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Faithful meiotic chromosome segregation and fertility require meiotic recombination between homologous chromosomes rather than the equally available sister chromatid, a bias that in Saccharomyces cerevisiae depends on the meiotic kinase, Mek1. Mek1 is thought to mediate repair template bias by specifically suppressing sister-directed repair. Instead, we found that when Mek1 persists on closely paired (synapsed) homologues, DNA repair is severely delayed, suggesting that Mek1 suppresses any proximal repair template. Accordingly, Mek1 is excluded from synapsed homologues in wild-type cells. Exclusion requires the AAA+-ATPase Pch2 and is directly coupled to synaptonemal complex assembly. Stage-specific depletion experiments further demonstrate that DNA repair in the context of synapsed homologues requires Rad54, a repair factor inhibited by Mek1. These data indicate that the sister template is distinguished from the homologue primarily by its closer proximity to inhibitory Mek1 activity. We propose that once pairing or synapsis juxtaposes homologues, exclusion of Mek1 is necessary to avoid suppression of all templates and accelerate repair progression. [ABSTRACT FROM AUTHOR]

Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates. [ABSTRACT FROM AUTHOR]

In humans, meiotic chromosome segregation errors increase dramatically as women age, but the molecular defects responsible are largely unknown. Cohesion along the arms of meiotic sister chromatids provides an evolutionarily conserved mechanism to keep recombinant chromosomes associated until anaphase I. One attractive hypothesis to explain agedependent nondisjunction (NDJ) is that loss of cohesion over time causes recombinant homologues to dissociate prematurely and segregate randomly during the first meiotic division. Using Drosophila as a model system, we have tested this hypothesis and observe a significant increase in meiosis I NDJ in experimentally aged Drosophila oocytes when the cohesin protein SMC1 is reduced. Our finding that missegregation of recombinant homologues increases with age supports the model that chiasmata are destabilized by gradual loss of cohesion over time. Moreover, the stage at which Drosophila oocytes are most vulnerable to age-related defects is analogous to that at which human oocytes remain arrested for decades. Our data provide the first demonstration in any organism that, when meiotic cohesion begins intact, the aging process can weaken it sufficiently and cause missegregation of recombinant chromosomes. One major advantage of these studies is that we have reduced but not eliminated the SMC1 subunit. Therefore, we have been able to investigate how aging affects normal meiotic cohesion. Our findings that recombinant chromosomes are at highest risk for loss of chiasmata during diplotene argue that human oocytes are most vulnerable to age-induced loss of meiotic cohesion at the stage at which they remain arrested for several years. [ABSTRACT FROM AUTHOR]

The decline in dengue incidence and/or prevalence during the COVID-19 pandemic (2020-22) appears to be attributed to reduced treatment-seeking rates, under-reporting, misdiagnosis, disrupted health services and reduced exposure to mosquito vectors due to prevailing lockdowns. There is limited scientific data on dengue virus (DENV) disease during the COVID-19 pandemic. Here, we conducted a community-based, cross-sectional, cluster-randomized survey to assess anti-DENV and anti-SARS-CoV-2 seroprevalence, and also estimated the spatial distribution of DENV-positive aedine mosquito vectors during the COVID-19 pandemic across all the 38 districts of Tamil Nadu, India. Using real-time PCR, the prevalence of DENV in mosquito pools during 2021 was analyzed and compared with the previous and following years of vector surveillance, and correlated with anti-DENV IgM and IgG levels in the population. Results implicate that both anti-DENV IgM and IgG seroprevalence and DENV positivity in mosquito pools were reduced across all the districts. A total of 13464 mosquito pools and 5577 human serum samples from 186 clusters were collected. Of these, 3.76% of the mosquito pools were positive for DENV. In the human sera, 4.12% were positive for anti-DENV IgM and 6.4% for anti-DENV IgG. While the anti-SARS-CoV-2 levels significantly correlated with overall DENV seropositivity, COVID-19 vaccination status significantly correlated with anti-DENV IgM levels. The study indicates a profound impact of anti-SARS-CoV-2 levels on DENV-positive mosquito pools and seropositivity. Continuous monitoring of anti-DENV antibody levels, especially with the evolving variants of SARS-CoV-2 and the surge in COVID-19 cases will shed light on the distribution, transmission and therapeutic attributes of DENV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Selvavinayagam et al. This 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.)

Introduction: While a number of studies have examined the nutritional impacts of agroecological interventions, few have examined impacts on child development, maternal and child anemia, and men's dietary diversity. Moreover, there have been few such evaluations at scale. We evaluated the impact of a large-scale, multi-component food-based nutrition intervention involving homestead food production, nutrition counselling, cooking demonstrations, and crop planning exercises., Methods: A cross-sectional assessment was conducted in 2021-2022 of 50 intervention villages where the nutrition-sensitive agroecology program had been implemented since 2018 and 79 control villages where only the agroecology program had been implemented. Data on self-reported dietary intake, caregiver-reported early child development, anthropometric measurements, and hemoglobin concentrations were collected using standardized procedures by trained Nutrition Farming Fellows, who were also responsible for implementing the program., Results: A sample of 3,511 households (1,121 intervention and 2,390 control) participated in the survey. Dietary diversity scores (DDS) among women and men were mean (SD) 6.53 (±1.62) and 6.16 (±1.65), respectively, in intervention villages and 5.81 (±1.58) and 5.39 (±1.61), respectively, in control villages (p<0.01). DDS among children 6-24 months of age in intervention and control villages was 2.99 (±1.52) and 2.73 (±1.62), respectively (p<0.01). Children <2 years of age were less likely to be anemic in intervention versus control villages (59% versus 69%, p<0.01). Children 18-35 months age in intervention villages had higher child development scores than children in control villages (all p<0.05)., Conclusion: Nutrition-sensitive agroecological programs may be effective in improving diets, nutrition, and child development in rural India., Competing Interests: LD, VK, VJ, VC and SY were affiliates of the government organization responsible for implementing the program evaluated in this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Ch et al. This 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.)

Candida albicans form biofilm by associating with biotic and abiotic surfaces. Biofilm formation by C. albicans is relevant and significant as the organisms residing within, gain resistance to conventional antimycotics and are therefore difficult to treat. This study targeted the potential of spice-based antimycotics to control C. albicans biofilms. Ten clinical isolates of C. albicans along with a standard culture MTCC-3017 (ATCC-90028) were screened for their biofilm-forming ability. C. albicans M-207 and C. albicans S-470 were identified as high biofilm formers by point inoculation on Trypticase Soy Agar (TSA) medium as they formed a lawn within 16 h and exhibited resistance to fluconazole and caspofungin at 25 mcg and 8 mcg respectively. Aqueous and organic spice extracts were screened for their antimycotic activity against C. albicans M-207 and S-470 by agar and disc diffusion and a Zone of Inhibition was observed. Minimal Inhibitory Concentration was determined based on growth absorbance and cell viability measurements. The whole aqueous extract of garlic inhibited biofilms of C. albicans M-207, whereas whole aqueous extracts of garlic, clove, and Indian gooseberry were effective in controlling C. albicans S-470 biofilm within 12 h of incubation. The presence of allicin, ellagic acid, and gallic acid as dominant compounds in the aqueous extracts of garlic, clove, and Indian gooseberry respectively was determined by High-Performance Thin Layer Chromatography and Liquid Chromatography-Mass Spectrometry. The morphology of C. albicans biofilm at different growth periods was also determined through bright field microscopy, phase contrast microscopy, and fluorescence microscopy. The results of this study indicated that the alternate approach in controlling high biofilm-forming, multi-drug resistant clinical isolates of C. albicans M-207 and S-470 using whole aqueous extracts of garlic, clove, and Indian gooseberry is a safe, potential, and cost-effective one that can benefit the health care needs with additional effective therapeutics to treat biofilm infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sadanandan et al. This 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.)

Background: We retrospectively data-mined the case records of Reverse Transcription Polymerase Chain Reaction (RT-PCR) confirmed COVID-19 patients hospitalized to a tertiary care centre to derive mortality predictors and formulate a risk score, for prioritizing admission., Methods and Findings: Data on clinical manifestations, comorbidities, vital signs, and basic lab investigations collected as part of routine medical management at admission to a COVID-19 tertiary care centre in Chengalpattu, South India between May and November 2020 were retrospectively analysed to ascertain predictors of mortality in the univariate analysis using their relative difference in distribution among 'survivors' and 'non-survivors'. The regression coefficients of those factors remaining significant in the multivariable logistic regression were utilised for risk score formulation and validated in 1000 bootstrap datasets. Among 746 COVID-19 patients hospitalised [487 "survivors" and 259 "non-survivors" (deaths)], there was a slight male predilection [62.5%, (466/746)], with a higher mortality rate observed among 40-70 years age group [59.1%, (441/746)] and highest among diabetic patients with elevated urea levels [65.4% (68/104)]. The adjusted odds ratios of factors [OR (95% CI)] significant in the multivariable logistic regression were SaO2<95%; 2.96 (1.71-5.18), Urea ≥50 mg/dl: 4.51 (2.59-7.97), Neutrophil-lymphocytic ratio (NLR) >3; 3.01 (1.61-5.83), Age ≥50 years;2.52 (1.45-4.43), Pulse Rate ≥100/min: 2.02 (1.19-3.47) and coexisting Diabetes Mellitus; 1.73 (1.02-2.95) with hypertension and gender not retaining their significance. The individual risk scores for SaO2<95-11, Urea ≥50 mg/dl-15, NLR >3-11, Age ≥50 years-9, Pulse Rate ≥100/min-7 and coexisting diabetes mellitus-6, acronymed collectively as 'OUR-ARDs score' showed that the sum of scores ≥ 25 predicted mortality with a sensitivity-90%, specificity-64% and AUC of 0.85., Conclusions: The 'OUR ARDs' risk score, derived from easily assessable factors predicting mortality, offered a tangible solution for prioritizing admission to COVID-19 tertiary care centre, that enhanced patient care but without unduly straining the health system., Competing Interests: The authors have declared that no competing interests exist.

BCG vaccination is known to induce innate immune memory, which confers protection against heterologous infections. However, the effect of BCG vaccination on the conventional adaptive immune cells subsets is not well characterized. We investigated the impact of BCG vaccination on the frequencies of T cell subsets and common gamma c (γc) cytokines in a group of healthy elderly individuals (age 60-80 years) at one month post vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrate that BCG vaccination induced enhanced frequencies of central (p<0.0001) and effector memory (p<0.0001) CD4+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001), stem cell memory (p = 0.0001) CD4+ T cells and regulatory T cells. In addition, BCG vaccination induced enhanced frequencies of central (p = 0.0008), effector (p<0.0001) and terminal effector memory (p<0.0001) CD8+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001) and stem cell memory (p = 0.0034) CD8+T cells. BCG vaccination also induced enhanced plasma levels of IL-7 (p<0.0001) and IL-15 (p = 0.0020) but diminished levels of IL-2 (p = 0.0033) and IL-21 (p = 0.0020). Thus, BCG vaccination was associated with enhanced memory T cell subsets as well as memory enhancing γc cytokines in elderly individuals, suggesting its ability to induce non-specific adaptive immune responses., Competing Interests: The authors have declared that no competing interests exist.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Though immune checkpoint inhibitors (ICIs) have revolutionized lung cancer therapy in recent years, there are several factors limiting the therapeutic efficacy of ICI-based immunotherapy in lung cancer. Recent evidence suggests that one such mechanism is the phenotypic shift of tumor-infiltrating macrophages away from an anti-tumor M1 phenotype and towards an anti-inflammatory and tumor-permissive M2 phenotype. Though this phenomenon is well documented, the means through which the lung tumor microenvironment (TME) usurps macrophage function are poorly described. Hepatocyte growth factor (HGF) is a known driver of both lung cancer pathobiology as well as M2 polarization, and its signaling is antagonized by the tumor suppressor gene HAI-1 (SPINT1). Using a combination of genomic databases, primary NSCLC specimens, and in vitro models, we determined that patients with loss of HAI-1 have a particularly poor prognosis, hallmarked by increased HGF expression and an M2-dominant immune infiltrate. Similarly, conditioned media from HAI-1-deficient tumor cells led to a loss of M1 and increased M2 polarization in vitro, and patient NSCLC tissues with loss of HAI-1 showed a similar loss of M1 macrophages. Combined, these results suggest that loss of HAI-1 is a potential means through which tumors acquire an immunosuppressive, M2-dominated TME, potentially through impaired M1 macrophage polarization. Hence, HAI-1 status may be informative when stratifying patients that may benefit from therapies targeting the HGF pathway, particularly as an adjuvant to ICI-based immunotherapy., Competing Interests: The authors have declared that no competing interests exist.

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart, small intestine and dorsal skin were collected for tissue analysis. Female APCHi mice showed the most severe radiation-induced deposition of cardiac collagens but were protected against a radiation-induced loss of microvascular density. Both male and female APCHi mice were protected against a radiation induced upregulation of toll-like receptor 4 in the heart, but this did not translate into a clear protection against immune cell infiltration. In the small intestine, the APCHi genotype had no effect on an increase in the number of myeloperoxidase positive cells (seen mostly in females) or an increase in the expression of T-cell marker CD2 (males). Lastly, both male and female APCHi mice were protected against radiation-induced epidermal thickening and increase in 3-nitrotyrosine positive keratinocytes. In conclusion, prolonged high levels of APC in a transgenic mouse model had little effects on indicators of DEARE in the heart, small intestine and skin, with some differential effects in male compared to female mice., Competing Interests: The authors have declared that no competing interests exist.

Karnataka, a state in south India, reported its first case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection on March 8, 2020, more than a month after the first case was reported in India. We used a combination of contact tracing and genomic epidemiology to trace the spread of SARS-CoV-2 in the state up until May 21, 2020 (1578 cases). We obtained 91 genomes of SARS-CoV-2 which clustered into seven lineages (Pangolin lineages-A, B, B.1, B.1.80, B.1.1, B.4, and B.6). The lineages in Karnataka were known to be circulating in China, Southeast Asia, Iran, Europe and other parts of India and are likely to have been imported into the state both by international and domestic travel. Our sequences grouped into 17 contact clusters and 24 cases with no known contacts. We found 14 of the 17 contact clusters had a single lineage of the virus, consistent with multiple introductions and most (12/17) were contained within a single district, reflecting local spread. In most of the 17 clusters, the index case (12/17) and spreaders (11/17) were symptomatic. Of the 91 sequences, 47 belonged to the B.6 lineage, including eleven of 24 cases with no known contact, indicating ongoing transmission of this lineage in the state. Genomic epidemiology of SARS-CoV-2 in Karnataka suggests multiple introductions of the virus followed by local transmission in parallel with ongoing viral evolution. This is the first study from India combining genomic data with epidemiological information emphasizing the need for an integrated approach to outbreak response., Competing Interests: FH is an employee of TruFactor-InMobi group company, however, he was permitted to participate as a volunteer in this study and his employer neither had access to data, nor any say in the design of the study or the decision to publish. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors are employees of state (PD, PK) or central government. The employers had no role in the design of the study or the decision to publish. The authors declare that they do not have any other financial or non-financial relationships that could present a conflict of interest.

During deep space missions, astronauts will be exposed to low doses of charged particle irradiation. The long-term health effects of these exposures are largely unknown. We previously showed that low doses of oxygen ion (16O) irradiation induced acute damage to the hematopoietic system, including hematopoietic progenitor and stem cells in a mouse model. However, the chronic effects of low dose 16O irradiation remain undefined. In the current study, we investigated the long-term effects of low dose 16O irradiation on the mouse hematopoietic system. Male C57BL/6J mice were exposed to 0.05 Gy, 0.1 Gy, 0.25 Gy and 1.0 Gy whole body 16O (600 MeV/n) irradiation. The effects of 16O irradiation on bone marrow (BM) hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) were examined three months after the exposure. The results showed that the frequencies and numbers of BM HPCs and HSCs were significantly reduced in 0.1 Gy, 0.25 Gy and 1.0 Gy irradiated mice compared to 0.05 Gy irradiated and non-irradiated mice. Exposure of mice to low dose 16O irradiation also significantly reduced the clongenic function of BM HPCs determined by the colony-forming unit assay. The functional defect of irradiated HSCs was detected by cobblestone area-forming cell assay after exposure of mice to 0.1 Gy, 0.25 Gy and 1.0 Gy of 16O irradiation, while it was not seen at three months after 0.5 Gy and 1.0 Gy of γ-ray irradiation. These adverse effects of 16O irradiation on HSCs coincided with an increased intracellular production of reactive oxygen species (ROS). However, there were comparable levels of cellular apoptosis and DNA damage between irradiated and non-irradiated HPCs and HSCs. These data suggest that exposure to low doses of 16O irradiation induces long-term hematopoietic injury, primarily via increased ROS production in HSCs.

Purpose: Space travel is associated with an exposure to low-dose rate ionizing radiation and the microgravity environment, both of which may lead to impairments in cardiac function. We used a mouse model to determine short- and long-term cardiac effects to simulated microgravity (hindlimb unloading; HU), continuous low-dose rate γ-irradiation, or a combination of HU and low-dose rate γ-irradiation., Methods: Cardiac tissue was obtained from female, C57BL/6J mice 7 days, 1 month, 4 months, and 9 months following the completion of a 21 day exposure to HU or a 21 day exposure to low-dose rate γ-irradiation (average dose rate of 0.01 cGy/h to a total of 0.04 Gy), or a 21 day simultaneous exposure to HU and low-dose rate γ-irradiation. Immunoblot analysis, rt-PCR, high-performance liquid chromatography, and histology were used to assess inflammatory cell infiltration, cardiac remodeling, oxidative stress, and the methylation potential of cardiac tissue in 3 to 6 animals per group., Results: The combination of HU and γ-irradiation demonstrated the strongest increase in reduced to oxidized glutathione ratios 7 days and 1 month after treatment, but a difference was no longer apparent after 9 months. On the other hand, no significant changes in 4-hydroxynonenal adducts was seen in any of the groups, at the measured endpoints. While manganese superoxide dismutase protein levels decreased 9 months after low-dose γ-radiation, no changes were observed in expression of catalase or Nrf2, a transcription factor that determines the expression of several antioxidant enzymes, at the measured endpoints. Inflammatory marker, CD-2 protein content was significantly decreased in all groups 4 months after treatment. No significant differences were observed in α-smooth muscle cell actin protein content, collagen type III protein content or % total collagen., Conclusions: This study has provided the first and relatively broad analysis of small molecule and protein markers of oxidative stress, T-lymphocyte infiltration, and cardiac remodeling in response to HU with simultaneous exposure to low-dose rate γ-radiation. Results from the late observation time points suggest that the hearts had mostly recovered from these two experimental conditions. However, further research is needed with larger numbers of animals for a more robust statistical power to fully characterize the early and late effects of simulated microgravity combined with exposure to low-dose rate ionizing radiation on the heart.

Background: Chikungunya virus (CHIKV), a reemerging pathogen causes a self limited illness characterized by fever, headache, myalgia and arthralgia. However, 10-20% affected individuals develop persistent arthralgia which contributes to considerable morbidity. The exact molecular mechanisms underlying these manifestations are not well understood. The present study investigated the possible occurrence of molecular mimicry between CHIKV E1 glycoprotein and host human components., Methodology: Bioinformatic tools were used to identify peptides of CHIKV E1 exhibiting similarity to host components. Two peptides (A&B) were identified using several bioinformatic tools, synthesised and used to validate the results obtained in silico. An ELISA was designed to assess the immunoreactivity of serum samples from CHIKV patients to these peptides. Further, experiments were conducted in a C57BL/6J experimental mouse model to investigate if peptide A and peptide B were indeed capable of inducing pathology., Findings: The serum samples showed reactivity of varying degrees, indicating that these peptides are indeed being recognized by the host immune system during CHIKV infection. Further, these peptides when injected into C57BL/6J mice were able to induce significant inflammation in the muscles of C57BL/6J mice, similar to that observed in animals that were injected with CHIKV alone. Additionally, animals that were primed initially with CHIKV followed by a subsequent injection of the CHIKV peptides exhibited enhanced inflammatory pathology in the skeletal muscles as compared to animals that were injected with peptides or virus alone. Collectively these observations validate the hypothesis that molecular mimicry between CHIKV E1 protein and host proteins does contribute to pathology in CHIKV infection., Competing Interests: The authors have declared that no competing interests exist.

Offspring of older mothers are at increased risk of adverse birth outcomes, childhood cancers, type 1 diabetes, and neurodevelopmental disorders. The underlying biologic mechanisms for most of these associations remain obscure. One possibility is that maternal aging may produce lasting changes in the epigenetic features of a child's DNA. To test this, we explored the association of mothers' age at pregnancy with methylation in her offspring, using blood samples from 890 Norwegian newborns and measuring DNA methylation at more than 450,000 CpG sites across the genome. We examined replication of a maternal-age finding in an independent group of 1062 Norwegian newborns, and then in 200 US middle-aged women. Older maternal age was significantly associated with reduced methylation at four adjacent CpGs near the 2nd exon of KLHL35 in newborns (p-values ranging from 3x10-6 to 8x10-7). These associations were replicated in the independent set of newborns, and replicated again in women 40 to 60 years after their birth. This study provides the first example of parental age permanently affecting the epigenetic profile of offspring. While the specific functions of the affected gene are unknown, this finding opens the possibility that a mother's age at pregnancy could affect her child's health through epigenetic mechanisms.

One of the major health risks to astronauts is radiation on long-duration space missions. Space radiation from sun and galactic cosmic rays consists primarily of 85% protons, 14% helium nuclei and 1% high-energy high-charge (HZE) particles, such as oxygen (16O), carbon, silicon, and iron ions. HZE particles exhibit dense linear tracks of ionization associated with clustered DNA damage and often high relative biological effectiveness (RBE). Therefore, new knowledge of risks from HZE particle exposures must be obtained. In the present study, we investigated the acute effects of low doses of 16O irradiation on the hematopoietic system. Specifically, we exposed C57BL/6J mice to 0.1, 0.25 and 1.0 Gy whole body 16O (600 MeV/n) irradiation and examined the effects on peripheral blood (PB) cells, and bone marrow (BM) hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) at two weeks after the exposure. The results showed that the numbers of white blood cells, lymphocytes, monocytes, neutrophils and platelets were significantly decreased in PB after exposure to 1.0 Gy, but not to 0.1 or 0.25 Gy. However, both the frequency and number of HPCs and HSCs were reduced in a radiation dose-dependent manner in comparison to un-irradiated controls. Furthermore, HPCs and HSCs from irradiated mice exhibited a significant reduction in clonogenic function determined by the colony-forming and cobblestone area-forming cell assays. These acute adverse effects of 16O irradiation on HSCs coincided with an increased production of reactive oxygen species (ROS), enhanced cell cycle entry of quiescent HSCs, and increased DNA damage. However, none of the 16O exposures induced apoptosis in HSCs. These data suggest that exposure to low doses of 16O irradiation induces acute BM injury in a dose-dependent manner primarily via increasing ROS production, cell cycling, and DNA damage in HSCs. This finding may aid in developing novel strategies in the protection of the hematopoietic system from space radiation.

Oral Tongue Squamous cell carcinoma (OTSCC), the most frequently affected oral cancer sub-site, is associated with a poor therapeutic outcome and survival despite aggressive multi- modality management. Till date, there are no established biomarkers to indicate prognosis and outcome in patients presenting with tongue cancer. There is an urgent need for reliable molecular prognostic factors to enable identification of patients with high risk of recurrence and treatment failure in OTSCC management. In the current study, we present the meta-analysis of OTSCC microarray based gene expression profiles, deriving a comprehensive molecular portrait of tongue cancer biology, showing the relevant genes and pathways which can be pursued further to derive novel, tailored therapeutics as well as for prognostication. We have studied 5 gene expression profiling data sets available on exclusively oral tongue subsite comprising of sample size; n = 190, consisting of 111 tumors and 79 normals. The meta- analysis results showed 2405 genes differentially regulated comparing OTSCC tumor and normal. The top up regulated genes were found to be involved in Extracellular matrix degradation (ECM) and Epithelial to mesenchymal transition (EMT) pathways. The top down regulated genes were found to be involved in detoxication pathways. We validated the results in clinical samples (n = 206), comprising of histologically normals (n = 10), prospective (n = 29) and retrospective (n = 167) OTSCC by evaluating MMP9 and E-cadherin gene expression by qPCR and immunohistochemistry. Consistent with meta-analysis results, MMP9 mRNA expression was significantly up regulated in OTSCC primary tumors compared to normals. MMP9 protein over expression was found to be a significant predictor of poor prognosis, disease recurrence and poor Disease Free Survival (DFS) in OTSCC patients. Analysis by univariate and multivariate Cox proportional hazard model showed patients with loss of E-cadherin expression in OTSCC tumors having a poorer DFS (HR = 1.566; P value = 0.045) and poorer Overall Survival (OS) (HR = 1.224; P value = 0.003) respectively. Combined over-expression of MMP9 and loss of E-cadherin membrane positivity in the invasive tumor front (ITF) of OTSCC had a significant association with poorer DFS (Log Rank = 16.040; P value = 0.001). These results suggest that along with known clinical indicators of prognosis like occult node positivity, assessment of MMP9 and E-cadherin expression at ITF can be useful to identify patients at high risk and requiring a more intensive treatment strategy for OTSCC. Meta-analysis study of gene expression profiles indicates that OTSCC is a disease of ECM degradation leading to activated EMT processes implying the aggressive nature of the disease. The triggers for these processes should be studied further. Newer clinical application with agents that can inhibit the mediators of ECM degradation may be a key to achieving clinical control of invasion and metastasis of OTSCC.

The pleiotropic role of human secretin (hSCT) validates its potential use as a therapeutic agent. Nevertheless, the structure of secretin in complex with its receptor is necessary to develop a suitable therapeutic agent. Therefore, in an effort to design a three-dimensional virtual homology model and identify a peptide agonist and/or antagonist for the human secretin receptor (hSR), the significance of the primary sequence of secretin peptides in allosteric binding and activation was elucidated using virtual docking, FRET competitive binding and assessment of the cAMP response. Secretin analogs containing various N- or C-terminal modifications were prepared based on previous findings of the role of these domains in receptor binding and activation. These analogs exhibited very low or no binding affinity in a virtual model, and were found to neither exhibit in vitro binding nor agonistic or antagonistic properties. A parallel analysis of the analogs in the virtual model and in vitro studies revealed instability of these peptide analogs to bind and activate the receptor.

Background: Existing reading schemes for chest X-ray (CXR) used to grade the extent of disease severity at diagnosis in patients with pulmonary tuberculosis (PTB) are often based on numerical scores that summate specific radiographic features. However, since PTB is known to exhibit a wide heterogeneity in pathology, certain features might be differentially associated with clinical parameters of disease severity., Objective: We aimed to grade disease severity in PTB patients at diagnosis and after completion of DOTS treatment by developing a reading scheme based on five different radiographic manifestations and analyze their association with the clinical parameters of systemic involvement and infectivity., Methods: 141 HIV-negative adults with newly diagnosed sputum smear-positive PTB were enrolled in a prospective observational study in Hyderabad, India. The presence and extent on CXRs of five radiographic manifestations, i.e., lung involvement, alveolar infiltration, cavitation, lymphadenopathy and pleural effusion, were classified using the new reading scheme by using a four-quadrant approach. We evaluated the inter-reader reliability of each manifestation, and its association with BMI and sputum smear positivity at diagnosis. The presence and extent of these radiographic manifestations were further compared with CXRs on completion of DOTS treatment., Results: At diagnosis, an average lung area of 51.7% +/- 23.3% was affected by radiographic abnormalities. 94% of the patients had alveolar infiltrates, with 89.4% located in the upper quadrants, suggesting post primary PTB and in 34.8% of patients cavities were found. We further showed that the extent of affected lung area was a negative predictor of BMI (β value -0.035, p 0.019). No significant association of BMI with any of the other CXR features was found. The extent of alveolar infiltrates, along with the presence of cavitation, were strongly associated with sputum smear positivity. The microbiological cure rate in our cohort after 6 months of DOTS treatment was 95%. The extent of the affected lung area in these patients decreased from 56.0% +/- 21.5% to 31.0 +/- 20% and a decrease was also observed in the extent of alveolar infiltrates from 98.4% to 25.8% in at least one quadrant, presence of cavities from 34.8% to 1.6%, lymphadenopathy from 46.8% to 16.1%, and pleural effusion from 19.4% to 6.5%., Conclusions: We established a new assessment scheme for grading disease severity in PTB by specifically considering five radiographic manifestations which were differently associated with the BMI and sputum smear positivity, changed to a different extent after 6 months of treatment and exhibited an excellent agreement between radiologists. Our results suggest that this reading scheme might contribute to the estimation of disease severity with respect to differences in disease pathology. Further studies are needed to determine a correlation with short and long-term pulmonary function impairment and whether there would be any benefit in lengthening or modulating therapy based on this CXR severity assessment.

Background: Household contacts of diagnostically established tuberculosis (TB) patients are highly susceptible to disease development. It is surmised that cytokines perhaps play a synergistic and a prognostic role in the activation of the otherwise latent infection in these house hold contacts. Evaluation of the cytokines and any of their inherent polymorphisms might provide a useful diagnostic tool in evaluating the immune regulation and the progression of the disease. The cytokines thus released in a paracrine manner in serum may also provide an indirect measure of the cytokine function., Objective: The present study was aimed to evaluate the levels of TNF-α, IL-10 & IL-6 cytokines and their correlation with genotype variants amongst tuberculosis patients and their household contacts., Methods: The cytokine levels were estimated in serum by enzyme-linked immunosorbent assay (ELISA) and their polymorphisms were studied by amplification refractory mutation system polymerase chain reaction (ARMs PCR) in active pulmonary tuberculosis patients (APTB = 150), household contacts (HHC = 190), and healthy controls (HC = 150)., Results: The median values of TNF-α cytokine were significantly high among APTB and HHC compared to HCs (P< 0.0001 and 0.0001). IL-6 levels also were elevated among APTB compared to HHC and HC, and a significant difference was observed between APTB and HHC at P<0.0001; APTB & HC at P< 0.04; HHC & HC at P< 0.01. The IL-10 levels were low in APTB compared to HHC and HCs and no significant difference was observed. TNF-α/IL-10 ratio was significant and indicated Th1 predominance in APTB and HHC. IL-6/IL-10 showed pronounced Th1 expression in APTB and Th2 in HHC and HC. The ROC analysis indicated that both IL-10 and IL-6 can be used to decide the risk of exposed individual to a disease. The results of multivariate analysis indicate that IL-10 (-1082) GA genotype was significantly associated with p<0.028 in APTB. No significant association was observed between genotypes, other serum cytokine levels and clinical characteristics between APTB, HHC and HCs., Conclusion: Large sample size with follow-up at different time points may further illuminate the role of IL-10 and IL-6 cytokines as a prognostic marker in house hold contacts.

In a heterogeneous wireless network, handover techniques are designed to facilitate anywhere/anytime service continuity for mobile users. Consistent best-possible access to a network with widely varying network characteristics requires seamless mobility management techniques. Hence, the vertical handover process imposes important technical challenges. Handover decisions are triggered for continuous connectivity of mobile terminals. However, bad network selection and overload conditions in the chosen network can cause fallout in the form of handover failure. In order to maintain the required Quality of Service during the handover process, decision algorithms should incorporate intelligent techniques. In this paper, a new and efficient vertical handover mechanism is implemented using a dynamic programming method from the operation research discipline. This dynamic programming approach, which is integrated with the Technique to Order Preference by Similarity to Ideal Solution (TOPSIS) method, provides the mobile user with the best handover decisions. Moreover, in this proposed handover algorithm a deterministic approach which divides the network into zones is incorporated into the network server in order to derive an optimal solution. The study revealed that this method is found to achieve better performance and QoS support to users and greatly reduce the handover failures when compared to the traditional TOPSIS method. The decision arrived at the zone gateway using this operational research analytical method (known as the dynamic programming knapsack approach together with Technique to Order Preference by Similarity to Ideal Solution) yields remarkably better results in terms of the network performance measures such as throughput and delay.

In utero exposure to diethylstilbestrol (DES) has been associated with increased risk of adverse health outcomes such as fertility problems and vaginal as well as breast cancer. Animal studies have linked prenatal DES exposure to lasting DNA methylation changes. We investigated genome-wide DNA methylation and in utero DES exposure in a sample of non-Hispanic white women aged 40-59 years from the Sister Study, a large United States cohort study of women with a family history of breast cancer. Using questionnaire information from women and their mothers, we selected 100 women whose mothers reported taking DES while pregnant and 100 control women whose mothers had not taken DES. DNA methylation in blood was measured at 485,577 CpG sites using the Illumina HumanMethylation450 BeadChip. Associations between CpG methylation and DES exposure status were analyzed using robust linear regression with adjustment for blood cell composition and multiple comparisons. Although four CpGs had p<105, after accounting for multiple comparisons using the false discovery rate (FDR), none reached genome-wide significance. In conclusion, adult women exposed to DES in utero had no evidence of large persistent changes in blood DNA methylation.

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

Breast cancer is the most common malignancy and the leading cause of cancer deaths in women worldwide. While specific genetic mutations have been linked to 5-10% of breast cancer cases, other environmental and epigenetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive breast cancer molecular profile is needed to develop more effective target therapies. Until recently, identifying genetic cancer mutations via personalized DNA sequencing was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 105 human breast cancer samples. The sequencing analysis revealed missense mutations in PIK3CA, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

Insulin resistance is a characteristic feature of Type 2 diabetes. Insulin resistance has also been implicated in the pathogenesis of cardiovascular disease. Currently used thiazolidinedione (TZD) insulin sensitizers although effective, have adverse side effects of weight gain, fluid retention and heart failure. Using fat cell-based phenotypic drug discovery approach we identified P1736, a novel antidiabetic molecule that has completed Phase II clinical trials. The present study evaluated the in vitro and in vivo pharmacological properties of P1736. P1736 is a non-TZD and it did not activate human PPAR(Peroxisome Proliferator Activated Receptor Gamma )receptors. P1736 caused dose dependent increase in glucose uptake (EC50-400 nM) in the insulin resistant 3T3 adipocytes. The compound (10 µM) induced translocation of GLUT-4 (Glucose Transporter type 4) transporters in these adipocytes while metformin (1.0mM) was inactive. In diabetic db/db mice, P1736 (150 mg/kg) was more efficacious than metformin in lowering plasma glucose (35% vs 25%) and triglyceride levels (38% vs 31%). P1736 tested at 5mg/kg, twice daily doses, reduced glucose by 41% and triglycerides by 32%, in db/db mice. These effects were not associated with adverse effects on body weight or liver function. Rosiglitazone (5mg/kg, twice daily) caused 60% and 40 % decreases in glucose and triglyceride levels, respectively. However, rosiglitazone induced 13% weight gain (p<0.05) in db/db mice. P1736 was also efficacious in ob/ob mice wherein 30-35% decrease in glucose and significant improvement in hyperinsulinemia were observed. Administration of P1736 to ob/ob mice resulted in 70% increase in glucose uptake in soleus muscles while metformin caused 38% increase. P1736 exhibited excellent safety profile and was weight neutral in all preclinical models of diabetes. Thus, P1736 with its unique pharmacology coupled with PPAR- independent mode of action could represent an alternative option in the management of insulin resistant Type 2 diabetic patients.

Background: Leber congenital amaurosis (LCA) is the most severe form of inherited retinal visual impairment in children. So far, mutations in more than 20 genes have been known to cause LCA and among them, RPE65 is a suitable candidate for gene therapy. The mutational screenings of RPE65 and other LCA genes are requisite in support of emerging gene specific therapy for LCA. Therefore, we have carried out a comprehensive LCA genes screening using a combined approach of direct sequencing and DNA microarray based Asper chip analysis., Methodology/principal Findings: Thirty clinically diagnosed index LCA cases from Southern India were screened for coding and flanking intronic regions of RPE65 through direct sequencing. Among thirty, 25 cases excluded from RPE65 mutations were subjected to Asper chip analysis, testing 784 known pathogenic variations in 15 major LCA genes. In RPE65 screening, four different pathogenic variations including two novel (c.361insT & c.939T>A) and two known (c.394G>A & c.361delT) mutations were identified in five index cases. In the chip analysis, seven known pathogenic mutations were identified in six index cases, involving genes GUCY2D, RPGRIP1, AIPL1, CRX and IQCB1. Overall, 11 out of 30 LCA cases (36.6%) revealed pathogenic variations with the involvement of RPE65 (16.6%), GUCY2D (10%), RPGRIP1 (3.3%), AIPL1 (3.3%) and CRX & IQCB1 (3.3%)., Conclusions/significance: Our study suggests that such combined screening approach is productive and cost-effective for mutation detection and can be applied in Indian LCA cohort for molecular diagnosis and genetic counselling.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.

Radiation therapy in the treatment of cancer is dose limited by radiation injury in normal tissues such as the intestine and the heart. To identify the mechanistic involvement of transforming growth factor-beta 1 (TGF-β1) in intestinal and cardiac radiation injury, we studied the influence of pharmacological induction of TGF-β1 with xaliproden (SR 57746A) in rat models of radiation enteropathy and radiation-induced heart disease (RIHD). Because it was uncertain to what extent TGF-β induction may enhance radiation injury in heart and intestine, animals were exposed to irradiation schedules that cause mild to moderate (acute) radiation injury. In the radiation enteropathy model, male Sprague-Dawley rats received local irradiation of a 4-cm loop of rat ileum with 7 once-daily fractions of 5.6 Gy, and intestinal injury was assessed at 2 weeks and 12 weeks after irradiation. In the RIHD model, male Sprague-Dawley rats received local heart irradiation with a single dose of 18 Gy and were followed for 6 months after irradiation. Rats were treated orally with xaliproden starting 3 days before irradiation until the end of the experiments. Treatment with xaliproden increased circulating TGF-β1 levels by 300% and significantly induced expression of TGF-β1 and TGF-β1 target genes in the irradiated intestine and heart. Various radiation-induced structural changes in the intestine at 2 and 12 weeks were significantly enhanced with TGF-β1 induction. Similarly, in the RIHD model induction of TGF-β1 augmented radiation-induced changes in cardiac function and myocardial fibrosis. These results lend further support for the direct involvement of TGF-β1 in biological mechanisms of radiation-induced adverse remodeling in the intestine and the heart.

Radiation-induced heart disease (RIHD) is a long-term side effect of radiotherapy of intrathoracic, chest wall and breast tumors when radiation fields encompass all or part of the heart. Previous studies have shown that pentoxifylline (PTX) in combination with α-tocopherol reduced manifestations of RIHD in rat models of local heart irradiation. The relative contribution of PTX and α-tocopherol to these beneficial effects are not known. This study examined the effects of PTX alone or in combination with tocotrienols, forms of vitamin E with potential potent radiation mitigation properties. Rats received localized X-irradiation of the heart with an image-guided irradiation technique. At 3 months after irradiation rats received oral treatment with vehicle, PTX, or PTX in combination with a tocotrienol-enriched formulation. At 6 months after irradiation, PTX-treated rats showed arrhythmia in 5 out of 14 animals. PTX alone or in combination with tocotrienols did not alter cardiac radiation fibrosis, left ventricular protein expression of the endothelial markers von Willebrand factor and neuregulin-1, or phosphorylation of the signal mediators Akt, Erk1/2, or PKCα. On the other hand, tocotrienols reduced cardiac numbers of mast cells and macrophages, but enhanced the expression of tissue factor. While this new rat model of localized heart irradiation does not support the use of PTX alone, the effects of tocotrienols on chronic manifestations of RIHD deserve further investigation.

Unlike other metazoan mRNAs, replication-dependent histone gene transcripts are not polyadenylated but instead have a conserved stem-loop structure at their 3' end. Our previous work has shown that under certain conditions replication-dependent histone genes can produce alternative transcripts that are polyadenylated at the 3' end and, in some cases, spliced. A number of microarray studies examining the expression of polyadenylated mRNAs identified changes in the levels of histone transcripts e.g. during differentiation and tumorigenesis. However, it remains unknown which histone genes produce polyadenylated transcripts and which conditions regulate this process. In the present study we examined the expression and polyadenylation of the human histone H2B gene complement in various cell lines. We demonstrate that H2B genes display a distinct expression pattern that is varies between different cell lines. Further we show that the fraction of polyadenylated HIST1H2BD and HIST1H2AC transcripts is increased during differentiation of human mesenchymal stem cells (hMSCs) and human fetal osteoblast (hFOB 1.19). Furthermore, we observed an increased fraction of polyadenylated transcripts produced from the histone genes in cells following ionizing radiation. Finally, we show that polyadenylated transcripts are transported to the cytoplasm and found on polyribosomes. Thus, we propose that the production of polyadenylated histone mRNAs from replication-dependent histone genes is a regulated process induced under specific cellular circumstances.

Background: Development of model systems have helped to a large extent, in bridging gap to understand the mechanism(s) of disease including diabetes. Interestingly, WNIN/GR-Ob rats (Mutants), established at National Centre for Laboratory Animals (NCLAS) of National Institute of Nutrition (NIN), form a suitable model system to study obesity with Type 2 diabetes (T2D) demonstrating several secondary complications (cataract, cardiovascular complications, infertility, nephropathy etc). The present study has been carried out to explore the potent application(s) of multipotent stem cells such as bone marrow mesenchymal stem cells (BM-MSCs), to portray features of pre-diabetic/T2D vis-à-vis featuring obesity, with impaired glucose tolerance (IGT), hyperinsulinemia (HI) and insulin resistance (IR) seen with Mutant rats akin to human situation., Methodology/principal Findings: Primary cultures of BM-MSCs (third passage) from Mutants, its lean littermate (Lean) and parental control (Control) were characterized for: proliferation markers, disease memory to mark obesity/T2D/HI/IR which included phased gene expression studies for adipogenic/pancreatic lineages, inflammatory markers and differentiation ability to form mature adipocytes/Insulin-like cellular aggregates (ILCAs). The data showed that BM-MSCs from Mutant demonstrated a state of disease memory, depicted by an upregulated expression of inflammatory markers (IL-6, TNFα); increased stem cell recruitment (Oct-4, Sox-2) and proliferation rates (CD90+/CD29+, PDA, 'S' phase of cell cycle by FACS and BrdU incorporation); accelerated preadipocyte induction (Dact-1, PPARγ2) with a quantitative increase in mature adipocyte formation (Leptin); ILCAs, which were non-responsive to high glucose did confer the Obese/T2D memory in Mutants. Further, these observations were in compliance with the anthropometric data., Conclusions: Given the ease of accessibility and availability of MSCs, the present study form the basis to report for the first time, application of BM-MSCs as a feasible in vitro model system to portray the disease memory of pre-clinical/T2D with IR - a major metabolic disorder of global concern.

Background: Japanese encephalitis (JE), the most common form of viral encephalitis occurs periodically in endemic areas leading to high mortality and neurological deficits in survivors. It is caused by a flavivirus, Japanese encephalitis virus (JEV), which is transmitted to humans through mosquitoes. No effective cure exists for reducing mortality and morbidity caused by JEV infection, which is primarily due to excessive inflammatory response. Fenofibrate, a peroxisome proliferator-activated receptor-α (PPARα) agonist is known to resolve inflammation by repressing nuclear factor-κB (NF-κB) and enhancing transcription of anti-oxidant and anti-inflammatory genes. In addition, fenofibrate also up-regulates a class of proteins, cytochrome P4504Fs (Cyp4fs), which are involved in detoxification of the potent pro-inflammatory eicosanoid, leukotriene B(4) (LTB(4)) to 20-hydroxy LTB(4)., Methodology/principal Findings: The neuroprotective effect of fenofibrate was examined using in vitro (BV-2 microglial cell line) and in vivo (BALB/c mice) models of JEV infection. Mice were treated with fenofibrate for 2 or 4 days prior to JEV exposure. Pretreatment with fenofibrate for 4 but not 2 days reduced mortality by 80% and brain LTB(4) levels decreased concomitantly with the induction of Cyp4f15 and 4f18, which catalyze detoxification of LTB(4) through hydroxylation. Expression of cytokines and chemokine decreased significantly as did microglial activation and replication of the JEV virus., Conclusions/significance: Fenofibrate confers neuroprotection against Japanese encephalitis, in vivo, in mouse model of JEV infection. Thus, fenofibrate, a PPARα agonist that is commonly used as a hypolipidemic drug could potentially be used for prophylaxis during JE epidemics to reduce mortality and morbidity.

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1(-) CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and "ancestral," were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.

The enzyme Polyphosphate Kinase (PPK) catalyses the reversible transfer of the terminal γ-Pi of ATP to form a long chain Polyphosphate (PolyP). Using an IPTG inducible mycobacterial vector, the vulnerability of this gene has been evaluated by antisense knockdown experiments in M. tuberculosis. Expression profiling studies point to the fact that down regulation of PPK caused cidality during the late phase in contrast to its bacteriostatic mode immediately following antisense expression. PPK thus seems to be a suitable anti-tubercular drug target. The enzyme which is a tetramer has been cloned in E. coli and purified to homogeneity. An enzyme assay suitable for High Throughput Screening was optimized by using the statistical Taguchi protocol and the kinetic parameters determined. The enzyme displayed a strong product inhibition by ADP. In order to accurately estimate the product inhibition, progress curve analysis of the enzyme reaction was monitored. The kinetic equation describing the progress curve was suitably modified by taking into account the product inhibition. The reversible nature of the enzyme indicated a possibility of a two way ATP↔ADP switch operating in the bacteria as a response to its growth requirement.

Background: We previously identified a panel of genes associated with outcome of ovarian cancer. The purpose of the current study was to assess whether variants in these genes correlated with ovarian cancer risk., Methods and Findings: Women with and without invasive ovarian cancer (749 cases, 1,041 controls) were genotyped at 136 single nucleotide polymorphisms (SNPs) within 13 candidate genes. Risk was estimated for each SNP and for overall variation within each gene. At the gene-level, variation within MSL1 (male-specific lethal-1 homolog) was associated with risk of serous cancer (p = 0.03); haplotypes within PRPF31 (PRP31 pre-mRNA processing factor 31 homolog) were associated with risk of invasive disease (p = 0.03). MSL1 rs7211770 was associated with decreased risk of serous disease (OR 0.81, 95% CI 0.66-0.98; p = 0.03). SNPs in MFSD7, BTN3A3, ZNF200, PTPRS, and CCND1A were inversely associated with risk (p<0.05), and there was increased risk at HEXIM1 rs1053578 (p = 0.04, OR 1.40, 95% CI 1.02-1.91)., Conclusions: Tumor studies can reveal novel genes worthy of follow-up for cancer susceptibility. Here, we found that inherited markers in the gene encoding MSL1, part of a complex that modifies the histone H4, may decrease risk of invasive serous ovarian cancer.

Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, alpha-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of beta-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.