Computational interrogation of cis-regulatory elements of genes that are common targets of luteotropin and luteolysin in the primate corpus luteum

The rapid recent increase in microarray-based gene expression studies in the corpus luteum (CL) utilizing macaque models gathered increasing volume of data in publically accessible microarray expression databases. Examining gene pathways in different functional states of CL may help to understand the factors that control luteal function and hence human fertility. Co-regulation of genes in microarray experiments may imply common transcriptional regulation by sequence-specific DNA-binding transcriptional factors. We have computationally analyzed the transcription factor binding sites (TFBS) in a previously reported macaque luteal microarray gene set (n = 15) that are common targets of luteotropin (luteinizing hormone (LH) and human chorionic gonadotropin (hCG)) and luteolysin (prostaglandin (PG) F-2 alpha). This in silico approach can reveal transcriptional networks that control these important genes which are representative of the interplay between luteotropic and luteolytic factors in the control of luteal function. Our computational analyses revealed 6 matrix families whose binding sites are significantly over-represented in promoters of these genes. The roles of these factors are discussed, which might help to understand the transcriptional regulatory network in the control of luteal function. These factors might be promising experimental targets for investigation of human luteal insufficiency. (C) 2012 Elsevier B.V. All rights reserved.

Exploration of deleterious single nucleotide polymorphisms in the components of human P bodies: An in silico approach

P bodies are 100-300 nm sized organelles involved in mRNA silencing and degradation. A total of 60 human proteins have been reported to localize to P bodies. Several human SNPs contribute to complex diseases by altering the structure and function of the proteins. Also, SNPs alter various transcription factors binding, splicing and miRNA regulatory sites. Owing to the essential functions of P bodies in mRNA regulation, we explored computationally the functional significance of SNPs in 7 P body components such as XRN1, DCP2, EDC3, CPEB1, GEMIN5, STAU1 and TRIM71. Computational analyses of non-synonymous SNPs of these components was initiated using well utilized publicly available software programs such as the SIFT, followed by PolyPhen, PANTHER, MutPred, I-Mutant-2.0 and PhosSNP 1.0. Functional significance of noncoding SNPs in the regulatory regions were analysed using FastSNP. Utilizing miRSNP database, we explored the role of SNPs in the context that alters the miRNA binding sites in the above mentioned genes. Our in silico studies have identified various deleterious SNPs and this cataloguing is essential and gives first hand information for further analysis by in vitro and in vivo methods for a better understanding of maintenance, assembly and functional aspects of P bodies in both health and disease. (C) 2013 Elsevier B.V. All rights reserved.

Polyvinylidene fluoride film based nasal sensor to monitor human respiration pattern: An initial clinical study

Design and development of a piezoelectric polyvinylidene fluoride (PVDF) thin film based nasal sensor to monitor human respiration pattern (RP) from each nostril simultaneously is presented in this paper. Thin film based PVDF nasal sensor is designed in a cantilever beam configuration. Two cantilevers are mounted on a spectacle frame in such a way that the air flow from each nostril impinges on this sensor causing bending of the cantilever beams. Voltage signal produced due to air flow induced dynamic piezoelectric effect produce a respective RP. A group of 23 healthy awake human subjects are studied. The RP in terms of respiratory rate (RR) and Respiratory air-flow changes/alterations obtained from the developed PVDF nasal sensor are compared with RP obtained from respiratory inductance plethysmograph (RIP) device. The mean RR of the developed nasal sensor (19.65 +/- A 4.1) and the RIP (19.57 +/- A 4.1) are found to be almost same (difference not significant, p > 0.05) with the correlation coefficient 0.96, p < 0.0001. It was observed that any change/alterations in the pattern of RIP is followed by same amount of change/alterations in the pattern of PVDF nasal sensor with k = 0.815 indicating strong agreement between the PVDF nasal sensor and RIP respiratory air-flow pattern. The developed sensor is simple in design, non-invasive, patient friendly and hence shows promising routine clinical usage. The preliminary result shows that this new method can have various applications in respiratory monitoring and diagnosis.

Evaluation of Brugia malayi sheath protein (Shp-1) as a diagnostic antigen for human lymphatic filariasis

Lymphatic filariasis is the second leading cause of permanent long-term disability globally and control of this disease needs efficient diagnostic methods. In this study, abundantly expressing microfilarial sheath protein (Shp-1) from Brugia malayi was characterized as a filarial diagnostic candidate using samples from different clinical population. Monoclonal antibodies were developed against E. coil expressed recombinant Shp-1 in order to assess its efficiency in filarial antigen detection assay system. Endemic Normal (EN, n = 170), asymptomatic microfilaeremics (MF, n = 65), symptomatic chronic pathology (CP, n = 45) and non endemic normal (NEN, n = 10) sera were analyzed by antigen capture enzyme-linked immunosorbent assay. Of the 290 individuals, all MF individuals (both brugian and bancroftian) were positive in this assay followed by CP and EN. When compared with SXP-1 and Og4C3 antigen assays, all assays detected Wb MF correctly, Bm MF was detected by Shp-1 and SXP-1 assays, and only Shp-1 was able to detect EN (12%) and CP (29%). Results showed that this assay may be useful for monitoring prior to mass drug administration. (c) 2014 Elsevier Inc. All rights reserved.

Quantifying Vocal Mimicry in the Greater Racket-Tailed Drongo: A Comparison of Automated Methods and Human Assessment

Objective identification and description of mimicked calls is a primary component of any study on avian vocal mimicry but few studies have adopted a quantitative approach. We used spectral feature representations commonly used in human speech analysis in combination with various distance metrics to distinguish between mimicked and non-mimicked calls of the greater racket-tailed drongo, Dicrurus paradiseus and cross-validated the results with human assessment of spectral similarity. We found that the automated method and human subjects performed similarly in terms of the overall number of correct matches of mimicked calls to putative model calls. However, the two methods also misclassified different subsets of calls and we achieved a maximum accuracy of ninety five per cent only when we combined the results of both the methods. This study is the first to use Mel-frequency Cepstral Coefficients and Relative Spectral Amplitude - filtered Linear Predictive Coding coefficients to quantify vocal mimicry. Our findings also suggest that in spite of several advances in automated methods of song analysis, corresponding cross-validation by humans remains essential.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Modeling non-stationarity in intensity, duration and frequency of extreme rainfall over India

Significant changes are reported in extreme rainfall characteristics over India in recent studies though there are disagreements on the spatial uniformity and causes of trends. Based on recent theoretical advancements in the Extreme Value Theory (EVT), we analyze changes in extreme rainfall characteristics over India using a high-resolution daily gridded (1 degrees latitude x 1 degrees longitude) dataset. Intensity, duration and frequency of excess rain over a high threshold in the summer monsoon season are modeled by non-stationary distributions whose parameters vary with physical covariates like the El-Nino Southern Oscillation index (ENSO-index) which is an indicator of large-scale natural variability, global average temperature which is an indicator of human-induced global warming and local mean temperatures which possibly indicate more localized changes. Each non-stationary model considers one physical covariate and the best chosen statistical model at each rainfall grid gives the most significant physical driver for each extreme rainfall characteristic at that grid. Intensity, duration and frequency of extreme rainfall exhibit non-stationarity due to different drivers and no spatially uniform pattern is observed in the changes in them across the country. At most of the locations, duration of extreme rainfall spells is found to be stationary, while non-stationary associations between intensity and frequency and local changes in temperature are detected at a large number of locations. This study presents the first application of nonstationary statistical modeling of intensity, duration and frequency of extreme rainfall over India. The developed models are further used for rainfall frequency analysis to show changes in the 100-year extreme rainfall event. Our findings indicate the varying nature of each extreme rainfall characteristic and their drivers and emphasize the necessity of a comprehensive framework to assess resulting risks of precipitation induced flooding. (C) 2014 Elsevier B.V. All rights reserved.

Remarkable visible light-triggered cytotoxicity of mitochondria targeting mixed-ligand cobalt(III) complexes of curcumin and phenanthroline bases binding to human serum albumin

Six new mixed-ligand cobalt(III) complexes of formulation Co(N-N)(2)(O-O)](ClO4)(2) (1-6), where N-N is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 2), dipyrido3,2-d:2',3'-f] quinoxaline (dpq in 3, 4), and dipyrido3,2-a:2',3'-c]phenazine (dppz in 5, 6), O-O is acetylacetonate (acac in 1, 3, 5) or curcumin (bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione, cur in 2, 4, 6), have been synthesized and characterized. The X-ray crystal structures of complex 1 (as PF6- salt, 1a) and 3 show distorted octahedral geometries formed by the CoN4O2 core. The complexes 1, 3 and 5 having the simple acac ligand are prepared as control species to understand the role of curcumin. The optimized geometries and the frontier orbitals of the curcumin complexes 2, 4, and 6 are obtained from the DFT calculations. The complexes 2, 4, and 6 having the photoactive curcumin moiety display an absorption band in the visible region near 420 nm and show remarkable photocytotoxicity in HeLa cancer cells with respective IC50 values of 7.4 mu M, 5.1 mu M and 1.6 mu M while being much less toxic in dark. MTT assay using complex 6 shows that it is not significantly photocytotoxic to MCF-10A normal cells. The control complexes having the acac ligand are non-toxic both in the presence and absence of light. The cell death is apoptotic in nature and triggered by the photogeneration of reactive oxygen species. Fluorescence imaging experiments on HeLa cells reveals that complex 6 accumulated primarily inside the mitochondria. Human serum albumin (HSA) binding experiments show that the complexes bind HSA with good affinity, but 6 binds with the highest affinity, with a K-b value of 9.8 x 10(5) M-1. Thus, complex 6 with its negligible toxicity in the dark and in normal cells but remarkable toxicity in visible light holds significant photochemotherapeutic potential.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-beta

Actions of transforming growth factor-beta are largely context dependent. For instance, TGF-beta is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-beta also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-beta would change in the context of transformation, we have compared the differential gene regulation by TGF-beta in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-beta in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-beta is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-beta. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-beta in hFhTERT cells and TGF-beta conferred action of these cells was mediated by WNT4. While TGF-beta activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-beta which matched with the response of HT1080 cells to TGF-beta. These data suggest context dependent activation of non-canonical signaling by TGF-beta. (C) 2015 Published by Elsevier Inc.

Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8(+) T Cells

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+) and CD8(+) T cells secreting gamma interferon (IFN-gamma) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.