Sustained CD28 Expression Delays Multiple Features of Replicative Senescence in Human CD8 T Lymphocytes

CD28 costimulatory signal transduction in T lymphocytes is essential for optimal telomerase activity, stabilization of cytokine mRNAs, and glucose metabolism. During aging and chronic infection with HIV-1, there are increased proportions of CD8 T lymphocytes that lack CD28 expression and show additional features of replicative senescence. Moreover, the abundance of these cells correlates with decreased vaccine responsiveness, early mortality in the very old, and accelerated HIV disease progression. Here, we show that sustained expression of CD28, via gene transduction, retards the process of replicative senescence, as evidenced by enhanced telomerase activity, increased overall proliferative potential, and reduced secretion of pro-inflammatory cytokines. Nevertheless, the transduced cultures eventually do reach senescence, which is associated with increased CTLA-4 gene expression and a loss of CD28 cell surface expression. These findings further elucidate the central role of CD28 in the replicative senescence program, and may ultimately lead to novel therapies for diseases associated with replicative senescence

Analysis of Clostridium beijerinckii NCIMB 8052's transcriptional response to ferulic acid and its application to enhance the strain tolerance

Background: Plant-based cellulose presents the best source of renewable sugars for biofuel production. However, the lignin associated with plant cellulose presents a hurdle as hydrolysis of this component leads to the production of inhibitory compounds, such as ferulic acid. Results: The impacts of ferulic acid, a phenolic compound commonly found in lignin hydrolysates, on the growth, solvent production, and transcriptional responses of Clostridium beijerinckii NCIMB 8052 were determined. Addition of ferulic acid to growing cultures resulted in a decrease in the growth and solvent production by 30% and 25%, respectively, when compared to the control cultures. To better understand the toxicity of this compound, microarray analyses were performed using samples taken from these cultures at three different growth states. Several gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified showing significant change at each status, including ATP-binding cassette (ABC) transporters, two component system, and oxidoreductase activity. Moreover, genes related with efflux systems and heat shock proteins were also strongly up-regulated. Among these, expression of the groESL operon was induced by more than fourfold and was consequently selected to improve C. beijerinckii tolerance to ferulic acid. Real-time quantitative PCR (RT-qPCR) analysis confirmed that C. beijerinckii harboring the plasmid, pSAAT-ptb_Gro, had a two-to fivefold increased groESL operon expression during growth of these cultures. Moreover, this strain was more tolerant to ferulic acid as the growth of this recombinant strain and its bioconversion of glucose into solvents were both improved. Conclusions: Using transcriptomics, we identified numerous genes that are differentially expressed when C. beijerinckii cultures were exposed to ferulic acid for varying amounts of time. The operon expressing groESL was consistently up-regulated, suggesting that this gene cluster may contribute to strain tolerance. This was confirmed as recombinant cultures showed both an enhanced growth and solvent yield in the presence of 0.5 g/L ferulic acidopen00

Systems Biology of the qa Gene Cluster in Neurospora crassa

An ensemble of genetic networks that describe how the model fungal system, Neurospora crassa, utilizes quinic acid (QA) as a sole carbon source has been identified previously. A genetic network for QA metabolism involves the genes, qa-1F and qa-1S, that encode a transcriptional activator and repressor, respectively and structural genes, qa-2, qa-3, qa-4, qa-x, and qa-y. By a series of 4 separate and independent, model-guided, microarray experiments a total of 50 genes are identified as QA-responsive and hypothesized to be under QA-1F control and/or the control of a second QA-responsive transcription factor (NCU03643) both in the fungal binuclear Zn(II)2Cys6 cluster family. QA-1F regulation is not sufficient to explain the quantitative variation in expression profiles of the 50 QA-responsive genes. QA-responsive genes include genes with products in 8 mutually connected metabolic pathways with 7 of them one step removed from the tricarboxylic (TCA) Cycle and with 7 of them one step removed from glycolysis: (1) starch and sucrose metabolism; (2) glycolysis/glucanogenesis; (3) TCA Cycle; (4) butanoate metabolism; (5) pyruvate metabolism; (6) aromatic amino acid and QA metabolism; (7) valine, leucine, and isoleucine degradation; and (8) transport of sugars and amino acids. Gene products both in aromatic amino acid and QA metabolism and transport show an immediate response to shift to QA, while genes with products in the remaining 7 metabolic modules generally show a delayed response to shift to QA. The additional QA-responsive cutinase transcription factor-1β (NCU03643) is found to have a delayed response to shift to QA. The series of microarray experiments are used to expand the previously identified genetic network describing the qa gene cluster to include all 50 QA-responsive genes including the second transcription factor (NCU03643). These studies illustrate new methodologies from systems biology to guide model-driven discoveries about a core metabolic network involving carbon and amino acid metabolism in N. crassa

A strategy for constructing aneuploid yeast strains by transient nondisjunction of a target chromosome

<p>Abstract</p> <p>Background</p> <p>Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In <it>Saccharomyces cerevisiae</it>, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication.</p> <p>Results</p> <p>The centromere of each chromosome was replaced by a centromere that can be blocked by growth in galactose, and <it>ura3::HIS3</it>, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH) confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes that are known to be deleterious when overexpressed, including the beta-tubulin gene <it>TUB2</it>. To test whether a tubulin stoichiometry imbalance is necessary for the apparent lethality caused by an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene <it>TUB1</it>, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes.</p> <p>Conclusion</p> <p>This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate that a tubulin imbalance is necessary for disomic VI lethality, but it may not be the only dosage-dependent effect.</p

P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDP-glucose (< 30 μM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 μM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues

Метод лабораторного определения параметров устройства гидроимпульсного воздействия

Дана стаття описує лабораторний метод, що визначає: мету, умови, обсяг і порядок проведення досліджень параметрів пристрою гідроімпульсної дії.This article describes the laboratory method that defines: the purpose, conditions, effort and procedure of the researching the device settings of hydroimpulsive impact

Global Transcriptome and Deletome Profiles of Yeast Exposed to Transition Metals

A variety of pathologies are associated with exposure to supraphysiological concentrations of essential metals and to non-essential metals and metalloids. The molecular mechanisms linking metal exposure to human pathologies have not been clearly defined. To address these gaps in our understanding of the molecular biology of transition metals, the genomic effects of exposure to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium), and VB (arsenic) elements on the yeast Saccharomyces cerevisiae were examined. Two comprehensive sets of metal-responsive genomic profiles were generated following exposure to equi-toxic concentrations of metal: one that provides information on the transcriptional changes associated with metal exposure (transcriptome), and a second that provides information on the relationship between the expression of ∼4,700 non-essential genes and sensitivity to metal exposure (deletome). Approximately 22% of the genome was affected by exposure to at least one metal. Principal component and cluster analyses suggest that the chemical properties of the metal are major determinants in defining the expression profile. Furthermore, cells may have developed common or convergent regulatory mechanisms to accommodate metal exposure. The transcriptome and deletome had 22 genes in common, however, comparison between Gene Ontology biological processes for the two gene sets revealed that metal stress adaptation and detoxification categories were commonly enriched. Analysis of the transcriptome and deletome identified several evolutionarily conserved, signal transduction pathways that may be involved in regulating the responses to metal exposure. In this study, we identified genes and cognate signaling pathways that respond to exposure to essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanism by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways

The Intensity of IUGR-Induced Transcriptome Deregulations Is Inversely Correlated with the Onset of Organ Function in a Rat Model

A low-protein diet applied during pregnancy in the rat results in intrauterine growth restricted (IUGR) fetuses. In humans, IUGR is associated with increased perinatal morbidity, higher incidence of neuro-developmental defects and increased risk of adult metabolic anomalies, such as diabetes and cardiovascular disease. Development and function of many organs are affected by environmental conditions such as those inducing fetal and early postnatal growth restriction. This phenomenon, termed “fetal programming” has been studied unconnectedly in some organs, but very few studies (if any) have investigated at the same time several organs, on a more comparative basis. However, it is quite probable that IUGR affects differentially most organ systems, with possible persistent changes in gene expression. In this study we address transcriptional alterations induced by IUGR in a multi-organ perspective, by systematic analysis of 20-days rat fetuses. We show that (1) expressional alterations are apparently stronger in organs functioning late in foetal or postnatal life than in organs that are functioning early (2) hierarchical classification of the deregulations put together kidney and placenta in one cluster, liver, lungs and heart in another; (3) the epigenetic machinery is set up especially in the placenta, while its alterations are rather mild in other organs; (4) the genes appear deregulated in chromosome clusters; (5) the altered expression cascades varies from organ to organ, with noticeably a very significant modification of the complement and coagulation cascades in the kidney; (6) we found a significant increase in TF binding site for HNF4 proteins specifically for liver genes that are down-regulated in IUGR, suggesting that this decrease is achieved through the action of HNF transcription factors, that are themselves transcriptionnally induced in the liver by IUGR (x 1.84 fold). Altogether, our study suggests that a combination of tissue-specific mechanisms contributes to bring about tissue-driven modifications of gene cascades. The question of these cascades being activated to adapt the organ to harsh environmental condition, or as an endpoint consequence is still raised

Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics

Comparative Developmental Expression Profiling of Two C. elegans Isolates

Gene expression is known to change during development and to vary among genetically diverse strains. Previous studies of temporal patterns of gene expression during C. elegans development were incomplete, and little is known about how these patterns change as a function of genetic background. We used microarrays that comprehensively cover known and predicted worm genes to compare the landscape of genetic variation over developmental time between two isolates of C. elegans. We show that most genes vary in expression during development from egg to young adult, many genes vary in expression between the two isolates, and a subset of these genes exhibit isolate-specific changes during some developmental stages. This subset is strongly enriched for genes with roles in innate immunity. We identify several novel motifs that appear to play a role in regulating gene expression during development, and we propose functional annotations for many previously unannotated genes. These results improve our understanding of gene expression and function during worm development and lay the foundation for linkage studies of the genetic basis of developmental variation in gene expression in this important model organism