting target of scientific research. In this thesis a protein-protein interaction strategy was used to gain insight in the cell wall biosynthesis of Arabidopsis thaliana and to identif...

  18. Dynamical complexity and temporal plasticity in pancreatic -cells

    Indian Academy of Sciences (India)

    Richard Bertram; Arthur Sherman

    2000-06-01

    We discuss some of the biological and mathematical issues involved in understanding and modelling the bursting electrical activity in pancreatic -cells. These issues include single-cell versus islet behaviour, parameter heterogeneity, channel noise, the effects of hormones, neurotransmitters, and ions, and multiple slow biophysical processes. Some of the key experimental and modelling studies are described, and some of the major open questions are discussed.

  19. Enantiopure titanocene complexes - direct evidence for paraptosis in cancer cells.

    Science.gov (United States)

    Cini, Melchior; Williams, Huw; Fay, Mike W; Searle, Mark S; Woodward, Simon; Bradshaw, Tracey D

    2016-03-16

    Tolerated by normal tissues, anti-cancer therapies based on titanium compounds are limited by low efficacy/selectivity and lack of understanding of their mode(s) of action. In vitro antitumour activity and mode of cell death incurred by enantiopure TiCl2{η-C5H4CHEt(2-MeOPh)}2 (abbreviated Cp(R)2TiCl2) has been investigated. The in vitro anti-tumour activity of Cp(R)2TiCl2 is selective for cancer cells; in clonogenic assays, (S,S)-Cp(R)2TiCl2 was twice as effective at inhibiting colony formation than other stereoisomers after 24 h exposure. HPLC, MS and NMR techniques determined hydrolysis of Cp(R)2TiCl2; data strongly correlate with soluble [Cp(R)2Ti(OH)(OH2)](+) being the biological trigger. Treatment of cells with Cp(R)2TiCl2 provoked extensive cytoplasmic vacuolization, endoplasmic reticulum (ER) swelling and activation of MAPKinase signal transduction, consistent with ligand-induced paraptosis, type III cell death, which is morphologically distinct from, and independent of apoptosis. Indeed, distinct from cisplatin, Cp(R)2TiCl2 failed to perturb cell cycle dynamics, induce γH2AX foci or evoke apoptosis in MDA-MB-468 and HCT-116 cells. PMID:26806372

  20. Enhanced stem cell tracking via electrostatically assembled fluorescent SPION-peptide complexes

    International Nuclear Information System (INIS)

    For cellular MRI there is a need to label cells with superparamagnetic iron oxide nanoparticles (SPION) that have multiple imaging moieties that are nontoxic and have increased NMR relaxation properties to improve the detection and tracking of therapeutic cells. Although increases in the relaxation properties of SPION have been accomplished, detection of tagged cells is limited by either poor cell labeling efficiency or low intracellular iron content. A strategy via a complex formation with transfection agents to overcome these obstacles has been reported. In this paper, we report a complex formation between negatively charged fluorescent monodisperse SPION and positively charged peptides and use the complex formation to improve the MR properties of labeled stem cells. As a result, labeled stem cells exhibited a strong fluorescent signal and enhanced T 2*-weighted MR imaging in vitro and in vivo in a flank tumor model.

  1. Enhanced stem cell tracking via electrostatically assembled fluorescent SPION-peptide complexes

    Science.gov (United States)

    Lee, Jae-Ho; Smith, Melissa A.; Liu, Wei; Gold, Eric M.; Lewis, Bobbi; Song, Ho-Taek; Frank, Joseph A.

    2009-09-01

    For cellular MRI there is a need to label cells with superparamagnetic iron oxide nanoparticles (SPION) that have multiple imaging moieties that are nontoxic and have increased NMR relaxation properties to improve the detection and tracking of therapeutic cells. Although increases in the relaxation properties of SPION have been accomplished, detection of tagged cells is limited by either poor cell labeling efficiency or low intracellular iron content. A strategy via a complex formation with transfection agents to overcome these obstacles has been reported. In this paper, we report a complex formation between negatively charged fluorescent monodisperse SPION and positively charged peptides and use the complex formation to improve the MR properties of labeled stem cells. As a result, labeled stem cells exhibited a strong fluorescent signal and enhanced T 2*-weighted MR imaging in vitro and in vivo in a flank tumor model.

  2. Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells.

    Directory of Open Access Journals (Sweden)

    Britta Muster

    Full Text Available BACKGROUND: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2-3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10-24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. CONCLUSION/SIGNIFICANCE: Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.

  3. Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

    Directory of Open Access Journals (Sweden)

    Kendall K

    2013-05-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

  4. Nanoscaled carborane ruthenium(II-arene complex inducing lung cancer cells apoptosis

    Directory of Open Access Journals (Sweden)

    Yan Hong

    2011-02-01

    Full Text Available Abstract Background The new ruthenium(II-arene complex, which bearing a carborane unit, ruthenium and ferrocenyl functional groups, has a novel versatile synthetic chemistry and unique properties of the respective material at the nanoscale level. The ruthenium(II-arene complex shows significant cytotoxicity to cancer cells and tumor-inhibiting properties. However, ruthenium(II-arene complex of mechanism of anticancer activity are scarcely explored. Therefore, it is necessary to explore ruthenium(II-arene complex mechanism of anticancer activity for application in this area. Results In this study, the ruthenium(II-arene complex could significantly induce apoptosis in human lung cancer HCC827 cell line. At the concentration range of 5 μM-100 μM, ruthenium(II-arene complex had obvious cell cytotoxicity effect on HCC827 cells with IC50 values ranging 19.6 ± 5.3 μM. Additionally, our observations demonstrate that the ruthenium(II-arene complex can readily induce apoptosis in HCC827 cells, as evidenced by Annexin-V-FITC, nuclear fragmentation as well as DNA fragmentation. Treatment of HCC827 cells with the ruthenium(II-arene complex resulted in dose-dependent cell apoptosis as indicated by high cleaved Caspase-8,9 ratio. Besides ruthenium(II-arene complex caused a rapid induction of cleaved Caspase-3 activity and stimulated proteolytic cleavage of poly-(ADP-ribose polymerase (PARP in vitro and in vivo. Conclusion In this study, the ruthenium(II-arene complex could significantly induce apoptosis in human lung cancer HCC827 cell line. Treatment of HCC827 cells with the ruthenium(II-arene complex resulted in dose-dependent cell apoptosis as indicated by high cleaved Caspase-8,9 ratio. Besides ruthenium(II-arene complex caused a rapid induction of cleaved Caspase-3 activity and stimulated proteolytic cleavage of poly-(ADP-ribose polymerase (PARP in vitro and in vivo. Our results suggest that ruthenium(II-arene complex could be a candidate for further

  5. Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

    Science.gov (United States)

    Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

    2015-10-01

    The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. PMID:26049080

  6. Untangling a species complex of arid zone grasses (Triodia) reveals patterns congruent with co-occurring animals.

    Science.gov (United States)

    Anderson, Benjamin M; Barrett, Matthew D; Krauss, Siegfried L; Thiele, Kevin

    2016-08-01

    The vast Australian arid zone formed over the last 15million years, and gradual aridification as well as more extreme Pliocene and Pleistocene climate shifts have impacted the evolution of its biota. Understanding the evolutionary history of groups of organisms or regional biotas such as the Australian arid biota requires clear delimitation of the units of biodiversity (taxa). Here we integrate evidence from nuclear (ETS and ITS) and chloroplast (rps16-trnK spacer) regions and morphology to clarify taxonomic boundaries in a species complex of Australian hummock grasses (Triodia) to better understand the evolution of Australian arid zone plants and to evaluate congruence in distribution patterns with co-occurring organisms. We find evidence for multiple new taxa in the T. basedowii species complex, but also incongruence between data sets and indications of hybridization that complicate delimitation. We find that the T. basedowii complex has high lineage diversity and endemism in the biologically important Pilbara region of Western Australia, consistent with the region acting as a refugium. Taxa show strong geographic structure in the Pilbara, congruent with recent work on co-occurring animals and suggesting common evolutionary drivers across the biota. Our findings confirm recognition of the Pilbara as an important centre of biodiversity in the Australian arid zone, and provide a basis for future taxonomic revision of the T. basedowii complex and more detailed study of its evolutionary history and that of arid Australia. PMID:27179699

  7. Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

    NARCIS (Netherlands)

    Janssen, B.J.C.; Halff, E.F.; Lambris, J.D.; Gros, P.

    2007-01-01

    Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step

  8. Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

    Science.gov (United States)

    Rothwell, P. J.; Berger, S.; Kensch, O.; Felekyan, S.; Antonik, M.; Wöhrl, B. M.; Restle, T.; Goody, R. S.; Seidel, C. A. M.

    2003-01-01

    By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide. PMID:12578980

  9. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry

    DEFF Research Database (Denmark)

    Chen, Zhuo Angel; Jawhari, Anass; Fischer, Lutz;

    2010-01-01

    Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to...... extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The...... C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and...

  10. A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production

    Directory of Open Access Journals (Sweden)

    Emanuel Joseph Paul Nazareth

    2016-05-01

    Full Text Available Human pluripotent stem cells (hPSCs exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4 and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.

  11. A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production.

    Science.gov (United States)

    Nazareth, Emanuel Joseph Paul; Rahman, Nafees; Yin, Ting; Zandstra, Peter William

    2016-05-10

    Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives. PMID:27132889

  12. Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Zou, Ying; Beausejour, Christian; Kaminker, Patrick; Yannone, Steven M.; Campisi, Judith

    2007-10-02

    Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

  13. A Nanoprinted Model of Interstitial Cancer Migration Reveals a Link between Cell Deformability and Proliferation.

    Science.gov (United States)

    Panagiotakopoulou, Magdalini; Bergert, Martin; Taubenberger, Anna; Guck, Jochen; Poulikakos, Dimos; Ferrari, Aldo

    2016-07-26

    Metastatic progression of tumors requires the coordinated dissemination of cancerous cells through interstitial tissues and their replication in distant body locations. Despite their importance in cancer treatment decisions, key factors, such as cell shape adaptation and the role it plays in dense tissue invasion by cancerous cells, are not well understood. Here, we employ a 3D electrohydrodynamic nanoprinting technology to generate vertical arrays of topographical pores that mimic interstitial tissue resistance to the mesenchymal migration of cancerous cells, in order to determine the effect of nuclear size, cell deformability, and cell-to-substrate adhesion on tissue invasion efficiency. The high spatial and temporal resolution of our analysis demonstrates that the ability of cells to deform depends on the cell cycle phase, peaks immediately after mitosis, and is key to the invasion process. Increased pore penetration efficiency by cells in early G1 phase also coincided with their lower nuclear volume and higher cell deformability, compared with the later cell cycle stages. Furthermore, artificial decondensation of chromatin induced an increase in cell and nuclear deformability and improved pore penetration efficiency of cells in G1. Together, these results underline that along the cell cycle cells have different abilities to dynamically remodel their actin cytoskeleton and induce nuclear shape changes, which determines their pore penetration efficiency. Thus, our results support a mechanism in which cell proliferation and pore penetration are functionally linked to favor the interstitial dissemination of metastatic cells. PMID:27268411

  14. A cell complex structure for the space of heteroclines for a semilinear parabolic equation

    Directory of Open Access Journals (Sweden)

    Michael Robinson

    2009-01-01

    Full Text Available It is well known that for many semilinear parabolic equations there is a global attractor which has a cell complex structure with finite dimensional cells. Additionally, many semilinear parabolic equations have equilibria with finite dimensional unstable manifolds. In this article, these results are unified to show that for a specific parabolic equation on an unbounded domain, the space of heteroclinic orbits has a cell complex structure with finite dimensional cells. The result depends crucially on the choice of spatial dimension and the degree of the nonlinearity in the parabolic equation, and thereby requires some delicate treatment.

  15. All-Atom Molecular Dynamics Simulations Reveal Significant Differences in Interaction between Antimycin and Conserved Amino Acid Residues in Bovine and Bacterial bc1 Complexes

    OpenAIRE

    Kokhan, Oleksandr; Shinkarev, Vladimir P.

    2011-01-01

    Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Qi site of the bacterial and bovine bc1 complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Qi pocket. We conclude that many of the dif...

  16. Cell cycle-related transformation of the E2F4-p130 repressor complex

    International Nuclear Information System (INIS)

    During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions

  17. BCG Vaccination Induces Robust CD4+ T Cell Responses to Mycobacterium tuberculosis Complex-Specific Lipopeptides in Guinea Pigs.

    Science.gov (United States)

    Kaufmann, Eva; Spohr, Christina; Battenfeld, Sibylle; De Paepe, Diane; Holzhauser, Thomas; Balks, Elisabeth; Homolka, Susanne; Reiling, Norbert; Gilleron, Martine; Bastian, Max

    2016-03-15

    A new class of highly antigenic, MHC-II-restricted mycobacterial lipopeptides that are recognized by CD4-positive T lymphocytes of Mycobacterium tuberculosis-infected humans has recently been described. To investigate the relevance of this novel class of mycobacterial Ags in the context of experimental bacille Calmette-Guérin (BCG) vaccination, Ag-specific T cell responses to mycobacterial lipid and lipopeptide-enriched Ag preparations were analyzed in immunized guinea pigs. Lipid and lipopeptide preparations as well as complex Ag mixtures, such as tuberculin, mycobacterial lysates, and culture supernatants, all induced a similar level of T cell proliferation. The hypothesis that lipopeptide-specific T cells dominate the early BCG-induced T cell response was corroborated in restimulation assays by the observation that Ag-expanded T cells specifically responded to the lipopeptide preparation. A comparative analysis of the responses to Ag preparations from different mycobacterial species revealed that the antigenic lipopeptides are specific for strains of the M. tuberculosis complex. Their intriguing conservation in pathogenic tuberculous bacteria and the fact that these highly immunogenic Ags seem to be actively released during in vitro culture and intracellular infection prompt the urgent question about their role in the fine-tuned interplay between the pathogen and its mammalian host, in particular with regard to BCG vaccination strategies. PMID:26889044

  18. A Systematic Analysis of Cell Cycle Regulators in Yeast Reveals That Most Factors Act Independently of Cell Size to Control Initiation of Division

    OpenAIRE

    Scott A Hoose; Jeremy A Rawlings; Kelly, Michelle M.; M Camille Leitch; Ababneh, Qotaiba O; Robles, Juan P.; David Taylor; Hoover, Evelyn M.; Bethel Hailu; McEnery, Kayla A.; S Sabina Downing; Deepika Kaushal; Yi Chen; Alex Rife; Kirtan A Brahmbhatt

    2012-01-01

    Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene dele...

  19. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin

    OpenAIRE

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G.

    2008-01-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an inc...

  20. Antheraea pernyi Silk Fibroin-Coated PEI/DNA Complexes for Targeted Gene Delivery in HEK 293 and HCT 116 Cells

    Directory of Open Access Journals (Sweden)

    Yu Liu

    2014-04-01

    Full Text Available Polyethylenimine (PEI has attracted much attention as a DNA condenser, but its toxicity and non-specific targeting limit its potential. To overcome these limitations, Antheraea pernyi silk fibroin (ASF, a natural protein rich in arginyl-glycyl-aspartic acid (RGD peptides that contains negative surface charges in a neutral aqueous solution, was used to coat PEI/DNA complexes to form ASF/PEI/DNA ternary complexes. Coating these complexes with ASF caused fewer surface charges and greater size compared with the PEI/DNA complexes alone. In vitro transfection studies revealed that incorporation of ASF led to greater transfection efficiencies in both HEK (human embryonic kidney 293 and HCT (human colorectal carcinoma 116 cells, albeit with less electrostatic binding affinity for the cells. Moreover, the transfection efficiency in the HCT 116 cells was higher than that in the HEK 293 cells under the same conditions, which may be due to the target bonding affinity of the RGD peptides in ASF for integrins on the HCT 116 cell surface. This result indicated that the RGD binding affinity in ASF for integrins can enhance the specific targeting affinity to compensate for the reduction in electrostatic binding between ASF-coated PEI carriers and cells. Cell viability measurements showed higher cell viability after transfection of ASF/PEI/DNA ternary complexes than after transfection of PEI/DNA binary complexes alone. Lactate dehydrogenase (LDH release studies further confirmed the improvement in the targeting effect of ASF/PEI/DNA ternary complexes to cells. These results suggest that ASF-coated PEI is a preferred transfection reagent and useful for improving both the transfection efficiency and cell viability of PEI-based nonviral vectors.

  1. Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells

    OpenAIRE

    Han, Lina; Qiu, Peng; Zeng, Zhihong; Jorgensen, Jeffrey L.; Mak, Duncan H; Burks, Jared K.; Schober, Wendy; McQueen, Teresa J; Cortes, Jorge; Tanner, Scott D; Roboz, Gail J; Kantarjian, Hagop M; Kornblau, Steven M.; Guzman, Monica L.; Andreeff, Michael

    2015-01-01

    Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which...

  2. Subtle spectral effects accompanying the assembly of bacteriochlorophylls into cyclic light harvesting complexes revealed by high-resolution fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Rätsep, Margus, E-mail: margus.ratsep@ut.ee; Pajusalu, Mihkel, E-mail: mihkel.pajusalu@ut.ee; Linnanto, Juha Matti, E-mail: juha.matti.linnanto@ut.ee [Institute of Physics, University of Tartu, Riia 142, 51014 Tartu (Estonia); Freiberg, Arvi, E-mail: arvi.freiberg@ut.ee [Institute of Physics, University of Tartu, Riia 142, 51014 Tartu, Estonia and Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu (Estonia)

    2014-10-21

    We have observed that an assembly of the bacteriochloropyll a molecules into B850 and B875 groups of cyclic bacterial light-harvesting complexes LH2 and LH1, respectively, results an almost total loss of the intra-molecular vibronic structure in the fluorescence spectrum, and simultaneously, an essential enhancement of its phonon sideband due to electron-phonon coupling. While the suppression of the vibronic coupling in delocalized (excitonic) molecular systems is predictable, as also confirmed by our model calculations, a boost of the electron-phonon coupling is rather unexpected. The latter phenomenon is explained by exciton self-trapping, promoted by mixing the molecular exciton states with charge transfer states between the adjacent chromophores in the tightly packed B850 and B875 arrangements. Similar, although less dramatic trends were noted for the light-harvesting complexes containing chlorophyll pigments.

  3. Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity.

    Science.gov (United States)

    Schmidt, Bryan H; Osheroff, Neil; Berger, James M

    2012-11-01

    Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset. PMID:23022727

  4. Subtle spectral effects accompanying the assembly of bacteriochlorophylls into cyclic light harvesting complexes revealed by high-resolution fluorescence spectroscopy

    International Nuclear Information System (INIS)

    We have observed that an assembly of the bacteriochloropyll a molecules into B850 and B875 groups of cyclic bacterial light-harvesting complexes LH2 and LH1, respectively, results an almost total loss of the intra-molecular vibronic structure in the fluorescence spectrum, and simultaneously, an essential enhancement of its phonon sideband due to electron-phonon coupling. While the suppression of the vibronic coupling in delocalized (excitonic) molecular systems is predictable, as also confirmed by our model calculations, a boost of the electron-phonon coupling is rather unexpected. The latter phenomenon is explained by exciton self-trapping, promoted by mixing the molecular exciton states with charge transfer states between the adjacent chromophores in the tightly packed B850 and B875 arrangements. Similar, although less dramatic trends were noted for the light-harvesting complexes containing chlorophyll pigments

  5. Intestinal crypt homeostasis revealed at single stem cell level by in vivo live-imaging

    Science.gov (United States)

    Zomer, Anoek; Snippert, Hugo J.; de Sauvage, Frederic J.; Simons, Benjamin D.; Clevers, Hans; van Rheenen, Jacco

    2014-01-01

    Summary The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt1. Alongside Lgr5, intestinal stem cells have been associated with various markers, which are expressed heterogeneously within the crypt base region1-6. Previous quantitative clonal fate analyses have proposed that homeostasis occurs as the consequence of neutral competition between dividing stem cells7-9. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here, we established the short-term dynamics of intestinal stem cells using a novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed ‘border cells’) can be passively displaced into the transit-amplifying (TA) domain, following division of proximate cells, implying that determination of stem cell fate can be uncoupled from division. Through the quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem cell maintenance in which a dynamically heterogeneous cell population is able to function long-term as a single stem cell pool. PMID:24531760

  6. Evolutionary history of black grouse major histocompatibility complex class IIB genes revealed through single locus sequence-based genotyping

    OpenAIRE

    Strand, Tanja; Wang, Biao; Meyer-Lucht, Yvonne; Höglund, Jacob

    2013-01-01

    Background: Gene duplications are frequently observed in the Major Histocompatibility Complex (MHC) of many species, and as a consequence loci belonging to the same MHC class are often too similar to tell apart. In birds, single locus genotyping of MHC genes has proven difficult due to concerted evolution homogenizing sequences at different loci. But studies on evolutionary history, mode of selection and heterozygosity correlations on the MHC cannot be performed before it is possible to analy...

  7. Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

    OpenAIRE

    Janssen, B.J.C.; Halff, E.F.; LAMBRIS, J. D.; Gros, P

    2007-01-01

    Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compsta...

  8. Muscular anatomy of an entoproct creeping-type larva reveals extraordinary high complexity and potential shared characters with mollusks

    OpenAIRE

    Merkel, Julia; Lieb, Bernhard; Wanninger, Andreas

    2015-01-01

    Background Entoprocta (Kamptozoa) is an enigmatic, acoelomate, tentacle-bearing phylum with indirect development, either via a swimming- or a creeping-type larva and still debated phylogenetic position within Lophotrochozoa. Recent morphological and neuro-anatomical studies on the creeping-type larva support a close relationship of Entoprocta and Mollusca, with a number of shared apomorphies including a tetraneurous nervous system and a complex serotonin-expressing apical organ. However, many...

  9. Uniaxial magnetic anisotropy of square-planar chromium(II) complexes revealed by magnetic and HF-EPR studies.

    Science.gov (United States)

    Deng, Yi-Fei; Han, Tian; Wang, Zhenxing; Ouyang, Zhongwen; Yin, Bing; Zheng, Zhiping; Krzystek, J; Zheng, Yan-Zhen

    2015-12-28

    Two mononuclear square-planar Cr(II) complexes are reported, exhibiting field-induced slow magnetic relaxation. The axial zero-field splitting parameter was unambiguously determined by both a high-frequency/field electron paramagnetic resonance (HF-EPR) technique and magnetic measurements. This result represents the first observed single-molecule-magnet behavior in the square planar coordination geometry of any metal ions. PMID:26587566

  10. Multiple Changes of Gene Expression and Function Reveal Genomic and Phenotypic Complexity in SLE-like Disease.

    OpenAIRE

    Maria Wilbe; Kozyrev, Sergey V.; Fabiana H. G. Farias; Bremer, Hanna D.; Anna Hedlund; Pielberg, Gerli R.; Seppälä, Eija H; Ulla Gustafson; Hannes Lohi; Örjan Carlborg; Göran Andersson; Helene Hansson-Hamlin; Kerstin Lindblad-Toh

    2015-01-01

    The complexity of clinical manifestations commonly observed in autoimmune disorders poses a major challenge to genetic studies of such diseases. Systemic lupus erythematosus (SLE) affects humans as well as other mammals, and is characterized by the presence of antinuclear antibodies (ANA) in patients' sera and multiple disparate clinical features. Here we present evidence that particular sub-phenotypes of canine SLE-related disease, based on homogenous (ANA(H)) and speckled ANA (ANA(S)) stain...

  11. Flux-based classification of reactions reveals a functional bow-tie organization of complex metabolic networks

    OpenAIRE

    Singh, Shalini; Samal, Areejit; Giri, Varun; Krishna, Sandeep; Raghuram, Nandula; Jain, Sanjay

    2012-01-01

    Unraveling the structure of complex biological networks and relating it to their functional role is an important task in systems biology. Here we attempt to characterize the functional organization of the large-scale metabolic networks of three microorganisms. We apply flux balance analysis to study the optimal growth states of these organisms in different environments. By investigating the differential usage of reactions across flux patterns for different environments, we observe a striking ...

  12. CGCI Investigators Reveal Comprehensive Landscape of Diffuse Large B-Cell Lymphoma (DLBCL) Genomes | Office of Cancer Genomics

    Science.gov (United States)

    Researchers from British Columbia Cancer Agency used whole genome sequencing to analyze 40 DLBCL cases and 13 cell lines in order to fill in the gaps of the complex landscape of DLBCL genomes. Their analysis, “Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing,” was published online in Blood on May 22. The authors are Ryan Morin, Marco Marra, and colleagues.  

  13. Ultrastructural characteristics of nurse cell-larva complex of four species of Trichinella in several hosts

    OpenAIRE

    Sacchi L.; Corona S.; Gajadhar A.A.; Pozio E.

    2001-01-01

    The nurse cell-larva complex of nematodes of the genus Trichinella plays an Important role in the survival of the larva in decaying muscles, frequently favouring the transmission of the parasite in extreme environmental conditions. The ultrastructure of the nurse cell-larva complex in muscles from different hosts infected with T. nativa (a walrus and a polar bear), T. spiralis (horses and humans), T. pseudospiralis (a laboratory mouse) and T. papuae (a laboratory mouse) were examined. Analysi...

  14. Dynamics of pre-replication complex proteins during the cell division cycle.

    OpenAIRE

    Prasanth, Supriya G.; Méndez, Juan; Prasanth, Kannanganattu V.; Stillman, Bruce

    2004-01-01

    Replication of the human genome every time a cell divides is a highly coordinated process that ensures accurate and efficient inheritance of the genetic information. The molecular mechanism that guarantees that many origins of replication fire only once per cell-cycle has been the area of intense research. The origin recognition complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre-replicative complex (pre-R...

  15. Two mechanisms that account for major histocompatibility complex restriction of T cells

    OpenAIRE

    Kranz, David M.

    2009-01-01

    In recent studies, two distinct mechanisms have been proposed to account for major histocompatibility complex (MHC) restriction of T-cell activity: (a) evolution-driven interactions between T-cell receptor (TCR) variable regions and MHC, and (b) a requirement for CD4 or CD8 binding to MHC to initiate signalling through the TCR complex. Both mechanisms are likely to be essential, but for different reasons.

  16. Characterization of Influenza Vaccine Hemagglutinin Complexes by Cryo-Electron Microscopy and Image Analyses Reveals Structural Polymorphisms

    Science.gov (United States)

    McCraw, Dustin M.; Gallagher, John R.

    2016-01-01

    Influenza virus afflicts millions of people worldwide on an annual basis. There is an ever-present risk that animal viruses will cross the species barrier to cause epidemics and pandemics resulting in great morbidity and mortality. Zoonosis outbreaks, such as the H7N9 outbreak, underscore the need to better understand the molecular organization of viral immunogens, such as recombinant influenza virus hemagglutinin (HA) proteins, used in influenza virus subunit vaccines in order to optimize vaccine efficacy. Here, using cryo-electron microscopy and image analysis, we show that recombinant H7 HA in vaccines formed macromolecular complexes consisting of variable numbers of HA subunits (range, 6 to 8). In addition, HA complexes were distributed across at least four distinct structural classes (polymorphisms). Three-dimensional (3D) reconstruction and molecular modeling indicated that HA was in the prefusion state and suggested that the oligomerization and the structural polymorphisms observed were due to hydrophobic interactions involving the transmembrane regions. These experiments suggest that characterization of the molecular structures of influenza virus HA complexes used in subunit vaccines will lead to better understanding of the differences in vaccine efficacy and to the optimization of subunit vaccines to prevent influenza virus infection. PMID:27074939

  17. Quaternary structure of the specific p53–DNA complex reveals the mechanism of p53 mutant dominance

    OpenAIRE

    Aramayo, Ricardo; Sherman, Michael B.; Brownless, Kathryne; Lurz, Rudi; Okorokov, Andrei L.; Orlova, Elena V

    2011-01-01

    The p53 tumour suppressor is a transcriptional activator that controls cell fate in response to various stresses. p53 can initiate cell cycle arrest, senescence and/or apoptosis via transactivation of p53 target genes, thus preventing cancer onset. Mutations that impair p53 usually occur in the core domain and negate the p53 sequence-specific DNA binding. Moreover, these mutations exhibit a dominant negative effect on the remaining wild-type p53. Here, we report the cryo electron microscopy s...

  18. Analyzing the complex machinery of cell wall biosynthesis

    NARCIS (Netherlands)

    Timmers, J.F.P.

    2009-01-01

    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a

  19. Arteriolar vascular smooth muscle cells: mechanotransducers in a complex environment.

    Science.gov (United States)

    Hill, Michael A; Meininger, Gerald A

    2012-09-01

    Contraction of small artery (diameters typically less than 250 μm) vascular smooth muscle cells (VSMCs) plays a critical role in local control of blood flow and arterial pressure through its affect on vascular caliber. Specifically, contraction of small arteries in response to increased intraluminal pressure is referred to as the myogenic response and represents an important role for mechanotransduction. Critical questions remain as to how changes in pressure are sensed by VSMCs and transduced across the cell membrane to tune the contractile state of the cell. Recent studies suggest a pivotal role for interactions between VSMCs and extracellular matrix (ECM) proteins. Thus, pressure-induced deformation of ECM proteins and their cell surface receptors (for example, integrins) may initiate contraction and cytoskeletal remodeling through modulation of ion channels, membrane depolarization, increased intracellular Ca(2+) and actomyosin crossbridge cycling. Importantly, it is argued that the contractile properties of small artery VSMCs reflect an intimate and integrated interaction with their extracellular environment and the three-dimensional structure of the vessel wall. PMID:22677491

  20. Modeling the Effect of APC Truncation on Destruction Complex Function in Colorectal Cancer Cells

    OpenAIRE

    Barua, Dipak; Hlavacek, William S

    2013-01-01

    In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β—catenin , which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β—catenin , serine/threonine kinases, and other proteins. The kinases and , which are recruited by Axin, mediate phosphorylation of β—catenin , which initiates its ubiquitination and proteosomal degradatio...

  1. Nanoscaled carborane ruthenium(II)-arene complex inducing lung cancer cells apoptosis

    OpenAIRE

    Yan Hong; Ye Hongde; Wu Chunhui; Zhang Gen; Wang Xuemei

    2011-01-01

    Abstract Background The new ruthenium(II)-arene complex, which bearing a carborane unit, ruthenium and ferrocenyl functional groups, has a novel versatile synthetic chemistry and unique properties of the respective material at the nanoscale level. The ruthenium(II)-arene complex shows significant cytotoxicity to cancer cells and tumor-inhibiting properties. However, ruthenium(II)-arene complex of mechanism of anticancer activity are scarcely explored. Therefore, it is necessary to explore rut...

  2. Fast Vesicle Fusion in Living Cells Requires at Least Three SNARE Complexes

    DEFF Research Database (Denmark)

    Mohrmann, Ralf; de Wit, Heidi; Verhage, Matthijs;

    2010-01-01

    relationship for fast (synchronous) fusion and a near-linear relationship for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, while slower release might occur with fewer. Heterogeneity in SNARE-complex number may...

  3. High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

    Directory of Open Access Journals (Sweden)

    Yo Saito

    Full Text Available Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S; therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

  4. A Self-Adaptive Steered Molecular Dynamics Method Based on Minimization of Stretching Force Reveals the Binding Affinity of Protein–Ligand Complexes

    Directory of Open Access Journals (Sweden)

    Junfeng Gu

    2015-10-01

    Full Text Available Binding affinity prediction of protein–ligand complexes has attracted widespread interest. In this study, a self-adaptive steered molecular dynamics (SMD method is proposed to reveal the binding affinity of protein–ligand complexes. The SMD method is executed through adjusting pulling direction to find an optimum trajectory of ligand dissociation, which is realized by minimizing the stretching force automatically. The SMD method is then used to simulate the dissociations of 19 common protein–ligand complexes which are derived from two homology families, and the binding free energy values are gained through experimental techniques. Results show that the proposed SMD method follows a different dissociation pathway with lower a rupture force and energy barrier when compared with the conventional SMD method, and further analysis indicates the rupture forces of the complexes in the same protein family correlate well with their binding free energy, which reveals the possibility of using the proposed SMD method to identify the active ligand.

  5. A new approach for determining phase response curves reveals that Purkinje cells can act as perfect integrators.

    Directory of Open Access Journals (Sweden)

    Elena Phoka

    2010-04-01

    Full Text Available Cerebellar Purkinje cells display complex intrinsic dynamics. They fire spontaneously, exhibit bistability, and via mutual network interactions are involved in the generation of high frequency oscillations and travelling waves of activity. To probe the dynamical properties of Purkinje cells we measured their phase response curves (PRCs. PRCs quantify the change in spike phase caused by a stimulus as a function of its temporal position within the interspike interval, and are widely used to predict neuronal responses to more complex stimulus patterns. Significant variability in the interspike interval during spontaneous firing can lead to PRCs with a low signal-to-noise ratio, requiring averaging over thousands of trials. We show using electrophysiological experiments and simulations that the PRC calculated in the traditional way by sampling the interspike interval with brief current pulses is biased. We introduce a corrected approach for calculating PRCs which eliminates this bias. Using our new approach, we show that Purkinje cell PRCs change qualitatively depending on the firing frequency of the cell. At high firing rates, Purkinje cells exhibit single-peaked, or monophasic PRCs. Surprisingly, at low firing rates, Purkinje cell PRCs are largely independent of phase, resembling PRCs of ideal non-leaky integrate-and-fire neurons. These results indicate that Purkinje cells can act as perfect integrators at low firing rates, and that the integration mode of Purkinje cells depends on their firing rate.

  6. Protein-binding, cytotoxicity in vitro and cell cycle arrest of ruthenium(II) polypyridyl complexes

    Science.gov (United States)

    Liu, Si-Hong; Zhu, Jian-Wei; Xu, Hui-Hua; Wang, Yan; Liu, Ya-Min; Liang, Jun-Bo; Zhang, Gui-Qiang; Cao, Di-Hua; Lin, Yang-Yang; Wu, Yong; Guo, Qi-Feng

    2016-05-01

    The cytotoxic activity of two Ru(II) complexes against A549, BEL-7402, HeLa, PC-12, SGC-7901 and SiHa cell lines was investigated by MTT method. Complexes 1 and 2 show moderate cytotoxicity toward BEL-7402 cells with an IC50 value of 53.9 ± 3.4 and 39.3 ± 2.1 μM. The effects of the complexes inducing apoptosis, cellular uptake, reactive oxygen species and mitochondrial membrane potential in BEL-7402 cells have been studied by fluorescence microscopy. The percentages of apoptotic and necrotic cells and cell cycle arrest were studied by flow cytometry. The BSA-binding behaviors were investigated by UV/visible and fluorescent spectra.

  7. Interactions of opsonized immune complexes with whole blood cells: binding to erythrocytes restricts complex uptake by leucocyte populations

    DEFF Research Database (Denmark)

    Nielsen, C H; Svehag, S E; Marquart, H V; Leslie, R G

    1994-01-01

    respect to IC binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d,g-covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC......The binding of opsonized, fluorescein-labelled bovine serum albumin (BSA)/rabbit anti-BSA complexes (IC) to washed human whole blood cells and isolated leucocytes in the presence of autologous serum was investigated by flow cytometry. In the presence of erythrocytes (E), the IC-binding to...... granulocytes (PMN), monocytes and lymphocytes was inhibited by up to 46%, 61% and 48%, respectively, depending on the incubation time and the IC-concentration tested. The E-mediated inhibition of the binding to PMN was found to correlate with the average numbers of CR1 per E during the initial 15 min of...

  8. Interactions of opsonized immune complexes with whole blood cells: binding to erythrocytes restricts complex uptake by leucocyte populations

    DEFF Research Database (Denmark)

    Nielsen, C H; Svehag, S E; Marquart, H V;

    1994-01-01

    The binding of opsonized, fluorescein-labelled bovine serum albumin (BSA)/rabbit anti-BSA complexes (IC) to washed human whole blood cells and isolated leucocytes in the presence of autologous serum was investigated by flow cytometry. In the presence of erythrocytes (E), the IC-binding to...... granulocytes (PMN), monocytes and lymphocytes was inhibited by up to 46%, 61% and 48%, respectively, depending on the incubation time and the IC-concentration tested. The E-mediated inhibition of the binding to PMN was found to correlate with the average numbers of CR1 per E during the initial 15 min of...... respect to IC binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d,g-covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC...

  9. Permeation of vanadium(III, IV, V)-dipicolinate complexes across MDCK cell monolayer and comparison with Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yue; YANG Xiaoda; WANG Kui

    2005-01-01

    The permeation and cytotoxicity of three insulin-mimetic vanadium(III, IV, V)-dipicolinate complexes were studied using the MDCK cell monolayer in comparison with the Caco-2 cells. On MDCK cell monolayer, the apparent permeation coefficients (Papp) were estimated to be (7.5 ± 1.0)×10-6, (1.0 ± 0.2)×10-6, (1.7±0.4)×10-6 cm/s for V(V), V(IV), and V(III)-dipic complexes, respectively. The permeability of V(V)-dipic complexes is much better than the others, which is in agreement with its better hypoglycemic effect in animal tests. On Caco-2 cell monolayer, Papp were found to be in the range of 1-3×10-6 cm/s and not to be affected by excessive amounts of dipicolinate ligand. By contrast, the permeability in the AP→BL direction across the MDCK monolayer increased greatly in the presence of free ligands, suggesting existence of active transport mechanism of vanadium complex anions on the MDCK cells. The cytotoxicity of the three complexes was found similar and the IC50 were measured in the range of 0.6―0.9 mmol/L for MDCK cells and 1.6―2 mmol/L for Caco-2 cells. The cytotoxicity of three vanadium complexes was conceivably in consistence with their permeability, suggesting that the toxicity, permeation and cellular metabolism of vanadium complexes are closely related.

  10. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Benjamin A Neely

    Full Text Available Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC. This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened

  11. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma

    Science.gov (United States)

    Wilkins, Christopher E.; Marlow, Laura A.; Malyarenko, Dariya; Kim, Yunee; Ignatchenko, Alexandr; Sasinowska, Heather; Sasinowski, Maciek; Nyalwidhe, Julius O.; Kislinger, Thomas; Copland, John A.; Drake, Richard R.

    2016-01-01

    Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from

  12. A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis.

    Science.gov (United States)

    Song, Jiazhe; Xue, Kai; She, Ji; Ding, Fangrong; Li, Song; Shangguan, Rulan; Dai, Yunping; Du, Liying; Li, Ning

    2014-09-10

    The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin(-)CD24(med)CD29(+) cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin(-)CD24(+)CD29(hi)), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin(-)CD24(med)CD29(+) cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin(-)CD24(med)CD29(+) cell population possessed low proportion of stem cells. In summary, heterogeneous Lin(-)CD24(med)CD29(+) cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment. PMID:24954407

  13. Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation.

    Science.gov (United States)

    Sato, Wataru; Hitaoka, Seiji; Inoue, Kaoru; Imai, Mizue; Saio, Tomohide; Uchida, Takeshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Yoshizawa, Kazunari; Ishimori, Koichiro

    2016-07-15

    Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO. PMID:27226541

  14. SSU rRNA reveals a sequential increase in shell complexity among the euglyphid testate amoebae (Rhizaria: Euglyphida)

    DEFF Research Database (Denmark)

    Lara, Enrique; Heger, Thierry J; Mitchell, Edward A D; Meisterfeld, Ralf; Ekelund, Flemming

    2007-01-01

    The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study...... aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows...

  15. The Cell Probe Complexity of Dynamic Range Counting

    DEFF Research Database (Denmark)

    Larsen, Kasper Green

    is specified by a point q = (x, y), and the goal is to report the sum of the weights assigned to the points dominated by q, where a point (x0, y0) is dominated by q if x0 x and y0 y. In addition to being the highest cell probe lower bound to date, our lower bound is also tight for data struc- tures...

  16. The Probabilistic Niche Model Reveals the Niche Structure and Role of Body Size in a Complex Food Web

    OpenAIRE

    Richard J Williams; Ananthi Anandanadesan; Drew Purves

    2010-01-01

    The niche model has been widely used to model the structure of complex food webs, and yet the ecological meaning of the single niche dimension has not been explored. In the niche model, each species has three traits, niche position, diet position and feeding range. Here, a new probabilistic niche model, which allows the maximum likelihood set of trait values to be estimated for each species, is applied to the food web of the Benguela fishery. We also developed the allometric niche model, in w...

  17. Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance

    Science.gov (United States)

    Mizutani, Tatsuaki; Neugebauer, Nina; Putz, Eva M.; Moritz, Nadine; Simma, Olivia; Zebedin-Brandl, Eva; Gotthardt, Dagmar; Warsch, Wolfgang; Eckelhart, Eva; Kantner, Hans-Peter; Kalinke, Ulrich; Lienenklaus, Stefan; Weiss, Siegfried; Strobl, Birgit; Müller, Mathias; Sexl, Veronika; Stoiber, Dagmar

    2012-01-01

    Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance. PMID:23170251

  18. agged tumor cells reveal regulatory steps during earliest stages of tumor progression and micrometastasis

    OpenAIRE

    Culp, L A; Lin, W.C.

    1999-01-01

    Histochemical marker genes were used to "tag" mouse fibrosarcoma or human neuroblastoma cells, providing a better understanding of their subsequent progression and metastasis mechanisms in nude mice. Micrometastases in the lung were initiated from clusters of 2-6 cells rather than single cells in most cases; tumor cells were also visualized binding to the endothelium of small blood vessels to initiate these micrometastases. Shortterm, these mechanisms relied ...

  19. Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry

    OpenAIRE

    Horowitz, Amir; Strauss-Albee, Dara M.; Leipold, Michael; Kubo, Jessica; Nemat-Gorgani, Neda; DOGAN, Ozge C.; Dekker, Cornelia L; Mackey, Sally; Maecker, Holden; Swan, Gary E.; Davis, Mark M.; Norman, Paul J.; Guethlein, Lisbeth A.; Desai, Manisha; Parham, Peter

    2013-01-01

    Natural Killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 35 parameters, including 28 NK cell receptors, on perip...

  20. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  1. Computational models reveal a passive mechanism for cell migration in the crypt.

    Directory of Open Access Journals (Sweden)

    Sara-Jane Dunn

    Full Text Available Cell migration in the intestinal crypt is essential for the regular renewal of the epithelium, and the continued upward movement of cells is a key characteristic of healthy crypt dynamics. However, the driving force behind this migration is unknown. Possibilities include mitotic pressure, active movement driven by motility cues, or negative pressure arising from cell loss at the crypt collar. It is possible that a combination of factors together coordinate migration. Here, three different computational models are used to provide insight into the mechanisms that underpin cell movement in the crypt, by examining the consequence of eliminating cell division on cell movement. Computational simulations agree with existing experimental results, confirming that migration can continue in the absence of mitosis. Importantly, however, simulations allow us to infer mechanisms that are sufficient to generate cell movement, which is not possible through experimental observation alone. The results produced by the three models agree and suggest that cell loss due to apoptosis and extrusion at the crypt collar relieves cell compression below, allowing cells to expand and move upwards. This finding suggests that future experiments should focus on the role of apoptosis and cell extrusion in controlling cell migration in the crypt.

  2. The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise

    NARCIS (Netherlands)

    H. Kempe; A. Schwabe; F. Crémazy; P.J. Verschure; F.J. Bruggeman

    2015-01-01

    Transcriptional stochasticity can be measured by counting the number of mRNA molecules per cell. Cell-to-cell variability is best captured in terms of concentration rather than molecule counts, because reaction rates depend on concentrations. We combined single-molecule mRNA counting with single-cel

  3. Genomic reconstruction of the history of extant populations of India reveals five distinct ancestral components and a complex structure.

    Science.gov (United States)

    Basu, Analabha; Sarkar-Roy, Neeta; Majumder, Partha P

    2016-02-01

    India, occupying the center stage of Paleolithic and Neolithic migrations, has been underrepresented in genome-wide studies of variation. Systematic analysis of genome-wide data, using multiple robust statistical methods, on (i) 367 unrelated individuals drawn from 18 mainland and 2 island (Andaman and Nicobar Islands) populations selected to represent geographic, linguistic, and ethnic diversities, and (ii) individuals from populations represented in the Human Genome Diversity Panel (HGDP), reveal four major ancestries in mainland India. This contrasts with an earlier inference of two ancestries based on limited population sampling. A distinct ancestry of the populations of Andaman archipelago was identified and found to be coancestral to Oceanic populations. Analysis of ancestral haplotype blocks revealed that extant mainland populations (i) admixed widely irrespective of ancestry, although admixtures between populations was not always symmetric, and (ii) this practice was rapidly replaced by endogamy about 70 generations ago, among upper castes and Indo-European speakers predominantly. This estimated time coincides with the historical period of formulation and adoption of sociocultural norms restricting intermarriage in large social strata. A similar replacement observed among tribal populations was temporally less uniform. PMID:26811443

  4. Cell-cell channels, viruses, and evolution: via infection, parasitism, and symbiosis toward higher levels of biological complexity.

    Science.gov (United States)

    Baluska, Frantisek

    2009-10-01

    Between prokaryotic cells and eukaryotic cells there is dramatic difference in complexity which represents a problem for the current version of the cell theory, as well as for the current version of evolution theory. In the past few decades, the serial endosymbiotic theory of Lynn Margulis has been confirmed. This results in a radical departure from our understanding of living systems: the eukaryotic cell represents de facto"cells-within-cell." Higher order "cells-within-cell" situations are obvious at the eukaryotic cell level in the form of secondary and tertiary endosymbiosis, or in the male and female gametophytes of higher plants. The next challenge of the current version of the cell theory is represented by the fact that the multicellular fungi and plants are, in fact, supracellular assemblies as their cells are not physically separated from each other. Moreover, there are also examples of alliances and mergings between multicellular organisms. Infection, especially the viral one, but also bacterial and fungal infections, followed by symbiosis, is proposed to act as the major force that drives the biological evolution toward higher complexity. PMID:19845631

  5. The presence of six cryptic species of the whitefly Bemisia tabaci complex in China as revealed by crossing experiments

    Institute of Scientific and Technical Information of China (English)

    Peng Wang; Di-Bing Sun; Bao-Li Qiu; Shu-Sheng Liu

    2011-01-01

    Recent phylogenetic analysis using mitochondrial cytochrome oxidase I(mtCOI)sequences of Bemisia tabaci worldwide indicates that the whitefly comprises at least 24 morphologically indistinguishable but genetically distinct cryptic species.While evidence of reproductive isolation has been reported for some of the putative species,more extensive crossing experiments are required to clarify the systematics of this species complex.In this study,we established laboratory cultures for six putative species of B.tabaci collected in China.We conducted 22 inter-species crosses among the six putative species.The data and those reported previously were collated,and the combined dataset covered all the 30 possible inter-species crosses among the six putative species.Intra-species controls always produced female and male progeny and the proportions of females in the first generation(F1)ranged from 56% to 70%.However,in inter-species crosses female progeny were rarely produced,and the few F1 females produced in four of the 30 inter-species crosses were either sterile or significantly weaker in viability.These results demonstrate a pattern of complete reproductive isolation among the six putative species and show that they are six cryptic species in the B.tabaci complex.

  6. Screening for active small molecules in mitochondrial complex I deficient patient's fibroblasts, reveals AICAR as the most beneficial compound.

    Directory of Open Access Journals (Sweden)

    Anna Golubitzky

    Full Text Available Congenital deficiency of the mitochondrial respiratory chain complex I (CI is a common defect of oxidative phosphorylation (OXPHOS. Despite major advances in the biochemical and molecular diagnostics and the deciphering of CI structure, function assembly and pathomechanism, there is currently no satisfactory cure for patients with mitochondrial complex I defects. Small molecules provide one feasible therapeutic option, however their use has not been systematically evaluated using a standardized experimental system. In order to evaluate potentially therapeutic compounds, we set up a relatively simple system measuring different parameters using only a small amount of patient's fibroblasts, in glucose free medium, where growth is highly OXPOS dependent. Ten different compounds were screened using fibroblasts derived from seven CI patients, harboring different mutations.5-Aminoimidazole-4-carboxamide ribotide (AICAR was found to be the most beneficial compound improving growth and ATP content while decreasing ROS production. AICAR also increased mitochondrial biogenesis without altering mitochondrial membrane potential (Δψ. Fluorescence microscopy data supported increased mitochondrial biogenesis and activation of the AMP activated protein kinase (AMPK. Other compounds such as; bezafibrate and oltipraz were rated as favorable while polyphenolic phytochemicals (resverastrol, grape seed extract, genistein and epigallocatechin gallate were found not significant or detrimental. Although the results have to be verified by more thorough investigation of additional OXPHOS parameters, preliminary rapid screening of potential therapeutic compounds in individual patient's fibroblasts could direct and advance personalized medical treatment.

  7. Complex network models reveal correlations among network metrics, exercise intensity and role of body changes in the fatigue process

    Science.gov (United States)

    Pereira, Vanessa Helena; Gama, Maria Carolina Traina; Sousa, Filipe Antônio Barros; Lewis, Theodore Gyle; Gobatto, Claudio Alexandre; Manchado-Gobatto, Fúlvia Barros

    2015-05-01

    The aims of the present study were analyze the fatigue process at distinct intensity efforts and to investigate its occurrence as interactions at distinct body changes during exercise, using complex network models. For this, participants were submitted to four different running intensities until exhaustion, accomplished in a non-motorized treadmill using a tethered system. The intensities were selected according to critical power model. Mechanical (force, peak power, mean power, velocity and work) and physiological related parameters (heart rate, blood lactate, time until peak blood lactate concentration (lactate time), lean mass, anaerobic and aerobic capacities) and IPAQ score were obtained during exercises and it was used to construction of four complex network models. Such models have both, theoretical and mathematical value, and enables us to perceive new insights that go beyond conventional analysis. From these, we ranked the influences of each node at the fatigue process. Our results shows that nodes, links and network metrics are sensibility according to increase of efforts intensities, been the velocity a key factor to exercise maintenance at models/intensities 1 and 2 (higher time efforts) and force and power at models 3 and 4, highlighting mechanical variables in the exhaustion occurrence and even training prescription applications.

  8. Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel.

    Science.gov (United States)

    Zhao, Haiyan; Speir, Jeffrey A; Matsui, Tsutomu; Lin, Zihan; Liang, Lingfei; Lynn, Anna Y; Varnado, Brittany; Weiss, Thomas M; Tang, Liang

    2016-01-01

    The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device. PMID:26882199

  9. Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel.

    Directory of Open Access Journals (Sweden)

    Haiyan Zhao

    Full Text Available The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.

  10. Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells.

    Science.gov (United States)

    Costello, M Joseph; Brennan, Lisa A; Mohamed, Ashik; Gilliland, Kurt O; Johnsen, Sönke; Kantorow, Marc

    2016-01-01

    An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12-15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 μm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 μm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 μm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial

  11. A novel molecule integrating therapeutic and diagnostic activities reveals multiple aspects of stem cell-based therapy.

    Science.gov (United States)

    Hingtgen, Shawn D; Kasmieh, Randa; van de Water, Jeroen; Weissleder, Ralph; Shah, Khalid

    2010-04-01

    Stem cells are promising therapeutic delivery vehicles; however pre-clinical and clinical applications of stem cell-based therapy would benefit significantly from the ability to simultaneously determine therapeutic efficacy and pharmacokinetics of therapies delivered by engineered stem cells. In this study, we engineered and screened numerous fusion variants that contained therapeutic (TRAIL) and diagnostic (luciferase) domains designed to allow simultaneous investigation of multiple events in stem cell-based therapy in vivo. When various stem cell lines were engineered with the optimized molecule, SRL(O)L(2)TR, diagnostic imaging showed marked differences in the levels and duration of secretion between stem cell lines, while the therapeutic activity of the molecule showed the different secretion levels translated to significant variability in tumor cell killing. In vivo, simultaneous diagnostic and therapeutic monitoring revealed that stem cell-based delivery significantly improved pharmacokinetics and anti-tumor effectiveness of the therapy compared to intravenous or intratumoral delivery. As treatment for highly malignant brain tumor xenografts, tracking SRL(O)L(2)TR showed stable stem cell-mediated delivery significantly regressed peripheral and intracranial tumors. Together, the integrated diagnostic and therapeutic properties of SRL(O)L(2)TR answer critical questions necessary for successful utilization of stem cells as novel therapeutic vehicles. PMID:20127797

  12. Lineage relationship of CD8+ T cell subsets is revealed by progressive changes in the epigenetic landscape

    Science.gov (United States)

    Crompton, Joseph G.; Narayanan, Manikandan; Cuddapah, Suresh; Roychoudhuri, Rahul; Ji, Yun; Yang, Wenjing; Patel, Shashank J.; Sukumar, Madhusudhanan; Palmer, Douglas C.; Peng, Weiqun; Wang, Ena; Marincola, Francesco M.; Klebanoff, Christopher A.; Zhao, Keji; Tsang, John S.; Gattinoni, Luca; Restifo, Nicholas P.

    2016-01-01

    To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8+ T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8+ T-cell subsets. Taken together, our results suggest that CD8+ lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory. PMID:25914936

  13. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    International Nuclear Information System (INIS)

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium

  14. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    Energy Technology Data Exchange (ETDEWEB)

    Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  15. Unraveling the Complexities of Androgen Receptor Signaling in Prostate Cancer Cells

    OpenAIRE

    Heemers, Hannelore V.; Tindall, Donald J.

    2009-01-01

    Androgen signaling is critical for proliferation of prostate cancer cells but cannot be fully inhibited by current androgen deprivation therapies. A study by Xu et al. in this issue of Cancer Cell provides insights into the complexities of androgen signaling in prostate cancer and suggests avenues to target a subset of androgen-sensitive genes.

  16. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay

    Science.gov (United States)

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. Electronic supplementary information (ESI) available: Materials, experimental methods and Fig. S1-S8. See DOI: 10.1039/c6nr00171h

  17. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

  18. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Gradua