The E-box DNA binding protein Sgc1p suppresses the gcr2 mutation, which is involved in transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

AbstractGlycolytic gene expression is mediated by the Gcr1p-Gcr2p transcriptional activation complex. A screen for multicopy suppressors of gcr2 yielded SGC1. SGC1’s suppression activity was specific to gcr2, it did not extend to gcr1. Disruption of SGC1 moderately affected glycolytic enzyme activities, although no growth defect was evident. Sgc1p exhibits a bHLH motif which is characteristic of E-box DNA-binding proteins. DNA footprinting experiments demonstrated Sgc1p’s ability to bind at an E-box. However, its binding specificity was less than 10-fold, which is also characteristic of E-box binding proteins. LexA fusion experiments demonstrated that Sgc1p has weak intrinsic activating activity independent of GCR1 and GCR2. We propose that Sgc1p binds at E-boxes of glycolytic genes and contributes to their activation

Non-homogeneous random walks, subdiffusive migration of cells and anomalous chemotaxis

This paper is concerned with a non-homogeneous in space and non-local in time random walk model for anomalous subdiffusive transport of cells. Starting with a Markov model involving a structured probability density function, we derive the non-local in time master equation and fractional equation for the probability of cell position. We show the structural instability of fractional subdiffusive equation with respect to the partial variations of anomalous exponent. We find the criteria under which the anomalous aggregation of cells takes place in the semi-infinite domain.Comment: 18 pages, accepted for publicatio

The degree of microbiome complexity influences the epithelial response to infection

<p>Abstract</p> <p>Background</p> <p>The human microflora is known to be extremely complex, yet most pathogenesis research is conducted in mono-species models of infection. Consequently, it remains unclear whether the level of complexity of a host's indigenous flora can affect the virulence potential of pathogenic species. Furthermore, it remains unclear whether the colonization by commensal species affects a host cell's response to pathogenic species beyond the direct physical saturation of surface receptors, the sequestration of nutrients, the modulation of the physico-chemical environment in the oral cavity, or the production of bacteriocins. Using oral epithelial cells as a model, we hypothesized that the virulence of pathogenic species may vary depending on the complexity of the flora that interacts with host cells.</p> <p>Results</p> <p>This is the first report that determines the global epithelial transcriptional response to co-culture with defined complex microbiota. In our model, human immortalized gingival keratinocytes (HIGK) were infected with mono- and mixed cultures of commensal and pathogenic species. The global transcriptional response of infected cells was validated and confirmed phenotypically. In our model, commensal species were able to modulate the expression of host genes with a broad diversity of physiological functions and antagonize the effect of pathogenic species at the cellular level. Unexpectedly, the inhibitory effect of commensal species was <it>not </it>correlated with its ability to inhibit adhesion or invasion by pathogenic species.</p> <p>Conclusion</p> <p>Studying the global transcriptome of epithelial cells to single and complex microbial challenges offers clues towards a better understanding of how bacteria-bacteria interactions and bacteria-host interactions impact the overall host response. This work provides evidence that the degree of complexity of a mixed microbiota <it>does </it>influence the transcriptional response to infection of host epithelial cells, and challenges the current dogma regarding the <it>potential </it>versus the <it>actual </it>pathogenicity of bacterial species. These findings support the concept that members of the commensal oral flora have evolved cellular mechanisms that directly modulate the host cell's response to pathogenic species and dampen their relative pathogenicity.</p

Identification of Cellular Genes Targeted by KSHV-Encoded MicroRNAs

MicroRNAs (miRNAs) are 19 to 23 nucleotide–long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma–associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-β. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-β activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis

Partitioning Transcript Variation in Drosophila: Abundance, Isoforms, and Alleles

Multilevel analysis of transcription is facilitated by a new array design that includes modules for assessment of differential expression, isoform usage, and allelic imbalance in Drosophila. The ∼2.5 million feature chip incorporates a large number of controls, and it contains 18,769 3′ expression probe sets and 61,919 exon probe sets with probe sequences from Drosophila melanogaster and 60,118 SNP probe sets focused on Drosophila simulans. An experiment in D. simulans identified genes differentially expressed between males and females (34% in the 3′ expression module; 32% in the exon module). These proportions are consistent with previous reports, and there was good agreement (κ = 0.63) between the modules. Alternative isoform usage between the sexes was identified for 164 genes. The SNP module was verified with resequencing data. Concordance between resequencing and the chip design was greater than 99%. The design also proved apt in separating alleles based upon hybridization intensity. Concordance between the highest hybridization signals and the expected alleles in the genotype was greater than 96%. Intriguingly, allelic imbalance was detected for 37% of 6579 probe sets examined that contained heterozygous SNP loci. The large number of probes and multiple probe sets per gene in the 3′ expression and exon modules allows the array to be used in D. melanogaster and in closely related species. The SNP module can be used for allele specific expression and genotyping of D. simulans

Microarray Analysis of Gene Expression Patterns during Healing of Rat Corneas after Excimer Laser Photorefractive Keratectomy

PURPOSE. To characterize changes over time in the genomic expression profile of rat corneas after excimer laser photorefractive keratectomy (PRK), in an effort to better understand the cellular response to injury and the dynamic changes that occur in gene expression patterns as a wound heals. METHODS. The corneal gene expression profile of 1176 genes at 3 and 7 days after PRK was determined and compared with untreated corneal gene expression patterns by interrogating commercially available cDNA arrays with labeled target cDNA prepared from pooled total RNA harvested from the respective treatment group of adult male rats. The gene expression patterns were inferred based on the hybridization intensities of the probes on the cDNA arrays. The hybridization signals were globally normalized and filtered. The data were analyzed by using hierarchical and k-means clustering algorithms before and after normalization of variances. RESULTS. Of the 1176 cDNA elements on the array, 588 consistently produced similar results in replicate experiments and comprised the data set analyzed in this work. In total, 73 genes were identified, with expression levels that differed by at least threefold at either 3 or 7 days after PRK. At 3 days after PRK, 70 genes were identified with expression levels that differed by more than threefold, compared with the expression level in untreated animals. The expression of 42 genes increased by threefold or more, whereas expression of 28 genes decreased by threefold or more. By day 7 after PRK, the number of genes displaying more than a threefold difference in expression pattern was reduced to 27 genes, 20 of which showed elevated levels, whereas 7 exhibited decreased levels. Hierarchical clustering of the 588 studied genes produced 10 clusters with correlation coefficients of 0.9 or greater. To determine whether any of the clusters were overrepresented by genes with related functions, the cumulative hypergeometric probability was calculated by obtaining the observed number of functionally related genes within each of the 10 clusters. Seven of the clusters were statistically overrepresented by one or more categories of functionally related genes, such as cell cycle regulators, transcription factors, and metabolic pathway genes. Clustering analysis of 56 genes generally considered to influence corneal wound healing produced 10 gene clusters with correlation coefficients of at least 0.9. Expression of 23 of these 56 genes increased at day 3, then decreased at day 7 to levels similar to those on day 0. These included several growth factors (VEGF, FGF, IGF-I), proteases (PAI-1, PAI-2A) and protease inhibitors (TIMP-2 and TIMP-3). Expression of nine genes increased on both days 3 and 7 compared with expression on day 0 (e.g., TGFB1, TGFBIIR, M6P/IGFR-2), and no genes decreased on both days 3 and 7, compared with day 0. CONCLUSIONS. Microarray analysis of 1176 identified 588 genes with reproducible patterns of expression in rat corneas on days 3 and 7 after PRK and 73 genes with a threefold change in expression compared with untreated corneas. Hierarchical clustering of these 588 genes identified 10 clusters of genes with very similar patterns of expression. Clustering of genes with similar patterns of expression implies a common regulatory pathway for the genes within a cluster, and identifies potential new targets for regulating corneal wound healing. (Invest Ophthalmol Vis Sci. 2002;43:1772-1782 C orneal wound healing is a complex biological process that involves the integrated actions of many genes that code for proteins with diverse functions, such as cytokines, growth factors, receptors, extracellular matrix proteins, integrins, proteases, inhibitors and cell cycle genes. 1,2 When expression of these genes is regulated correctly, corneal wounds heal appropriately. However, when intrinsic or extrinsic factors disrupt the expression of these genes, corneal wounds may either fail to heal adequately or may heal with excessive scar formation. Thus, there is a need to understand, as broadly as possible, how the expression of key genes changes during corneal wound healing so that therapies can be logically designed to prevent complications from occurring. Previous studies investigating gene expression during corneal wound healing have analyzed mRNA levels of a small number of selected growth factors, receptors, and extracellular matrix genes that are thought to regulate or participate in corneal wound healing. For example, quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) of rat corneas after photorefractive keratectomy (PRK) showed prolonged elevation of mRNAs for the three isoforms of transforming growth factor-␤ (TGF␤1, TGF␤2, TGF␤3), the TGF␤ type II receptor (TGF␤R-II), and extracellular matrix proteins (types I, III, and VI collagen and fibronectin). 3 Similar results were reported for mRNA levels for collagens I, III, IV, and V using semiquantitative RT-PCR in rat corneas after PRK. 5 Other groups have investigated changes in protein levels in corneas during wound healing. Immunohistochemical analysis of cat corneas after PRK indicated there were increased levels of TGF␤1, TGF␤2, and TGF␤3 and the TGF␤R-I and TGF␤R-II receptor proteins in the subepithelial tissue of laser-ablated regions at 4 weeks after injury. 7 Analyses of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) in rat and rabbit corneas after PRK found increased levels of TIMP-2 and MMP-9, -7, and -13 proteins and mRNAs in migrating basal epithelial cells. 8 -12 Immunofluorescence analysis of radial keratotomy (RK) wounds in human corneas detected several extracellular matrix proteins that were not present in the stroma of normal corneas, including collagen types III, VIII, and XIV; the ␣1-␣2 chains of type IV collagen; tenascin-C; and fibrillin-1. MATERIALS AND METHODS Animal Models Animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the animal protocol was approved by the University of Florida Animal Care and Use Committee. Rat corneas underwent excimer laser ablation as described previously. RNA Isolation from Rat Corneas Total RNA was extracted from each group of 10 pooled corneas using guanidine isothiocyanate and phenol-chloroform according to standard methods. Briefly, pooled corneal tissue was homogenized in 0.75 mL RNA extraction solution (TRIzol; Gibco, Grand Island, NY) using a 5-mL frosted glass-on-glass tissue grinder (Duall; Konte Scientific Glassware, Vineland, NJ), RNA was extracted with chloroform, precipitated with isopropanol, washed with ethanol, and dissolved in RNase-free water treated with 0.1% diethylpyrocarbonate. The material was then treated with 1 U/L DNase I at 37°C for 30 minutes to remove any contaminating genomic DNA, and then extracted once with phenolchloroform-isoamyl alcohol (25:24:1 pH 4.5), followed by extraction with chloroform, precipitated with 2 M sodium acetate and 95% ethanol, washed with 80% ethanol, and resuspended in RNase-free water. The concentration of RNA were measured spectrophotometrically at 260 nm (GeneQuant; Amersham Pharmacia Biotech, Uppsala, Sweden). Absorbance ratios of 280 to 260 nm were consistently more than 1.8 for all samples. DNA Arrays and Interrogations Rat cDNA expression arrays (Atlas 1.2; Clontech Laboratories, Palo Alto, CA) were used in this work to study gene expression patterns in corneas after PRK. Each DNA array was manufactured by spotting a nylon membrane with cDNA fragments representing 1176 known genes in addition to three plasmid and bacteriophage DNA sequences, which were included as a negative control, and 9 housekeeping gene sequences, which were included as a positive control. To minimize cross-hybridization and nonspecific binding, cDNA fragments bound on the array were selected, so that they ranged in size from 200 to 600 bp and they did not contain repetitive elements. All clones used to generate the immobilized gene probes were sequenced by the manufacturer to verify identity, and the size of PCR products generated from all clones by gene-specific primers was confirmed by gel electrophoresis. For DNA array interrogation, the arrays were interrogated with ␣ 32 P-labeled cDNA prepared by reverse transcribing pools of total corneal RNA isolated. Using the manufacturer&apos;s protocol (Clontech Laboratories), 5 g total RNA was reverse transcribed with genespecific primers and reagents provided with the cDNA expression array. The radiolabeled target cDNAs were purified by column chromatography, denatured under basic conditions, neutralized, and added to 10-mL aliquots of hybridization solution (ExpressHyb; Clontech Laboratories) at 68°C, containing 100 g/mL heat-denatured and sheared salmon testes DNA (Sigma, St. Louis, MO), to obtain a final probe concentration of 5 ϫ 10 6 cpm/mL. The radiolabeled cDNA hybridization solutions were applied to prehybridize the array membranes (30 minutes in ExpressHyb with salmon testes DNA at 68°C without labeled probe) and were hybridized overnight at 68°C in glass bottles with constant rotational mixing in a hybridization incubator. After hybridization, the membranes were washed four times with 140 mL 2ϫ SSC (300 mM sodium chloride, 330 mM sodium citrate, pH 7.0), and 1% SDS solution at 68°C for 30 minutes each, followed by one wash for 30 minutes in 140 mL 0.1% SSC and 0.5% SDS, at 68°C. Finally, the membranes were rinsed for 5 minutes in a solution of 2ϫ SSC at room temperature, and exposed to phosphor screens. Each sample was applied to a new membrane--that is, the membranes were not stripped and rehybridized

Gene expression changes in the human diaphragm after cardiothoracic surgery

ObjectiveWe examined the effects of cardiothoracic surgery, including cardiopulmonary bypass and controlled mechanical ventilation, on messenger RNA gene expression in human diaphragm. We hypothesized that genes responsible for stress response, redox regulation, protein turnover, energy metabolism, and contractile function would be altered by cardiothoracic surgery.MethodsPaired diaphragm biopsy samples were obtained from 5 male patients (67 ± 11 years) during cardiothoracic surgery, the first as soon as the diaphragm was exposed and the second as late in surgery as possible (4.9 ± 1.8 hours between samples). We profiled messenger RNA from 5 specimen pairs with microarray analysis (Hu U133 plus 2.0; Affymetrix UK Ltd, High Wycombe, UK). Quantitative reverse transcriptase polymerase chain reaction was performed with a select set of genes exhibiting differential expression for validation.ResultsMicroarray analysis identified 779 differentially expressed (early vs late samples) unique gene products (P < .005). Postoperatively, genes related to stress response and redox regulation were upregulated. Additionally, we found significantly upregulated expression of cathepsin C (2.7-fold), cathepsin L1 (2.0-fold), various ubiquitin-conjugating enzymes (E2, approximately 1.8-fold), proinflammatory cytokine interleukin 6 (15.6-fold), and muscle-specific ubiquitin ligase (MuRF-1, 2.6-fold). Comparison of fold change values obtained by quantitative reverse transcriptase polymerase chain reaction and microarray yielded significant correlation (r = 0.95, P < .0001).ConclusionsCardiothoracic surgery results in rapid changes in human diaphragm gene expression in the operating room, including genes related to stress response, inflammation, redox regulation, and proteolysis. These results may provide insight into diaphragm muscle biology after prolonged cardiothoracic procedures

A pivotal role for starch in the reconfiguration of 14C-partitioning and allocation in Arabidopsis thaliana under short-term abiotic stress.

Plant carbon status is optimized for normal growth but is affected by abiotic stress. Here, we used 14C-labeling to provide the first holistic picture of carbon use changes during short-term osmotic, salinity, and cold stress in Arabidopsis thaliana. This could inform on the early mechanisms plants use to survive adverse environment, which is important for efficient agricultural production. We found that carbon allocation from source to sinks, and partitioning into major metabolite pools in the source leaf, sink leaves and roots showed both conserved and divergent responses to the stresses examined. Carbohydrates changed under all abiotic stresses applied; plants re-partitioned 14C to maintain sugar levels under stress, primarily by reducing 14C into the storage compounds in the source leaf, and decreasing 14C into the pools used for growth processes in the roots. Salinity and cold increased 14C-flux into protein, but as the stress progressed, protein degradation increased to produce amino acids, presumably for osmoprotection. Our work also emphasized that stress regulated the carbon channeled into starch, and its metabolic turnover. These stress-induced changes in starch metabolism and sugar export in the source were partly accompanied by transcriptional alteration in the T6P/SnRK1 regulatory pathway that are normally activated by carbon starvation

Supporting thinking on sample sizes for thematic analyses: a quantitative tool

Thematic analysis is frequently used to analyse qualitative data in psychology, healthcare, social research and beyond. An important stage in planning a study is determining how large a sample size may be required, however current guidelines for thematic analysis are varied, ranging from around 2 to over 400 and it is unclear how to choose a value from the space in between. Some guidance can also not be applied prospectively. This paper introduces a tool to help users think about what would be a useful sample size for their particular context when investigating patterns across participants. The calculation depends on (a) the expected population theme prevalence of the least prevalent theme, derived either from prior knowledge or based on the prevalence of the rarest themes considered worth uncovering, e.g. 1 in 10, 1 in 100; (b) the number of desired instances of the theme; and (c) the power of the study. An adequately powered study will have a high likelihood of finding sufficient themes of the desired prevalence. This calculation can then be used alongside other considerations. We illustrate how to use the method to calculate sample size before starting a study and achieved power given a sample size, providing tables of answers and code for use in the free software, R. Sample sizes are comparable to those found in the literature, for example to have 80% power to detect two instances of a theme with 10% prevalence, 29 participants are required. Increasing power, increasing the number of instances or decreasing prevalence increases the sample size needed. We do not propose this as a ritualistic requirement for study design, but rather as a pragmatic supporting tool to help plan studies using thematic analysis