Search

Objective: To compare the diagnostic performance of the GenoType MRBDRplus assay with the gold standard phenotypic drug susceptibility testing in the detection of drug resistance among culture isolates obtained from patients in Karachi, Pakistan. Design: Mycobacterium tuberculosis isolates were obtained from 96 consecutive tuberculosis patients found to have resistance to isoniazid from two health centers in Karachi (January-November 2017). Isolates were tested for drug resistance against rifampin and isoniazid using the MTBDRplus assay. Results were compared with conventional drug-susceptibility testing and the frequency of specific mutations were reported. Results: The MTBDRplus assay had a sensitivity for rifampin resistance of 98.8% (95% CI: 93.4–100) and for isoniazid resistance of 90.6% (95% CI: 83.0–95.6). The MTBDRplus assay showed mutations in rpoB in 81 of the 96 (84.4%) isolates. Of the 87 isolates showing resistance to isoniazid via the MTBDRplus assay, 71 (74.0%) isolates had mutations in the katG gene only, 15 (15.6%) isolates had mutations in the inhA promoter region, and 1 (1.0%) showed mutations in both genes. Conclusion: The GenoType MTBDRplus assay in Pakistan can identify subgroups at high-risk of having isolates with mutations in the katG and/or inhA genes. Understanding the local burden of these mutations have implications for local diagnostic and treatment guidelines. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is the most common curable sexually transmitted infection (STI) worldwide. Although predominately asymptomatic, the disease spectrum of trichomoniasis in women is characterized primarily by signs and symptoms of vaginitis, including purulent discharge and localized vulvar pruritus and erythema. Several FDA-cleared nucleic acid amplification tests (NAATs) are available for the diagnosis of T. vaginalis infections, but laboratory developed tests (LDTs) are widely utilized and cost-effective solutions in both the research and clinical diagnostic settings. LDT diagnosis of T. vaginalis is particularly appealing since it can be performed using remnant specimens collected for other STI testing. Using a LDT implemented as part of this study, T. vaginalis was detected in 7% of participating Louisiana women (14/199). The mean T. vaginalis organism burden was 1.0x106 ± 4.5x105 organisms per mL of ThinPrep PreservCyt. Using DNA eluates obtained after HPV testing on the cobas 4800 system, the T. vaginalis LDT was characterized by excellent intra- and interassay reproducibility (coefficient of variation values all <3.5%). Compared with two commercially available NAATs from TIB MOLBIOL, the sensitivity and specificity of the LDT was 92.9 and 99.5%, respectively. Collectively, this study details the diagnostic and quantitative utility of a LDT for T. vaginalis. When applied in the clinical research setting, we confirmed the high prevalence of T. vaginalis, but also observed extraordinarily high organism burdens in the cervix. These findings highlight the unique host-pathogen relationship of T. vaginalis with lower reproductive tract tissues, and substantiate the need for continued investigation of this highly prevalent STI. [ABSTRACT FROM AUTHOR]

We previously characterized a clonal olfactory placode-derived cell line (OP6) as a model system for studying odorant receptor (OR) choice, where individual OP6 cells, similar to olfactory sensory neurons in vivo, transcribe one allele (“monoallelic”) of one OR gene (“monogenic”). The OP6 cell line provides a unique opportunity to investigate intrinsic properties of OR regulation that cannot easily be investigated in vivo. First, whereas OR-expressing cells in vivo are post-mitotic, OP6 cells are immortalized, raising interesting questions about the stability of epigenetic states associated with OR selection/silencing as OP6 cells progress through the cell cycle. Second, OP6 cells have been isolated away from extrinsic developmental cues, and therefore, any long-term OR selection biases are likely to arise from intrinsic epigenetic states that persist in the absence of developmental context. In this study, we investigated OR re-selection frequency and selection biases within clonal OP6 cell populations. We found no evidence of OR stability through the cell cycle: our results were most consistent with OR re-selection events transpiring at least once per cell division, suggesting that chromatin states associated with OR selection in this system might not be maintained in the subsequent generation. In contrast, we found strong evidence for OR selection biases maintained over prolonged culturing across a diverse set of OP6 cell lineages, suggesting the persistence of intrinsic epigenetic states that advantage some OR loci over others. Together, our data suggest that in the absence of instructive cues, intrinsic epigenetic states influencing OR eligibility, but not those determining OR choice, might persist through the cell cycle. [ABSTRACT FROM AUTHOR]

Background: Large and giant dog breeds have a high risk for gastric dilatation-volvulus (GDV) which is an acute, life-threatening condition. Previous work by our group identified a strong risk of GDV linked to specific alleles in innate and adaptive immune genes. We hypothesize that variation in the genes of the immune system act through modulation of the gut microbiome, or through autoimmune mechanisms, or both, to predispose dogs to this condition. Here, we investigate whether differences in the canine fecal microbiome are associated with GDV and are linked to previously identified risk alleles. Methodology/Principle findings: Fecal samples from healthy Great Danes (n = 38), and dogs with at least one occurrence of GDV (n = 37) were collected and analyzed by paired-end sequencing of the 16S rRNA gene. Dietary intake and temperament were estimated from a study-specific dietary and temperament questionnaire. Dogs with GDV had significantly more diverse fecal microbiomes than healthy control dogs. Alpha diversity was significantly increased in dogs with GDV, as well as dogs with at least one risk allele for DRB1 and TRL5. We found no significant association of dietary intake and GDV. Dogs with GDV showed a significant expansion of the rare lineage Actinobacteria (p = 0.004), as well as a significantly greater abundance of Firmicutes (p = 0.004) and a significantly lower abundance of Bacteroidetes (p<0.004). There was a significant difference in the abundance of 10 genera but after correction for multiple comparisons, none were significant. Bacterial phyla were significantly different between controls and dogs with GDV and at least one risk allele for DRB1 and TRL5. Actinobacteria were significantly higher in dogs with GDV and with one risk allele for DRB1 and TLR5 but not DLA88 genes. Furthermore, Collinsella was significantly increased in dogs with at least one risk allele for DRB1 and TLR5. Logistic regression showed that a model which included Actinobacteria, at least one risk allele,and temperament, explained 29% of the variation in risk of GDV in Great Danes. Conclusions: The microbiome in GDV was altered by an expansion of a minor lineage and was associated with specific alleles of both innate and adaptive immunity genes. These associations are consistent with our hypothesis that immune genes may play a role in predisposition to GDV by altering the gut microbiome. Further research will be required to directly test the causal relationships of immune genes, the gut microbiome and GDV. [ABSTRACT FROM AUTHOR]

Smut caused by biotrophic fungus Sporisorium scitamineum is a major disease of cultivated sugarcane that can cause considerable yield losses. It has been suggested in literature that there are at least two types of resistance mechanisms in sugarcane plants: an external resistance, due to chemical or physical barriers in the sugarcane bud, and an internal resistance governed by the interaction of plant and fungus within the plant tissue. Detailed molecular studies interrogating these two different resistance mechanisms in sugarcane are scarce. Here, we use light microscopy and global expression profiling with RNA-seq to investigate these mechanisms in sugarcane cultivar CP74-2005, a cultivar that possibly possesses both internal and external defence mechanisms. A total of 861 differentially expressed genes (DEGs) were identified in a comparison between infected and non-infected buds at 48 hours post-inoculation (hpi), with 457 (53%) genes successfully annotated using BLAST2GO software. This includes genes involved in the phenylpropanoid pathway, cell wall biosynthesis, plant hormone signal transduction and disease resistance genes. Finally, the expression of 13 DEGs with putative roles in S. scitamineum resistance were confirmed by quantitative real-time reverse transcription PCR (qRT-PCR) analysis, and the results were consistent with the RNA-seq data. These results highlight that the early sugarcane response to S. scitamineum infection is complex and many of the disease response genes are attenuated in sugarcane cultivar CP74-2005, while others, like genes involved in the phenylpropanoid pathway, are induced. This may point to the role of the different disease resistance mechanisms that operate in cultivars such as CP74-2005, whereby the early response is dominated by external mechanisms and then as the infection progresses, the internal mechanisms are switched on. Identification of genes underlying resistance in sugarcane will increase our knowledge of the sugarcane-S. scitamineum interaction and facilitate the introgression of new resistance genes into commercial sugarcane cultivars. [ABSTRACT FROM AUTHOR]

Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients’ cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism whereby telomerase deficiency and subsequent shortened telomeres initiate a DDR and create a pro-oxidant environment, especially in cells carrying the TINF2 mutations. Finally, the ameliorative effects of antioxidants in vitro suggest this could translate to therapeutic benefits in DC patients. [ABSTRACT FROM AUTHOR]

Background: Small cell lung cancer (SCLC) is an extremely aggressive disease, commonly displaying therapy-resistant relapse. We have previously identified neuroendocrine and epithelial phenotypes in SCLC tumours and the neuroendocrine marker, pro-opiomelanocortin (POMC), correlated with worse overall survival in patients. However, the effect of treatment on these phenotypes is not understood. The current study aimed to determine the effect of repeated irradiation treatment on SCLC cell phenotype, focussing on the neuroendocrine marker, POMC. Results: Human SCLC cells (DMS 79) were established as subcutaneous xenograft tumours in CBA nude mice and then exposed to repeated 2Gy irradiation. In untreated animals, POMC in the blood closely mirrored tumour growth; an ideal characteristic for a circulating biomarker. Following repeated localised irradiation in vivo, circulating POMC decreased (p< 0.01), in parallel with a decrease in tumour size, but remained low even when the tumours re-established. The excised tumours displayed reduced and distinctly heterogeneous expression of POMC compared to untreated tumours. There was no difference in the epithelial marker, cytokeratin. However, there were significantly more N-cadherin positive cells in the irradiated tumours. To investigate the tumour response to irradiation, DMS79 cells were repeatedly irradiated in vitro and the surviving cells selected. POMC expression was reduced, while mesenchymal markers N-cadherin, β1-integrin, fibroblast-specific protein 1, β-catenin and Zeb1 expression were amplified in the more irradiation-primed cells. There were no consistent changes in epithelial marker expression. Cell morphology changed dramatically with repeatedly irradiated cells displaying a more elongated shape, suggesting a switch to a more mesenchymal phenotype. Conclusions: In summary, POMC biomarker expression and secretion were reduced in SCLC tumours which regrew after irradiation and in repeatedly irradiation (irradiation-primed) cells. Therefore, POMC was no longer predictive of tumour burden. This highlights the importance of fully evaluating biomarkers during and after therapy to assess clinical utility. Furthermore, the gain in mesenchymal characteristics in irradiated cells could be indicative of a more invasive phenotype. [ABSTRACT FROM AUTHOR]

The fish subfamily Poeciliinae (sensu Parenti, 1981) is widely distributed across the Western Hemisphere and a dominant component of the fish communities of Central America. Poeciliids have figured prominently in previous studies on the roles of dispersal and vicariance in shaping current geographic distributions. Most recently, Hrbek et al. combined a DNA-based phylogeny of the family with geological models to provide a biogeographic perspective that emphasized the role of both vicariance and dispersal. Here we expand on that effort with a database enlarged in the quantity of sequence represented per species, in the number of species included, and in an enlarged and more balanced representation of the order Cyprinodontiformes. We combine a robust timetree based upon multiple fossil calibrations with enhanced biogeographic analyses that include ancestral area reconstructions to provide a detailed biogeographic history of this clade. Key features of our results are that the family originated in South America, but its major diversification dates to a later colonization of Central America. We also resolve additional colonizations among South, Central and North America and the Caribbean and consider how this reconstruction contributes to our understanding of the mechanisms of dispersal. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine the effect of a 40% high-fat diet (HFD) supplemented with a dietary attainable level of quercetin (0.02%) on body composition, adipose tissue (AT) inflammation, Non-Alcoholic Fatty-Liver Disease (NAFLD), and metabolic outcomes. Diets were administered for 16 weeks to C57BL/6J mice (n = 10/group) beginning at 4 weeks of age. Body composition and fasting blood glucose, insulin, and total cholesterol concentrations were examined intermittently. AT and liver mRNA expression (RT-PCR) of inflammatory mediators (F4/80, CD206 (AT only), CD11c (AT only) TLR-2 (AT only), TLR-4 (AT only), MCP-1, TNF-α, IL-6 (AT only), and IL-10 (AT only)) were measured along with activation of NFκB-p65, and JNK (western blot). Hepatic lipid accumulation, gene expression (RT-PCR) of hepatic metabolic markers (ACAC1, SREBP-1, PPAR-γ), protein content of Endoplasmic Reticulum (ER) Stress markers (BiP, phosphorylated and total EIF2α, phosphorylated and total IRE1α, CHOP), and hepatic oxidative capacity were assessed (western blot). Quercetin administration had no effect at mitigating increases in visceral AT, AT inflammation, hepatic steatosis, ER Stress, decrements in hepatic oxidative capacity, or the development of insulin resistance and hypercholesterolemia. In conclusion, 0.02% quercetin supplementation is not an effective therapy for attenuating HFD-induced obesity development. It is likely that a higher dose of quercetin supplementation is needed to elicit favorable outcomes in obesity. [ABSTRACT FROM AUTHOR]

Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an evolutionarily conserved calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor. While an important intracellular signaling protein for CD117+ mast cells (MCs), its roles in other immune cells is less clear. In this study, we identified a subset of in vivo-differentiated splenic CD117+ dendritic cells (DCs) in wild-type (WT) C57BL/6 mice that unexpectedly contained RasGRP4 mRNA and protein. In regard to the biologic significance of these data to innate immunity, LPS-treated splenic CD117+ DCs from WT mice induced natural killer (NK) cells to produce much more interferon-γ (IFN-γ) than comparable DCs from RasGRP4-null mice. The ability of LPS-responsive MCs to cause NK cells to increase their expression of IFN-γ was also dependent on this intracellular signaling protein. The discovery that RasGRP4 is required for CD117+ MCs and DCs to optimally induce acute NK cell-dependent immune responses to LPS helps explain why this signaling protein has been conserved in evolution. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband’s fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1. Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication. [ABSTRACT FROM AUTHOR]

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10–100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years. [ABSTRACT FROM AUTHOR]

To prevent the global health burdens of human immunodeficiency virus [HIV] and unintended/mistimed pregnancies, we developed an intravaginal ring [IVR] that delivers tenofovir [TFV] at ~10mg/day alone or with levonorgestrel [LNG] at ~20μg/day for 90 days. We present safety, pharmacokinetics, pharmacodynamics, acceptability and drug release data in healthy women. CONRAD A13-128 was a randomized, placebo controlled phase I study. We screened 86 women; 51 were randomized to TFV, TFV/LNG or placebo IVR [2:2:1] and 50 completed all visits, using the IVR for approximately 15 days. We assessed safety by adverse events, colposcopy, vaginal microbiota, epithelial integrity, mucosal histology and immune cell numbers and phenotype, cervicovaginal [CV] cytokines and antimicrobial proteins and changes in systemic laboratory measurements, and LNG and TFV pharmacokinetics in multiple compartments. TFV pharmacodynamic activity was measured by evaluating CV fluid [CVF] and tissue for antiviral activity using in vitro models. LNG pharmacodynamic assessments were timed based on peak urinary luteinizing hormone levels. All IVRs were safe with no significant colposcopic, mucosal, immune and microbiota changes and were acceptable. Among TFV containing IVR users, median and mean CV aspirate TFV concentrations remained above 100,000 ng/mL 4 hours post IVR insertion and mean TFV-diphosphate [DP] concentrations in vaginal tissue remained above 1,000 fmol/mg even 3 days post IVR removal. CVF of women using TFV-containing IVRs completely inhibited [94–100%] HIV infection in vitro. TFV/LNG IVR users had mean serum LNG concentrations exceeding 300 pg/mL within 1 hour, remaining high throughout IVR use. All LNG IVR users had a cervical mucus Insler score <10 and the majority [95%] were anovulatory or had abnormal cervical mucus sperm penetration. Estimated in vivo TFV and LNG release rates were within expected ranges. All IVRs were safe with the active ones delivering sustained high concentrations of TFV locally. LNG caused changes in cervical mucus, sperm penetration, and ovulation compatible with contraceptive efficacy. The TFV and TFV/LNG rings are ready for expanded 90 day clinical testing. Trial registration ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

The transcriptome can reveal insights into precancer biology. We recently conducted RNA-Seq analysis on liver RNA from male rats exposed to the carcinogen, aflatoxin B1 (AFB1), for 90 days prior to liver tumor onset. Among >1,000 differentially expressed transcripts, several novel, unannotated Cufflinks-assembled transcripts, or HAfTs (epatic latoxin ranscripts) were found. We hypothesized PCR-cloning and RACE (rapid amplification of cDNA ends) could further HAfT identification. Sanger data was obtained for 6 transcripts by PCR and 16 transcripts by 5’- and 3’-RACE. BLAST alignments showed, with two exceptions, HAfT transcripts were lncRNAs, >200nt without apparent long open reading frames. Six rat HAfT transcripts were classified as ‘novel’ without RefSeq annotation. Sequence alignment and genomic synteny showed each rat lncRNA had a homologous locus in the mouse genome and over half had homologous loci in the human genome, including at least two loci (and possibly three others) that were previously unannotated. While HAfT functions are not yet clear, coregulatory roles may be possible from their adjacent orientation to known coding genes with altered expression that include 8 HAfT-gene pairs. For example, a unique rat HAfT, homologous to Pvt1, was adjacent to known genes controlling cell proliferation. Additionally, PCR and RACE Sanger sequencing showed many alternative splice variants and refinements of exon sequences compared to Cufflinks assembled transcripts and gene prediction algorithms. Presence of multiple splice variants and short tandem repeats found in some HAfTs may be consequential for secondary structure, transcriptional regulation, and function. In summary, we report novel, differentially expressed lncRNAs after exposure to the genotoxicant, AFB1, prior to neoplastic lesions. Complete cloning and sequencing of such transcripts could pave the way for a new set of sensitive and early prediction markers for chemical hepatocarcinogens. [ABSTRACT FROM AUTHOR]

FKBP5 is a critical component of the Hypothalamic-Pituitary-Adrenal (HPA) axis, a system which regulates our response to stress. It forms part of a complex of chaperones, which inhibits binding of cortisol and glucocorticoid receptor translocation to the nucleus. Variations in both the HPA axis and FKBP5 have been associated with suicidal behavior. We developed a systematic, targeted sequencing approach to investigate coding and regulatory regions in or near FKBP5 in 476 bipolar disorder suicide attempters and 473 bipolar disorder non-attempters. Following stringent quality control checks, we performed single-variant, gene-level and haplotype tests on the resulting 481 variants. Secondary analyses investigated whether sex-specific variations in FKBP5 increased the risk of attempted suicide. One variant, rs141713011, showed an excess of minor alleles in suicide attempters that was statistically significant following correction for multiple testing (Odds Ratio = 6.65, P-value = 7.5 x 10−4, Permuted P-value = 0.038). However, this result could not be replicated in an independent cohort (Odds Ratio = 0.90, P-value = 0.78). Three female-specific and four male-specific variants of nominal significance were also identified (P-value < 0.05). The gene-level and haplotype association tests did not produce any significant results. This comprehensive study of common and rare variants in FKBP5 focused on both regulatory and coding regions in relation to attempted suicide. One rare variant remained significant following correction for multiple testing but could not be replicated. Further investigation is required in larger sample sets to fully elucidate the association of this variant with suicidal behavior. [ABSTRACT FROM AUTHOR]

A number of HIV-1 subtypes are identified in Pakistan by characterization of partial viral gene sequences. Little is known whether new recombinants are generated and how they disseminate since whole genome sequences for these viruses have not been characterized. Near full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes or next generation sequencing from 34 HIV-1-infected individuals in Pakistan. Phylogenetic tree analysis showed that the newly characterized sequences were 16 subtype As, one subtype C, and 17 A/G recombinants. Further analysis showed that all 16 subtype A1 sequences (47%), together with the vast majority of sequences from Pakistan from other studies, formed a tight subcluster (A1a) within the subtype A1 clade, suggesting that they were derived from a single introduction. More in-depth analysis of 17 A/G NFLG sequences showed that five shared similar recombination breakpoints as in CRF02 (15%) but were phylogenetically distinct from the prototype CRF02 by forming a tight subcluster (CRF02a) while 12 (38%) were new recombinants between CRF02a and A1a or a divergent A1b viruses. Unique recombination patterns among the majority of the newly characterized recombinants indicated ongoing recombination. Interestingly, recombination breakpoints in these CRF02/A1 recombinants were similar to those in prototype CRF02 viruses, indicating that recombination at these sites more likely generate variable recombinant viruses. The dominance and fast dissemination of new CRF02a/A1 recombinants over prototype CRF02 suggest that these recombinant have more adapted and may become major epidemic strains in Pakistan. [ABSTRACT FROM AUTHOR]

Obesity is one of the dramatic health issues affecting developed and developing nations, and exercise is a well-established intervention strategy. While exercise-by-genotype interactions have been shown in humans, overall little is known. Using the natural negative geotaxis of Drosophila melanogaster, an important model organism for the study of genetic interactions, a novel exercise machine, the TreadWheel, can be used to shed light on this interaction. The mechanism for inducing exercise with the TreadWheel is inherently gentle, thus minimizing possible confounding effects of other stressors. Using this machine, we were able to assess large cohorts of adult flies from eight genetic lines for their response to exercise after one week of training. We measured their triglyceride, glycerol, protein, glycogen, glucose content, and body weight, as well as their climbing ability and feeding behavior in response to exercise. Exercised flies showed decreased stored triglycerides, glycogen, and body weight, and increased stored protein and climbing ability. In addition to demonstrating an overall effect of TreadWheel exercise on flies, we found significant interactions of exercise with genotype, sex, or genotype-by-sex effects for most of the measured phenotypes. We also observed interaction effects between exercise, genotype, and tissue (abdomen or thorax) for metabolite profiles, and those differences can be partially linked to innate differences in the flies' persistence in maintaining activity during exercise bouts. In addition, we assessed gene expression levels for a panel of 13 genes known to be associated with respiratory fitness and found that many responded to exercise. With this study, we have established the TreadWheel as a useful tool to study the effect of exercise in flies, shown significant genotype-specific and sex-specific impacts of exercise, and have laid the ground work for more extensive studies of how genetics, sex, environment, and aging interact with exercise to influence metabolic fitness in Drosophila. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptor 4 (TLR4) is one of the best known TLR members expressed on the surface of several leukocytes and tissue cells and has a key function in detecting pathogen and danger-associated molecular patterns. The role of TLR4 in the pathophysiology of several age-related diseases is also well recognized, such as prostate cancer (PCa). TLR4 polymorphisms have been related to PCa risk, but the relationship between TLR4 genotypes and aggressive PCa risk has not been evaluated by any systematic reviews. Methods: We performed a systematic review and meta-analysis of candidate-gene and genome-wide association studies analyzing this relationship and included only white population. Considering appropriate criteria, only nine studies were analyzed in the meta-analysis, including 3,937 aggressive PCa and 7,382 controls. Results: Using random effects model, no significant association was found in the ten TLR4 SNPs reported by at least four included studies under any inheritance model (rs2737191, rs1927914, rs10759932, rs1927911, rs11536879, rs2149356, rs4986790, rs11536889, rs7873784, and rs1554973). Pooled estimates from another ten TLR4 SNPs reported by three studies also showed no significant association (rs10759930, rs10116253, rs11536869, rs5030717, rs4986791, rs11536897, rs1927906, rs913930, rs1927905, and rs7045953). Meta-regression revealed that study type was not a significant source of between-study heterogeneity. Conclusions: TLR4 polymorphisms were not significantly associated with the risk of aggressive PCa. [ABSTRACT FROM AUTHOR]

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer’s disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer’s disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid. [ABSTRACT FROM AUTHOR]

Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings' history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ∼3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463–65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of ≤1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined. [ABSTRACT FROM AUTHOR]