Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis

Structural Insights into the Recovery of Aldolase Activity in N -Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ-Thialysine by Using a Chemical Mutagenesis Strategy

Chemical modification has been used to introduce the unnatural amino acid γ‐thialysine in place of the catalytically important Lys165 in the enzyme N‐acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site‐directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ‐thialysine through dehydroalanine (Dha) as demonstrated by ESI‐MS. Initial kinetic characterisation showed that the protein containing γ‐thialysine regained 17 % of the wild‐type activity. To understand the reason for this lower activity, we solved X‐ray crystal structures of the wild‐type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165‐γ‐thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ‐thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH‐activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine‐containing enzyme showed almost 30 % of the activity of the wild‐type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid‐containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pKa of the residue at position 165 is perturbed by replacement with γ‐thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL

Enhanced production of IL-17A in patients with severe asthma is inhibited by 1α,25-dihydroxyvitamin D3 in a glucocorticoid-independent fashion

Background: TH17 cells are proposed to play a role in the pathology of asthma, including steroid-resistant (SR) disease. We previously identified a steroid-enhancing function of vitamin D in patients with SR asthma in restoring the impaired response to steroids for production of the anti-inflammatory cytokine IL-10. / Objective: We sought to investigate the production of the TH17-associated cytokines IL-17A and IL-22 in culture in patients with moderate-to-severe asthma defined on the basis of their clinical response to steroids and the susceptibility of this response to inhibition by steroids and the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3). / Methods: PBMCs were stimulated in culture with or without dexamethasone and 1,25(OH)2D3. A cytometric bead array, ELISA, and intracellular cytokine staining were used to assess cytokine production. The role of CD39 in inhibition of the TH17 response was studied by using quantitative real-time PCR, flow cytometry, and addition of the antagonist POM-1 to culture. / Results: Asthmatic patients synthesized much higher levels of IL-17A and IL-22 than nonasthmatic control subjects, with patients with SR asthma expressing the highest levels of IL-17A. Glucocorticoids did not inhibit IL-17A cytokine expression in patients and enhanced production in cultures from control subjects. Treatment with 1,25(OH)2D3 with or without dexamethasone significantly reduced both IL-17A and IL-22 levels. An antagonist of the ectonucleotidase CD39 reversed 1,25(OH)2D3-mediated inhibition of the IL-17A response. / Conclusion: Patients with severe asthma exhibit increased levels of TH17 cytokines, which are not inhibited by steroids. 1,25(OH)2D3 inhibits TH17 cytokine production in all patients studied, irrespective of their clinical responsiveness to steroids, identifying novel steroid-enhancing properties of vitamin D in asthmatic patients

Ethnic Variation in Inflammatory Profile in Tuberculosis

PMCID: PMC3701709This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Regulation of Epithelial Branching Morphogenesis and Cancer Cell Growth of the Prostate by Wnt Signaling

Although Wnt signaling has been shown to be important for embryonic morphogenesis and cancer pathogenesis of several tissues, its role in prostatic development and tumorigenesis is not well understood. Here we show that Wnt signaling regulated prostatic epithelial branching morphogenesis and luminal epithelial cell differentiation in developing rat prostate organ cultures. Specifically, Wnt signaling regulated the proliferation of prostate epithelial progenitor cells. Assessment of the expression levels of a Wnt pathway transcriptional target gene, Axin2, showed that the Wnt pathway was activated in the developing prostate, but was down-regulated in the adult. Castration resulted in an upregulation of Axin2 whereas androgen replacement resulted in a down regulation of Axin2. Such dynamic changes of Wnt activity was also confirmed in a BAT-gal transgenic mouse line in which β-galactosidase reporter is expressed under the control of β-catenin/T cell factor responsive elements. Furthermore, we evaluated the role of Wnt signaling in prostate tumorigenesis. Axin2 expression was found upregulated in the majority of human prostate cancer cell lines examined. Moreover, addition of a Wnt pathway inhibitor, Dickkopf 1 (DKK1), into the culture medium significantly inhibited prostate cancer cell growth and migration. These findings suggest that Wnt signaling regulates prostatic epithelial ductal branching morphogenesis by influencing cell proliferation, and highlights a role for Wnt pathway activation in prostatic cancer progression

Adaptation, compromise, and constraint: the development, morphometrics, and behavioral basis of a fighter-flier polymorphism in male Hoplothrips karnyi (Insecta: Thysanoptera)

Males of the colonial, wing-polymorphic thrips Hoplothrips karnyi (Hood) fight each other with their forelegs in defense of communal female oviposition areas. In this study, males were reared individually under varying conditions of food deprivation to investigate the developmental cues used in morph determination and the relationships between wing morph, developmental time in each instar, propupal weight, and five adult morphological characters associated with fighting ability and dispersal ability. Males deprived of food for five days midway through the second (final) larval instar had smaller propupal weights and were more likely to develop wings than males deprived of food in the first instar or control males. However, the mean propupal weight of all males that developed wings was not significantly less than that of wingless males. Wing morph of female parents had no measurable effect on this character in the offspring. Wingless males possess relatively larger fore-femora and prothoraces than do winged males, but winged males possess relatively larger pterothoraces (Fig. 1). Behavioral observations of wingless and winged males of similar weight as propupae showed that wingless males won fights and became dominant in oviposition areas. Thus, a trade-off exists between characters associated with male fighting and dispersal ability. The cost of wings, in terms of fore-femora size and prothorax size, increased with propupal weight. Wingless males that developed in the experimental treatment that produced a high proportion of winged males were relatively small in size, and were intermediate in body shape with respect to winged males and other wingless males (Fig. 2). This shape intermediacy indicates that there may be developmental constraints on alternative tactics of resource allocation. Total developmental time varied between wing morphs, but was not correlated with propupal weight or adult morphological characters of winged or wingless males. For wingless males that developed in the treatment that produced a high proportion of winged males, adult morphological characters were negatively correlated with the duration of the second instar. This correlation suggests that the development of small wingless males involves a compromise between the benefits of large adult size and the costs of prolonging the second instar to increase the probability of becoming larger.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46886/1/265_2004_Article_BF00299892.pd

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Rediscovering the value of families for psychiatric genetics research

As it is likely that both common and rare genetic variation are important for complex disease risk, studies that examine the full range of the allelic frequency distribution should be utilized to dissect the genetic influences on mental illness. The rate limiting factor for inferring an association between a variant and a phenotype is inevitably the total number of copies of the minor allele captured in the studied sample. For rare variation, with minor allele frequencies of 0.5% or less, very large samples of unrelated individuals are necessary to unambiguously associate a locus with an illness. Unfortunately, such large samples are often cost prohibitive. However, by using alternative analytic strategies and studying related individuals, particularly those from large multiplex families, it is possible to reduce the required sample size while maintaining statistical power. We contend that using whole genome sequence (WGS) in extended pedigrees provides a cost-effective strategy for psychiatric gene mapping that complements common variant approaches and WGS in unrelated individuals. This was our impetus for forming the “Pedigree-Based Whole Genome Sequencing of Affective and Psychotic Disorders” consortium. In this review, we provide a rationale for the use of WGS with pedigrees in modern psychiatric genetics research. We begin with a focused review of the current literature, followed by a short history of family-based research in psychiatry. Next, we describe several advantages of pedigrees for WGS research, including power estimates, methods for studying the environment, and endophenotypes. We conclude with a brief description of our consortium and its goals.This research was supported by National Institute of Mental Health grants U01 MH105630 (DCG), U01 MH105634 (REG), U01 MH105632 (JB), R01 MH078143 (DCG), R01 MH083824 (DCG & JB), R01 MH078111 (JB), R01 MH061622 (LA), R01 MH042191 (REG), and R01 MH063480 (VLN).UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí