👤 Chitrangda Srivastava

Various pathways and cytokines are implicated in pathogenesis of diabetic macular edema (DME). Computational imaging biomarkers (CIBs) of vessel tortuosity from ultra-widefield fluorescein angiography (UWFA) and texture patterns from OCT images have been associated with anti-vascular endothelial growth factor (VEGF) therapy treatment response in DME. This analysis was a radiogenomic assessment of the association between underlying cytokines, UWFA, and OCT-based DME CIBs. Biclustering analysis based on UWFA and OCT CIBs to identify a common imaging phenotype across patients with subsequent assessment of underlying cytokine signatures and treatment response attributes. The IMAGINE DME study was a post hoc study of cytokine expressions that included 24 eyes with sufficient baseline aqueous humor samples and an in-depth assessment of the imaging studies obtained during the phase I/II DmeAntiVEgf study (DAVE) that measured different cytokine expressions. A total of 151 graph or morphologic features quantifying leakage shape, size, density, interobject distance, and architecture of leakage spots and 5 vessel tortuosity features were extracted from the baseline UWFA scans, and 494 texture-based radiomics features were extracted from each of the fluid and retinal tissue compartments of OCT images. Biclustering enables simultaneous clustering of patients and features and was used to aggregate patients in terms of their commonality of phenotypes (based on similar imaging attributes) and to identify commonality in terms of cytokine expression and treatment response to anti-VEGF therapy. Identification of eyes with similar imaging phenotypes to evaluate commonalities of patterns and underlying cytokine expression. Strong correlations between VEGF and 7 UWFA leakage morphologic features (Pearson correlation coefficient [PCC], 0.45-0.51; This study identified groups of eyes with similar imaging phenotypes as defined by UWFA and OCT CIBs that demonstrated similar treatment response patterns and cytokine expression, including a strong association between VEGF with UWFA-derived leakage morphologic and vessel tortuosity features. Show less

Venous thrombo-embolism (VTE) is multi-factorial disease involving several genetic and acquired risk factors responsible for its onset. It may occur spontaneously upon climbing at High Altitude (HA). Several studies demonstrated that hypoxic conditions prevailing at HA pose an independent risk factor for VTE; however, molecular mechanism remains unknown. Present study aims to identify genes associated with HA-induced VTE pathophysiology using real time TaqMan Low-Density Array (TLDA) of known candidate genes. Gene expression of total 93 genes were studied and analyzed in patients of VTE from HA (HA-VTE) and from sea level (SL-VTE) in comparison to respective controls. Both HA-VTE and SL-VTE patients showed up-regulation of 37 genes involved in blood coagulation cascade, clot formation, platelet formation, endothelial response, angiogenesis, cell adhesion and calcium channel activity. Seven genes including ACE, EREG, C8A, DLG2, USF1, F2 and PCDHA7 were up-regulated in both HA-controls and VTE patients (both HA-VTE and SL-VTE) indicating their role during VTE event and also upon HA exposure. Ten genes; CDH18, FGA, EDNBR, GATA2, MAPK9, BCAR1, FRK, F11, PCDHA1 and ST8SIA4 were uniquely up-regulated in HA-VTE. The differentially expressed genes from the present study could be determining factors for HA-VTE susceptibility and provide insights into VTE occurrence at HA. Show less

Interleukin-27 (IL-27) is an immunoregulatory cytokine that suppresses inflammation through multiple mechanisms, including induction of IL-10, but the transcriptional network mediating its diverse functions remains unclear. Combining temporal RNA profiling with computational algorithms, we predict 79 transcription factors induced by IL-27 in T cells. We validate 11 known and discover 5 positive (Cebpb, Fosl2, Tbx21, Hlx, and Atf3) and 2 negative (Irf9 and Irf8) Il10 regulators, generating an experimentally refined regulatory network for Il10. We report two central regulators, Prdm1 and Maf, that cooperatively drive the expression of signature genes induced by IL-27 in type 1 regulatory T cells, mediate IL-10 expression in all T helper cells, and determine the regulatory phenotype of colonic Foxp3 Show less

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2. Show less

Incretin therapy is one of the most potential approaches in the treatment of diabetes. In contrast to markedly available drugs, the herbal incretin modulators have lesser side effects with low economic cost. The main aim of this work was to analyze the potential of previously reported DPPIV inhibitor, aqueous extract of Pueraria tuberosa tubers (PTY-2) as incretin hormones receptor agonist against streptozotocin (STZ)-induced diabetes. Chronic diabetes was induced with STZ (65mg/kg bw) in rats for 60days and grouped into diabetic control and PTY-2. Expression of genes was assessed by PCR, IHC, and ELISA. Morphological analysis of tissue was observed using H & E stain. In silico molecular docking approach has been used to see the interaction of active phytochemicals of PTY-2 on the basis of their binding energy [kcal/mol] and dissociation constant [pM] using YASARA software. Interactive visualization was done using Discovery studio 3.0. In comparison to diabetic control, the size and number of islet cells along with the plasma level of GLP-1, GIP, and pancreatic expressions of GLP-1R, GIP-R, Bcl2, and insulin were enhanced significantly after PTY-2 treatment. Through in silico molecular docking, tuberostan showed the best interaction for GLP-1R with binding energy at 8.15kcal/mol and dissociation constant at 1061624.125 pM. Puererone showed the best interaction for GIP-R with binding energy at 8.31kcal/mol and dissociation constant at 810381 pM. In addition to previously studied DPPIV inhibitor, PTY-2 also acts as incretin receptors agonist and protects against STZ-induced diabetes by down regulating β cells apoptosis. Show less

A number of clinical findings suggested HDL-raising as a plausible approach to treat residual risk of CVD. However, lack of CVD risk reduction by elevated HDL cholesterol (HDL-C) through cholesterol ester transfer protein (CETP) inhibition and enhanced risk reduction in apolipoprotein A-I Milano (apoAI-M) individuals with low HDL-C shifted the focus from HDL-C level to HDL function. In the present study, we investigated correlations between HDL-C, HDL function, fecal cholesterol excretion, and Show less

Coronary artery disease, the leading cause of death in the developed and developing countries, is prevalent in diabetes mellitus with 68% cardiovascular disease (CVD)-related mortality. Epidemiological studies suggested inverse correlation between HDL and CVD occurrence. Therefore, low HDL concentration observed in diabetic patients compared to non-diabetic individuals was thought to be one of the primary causes of increased risks of CVD. Efforts to raise HDL level via CETP inhibitors, Torcetrapib and Dalcetrapib, turned out to be disappointing in outcome studies despite substantial increases in HDL-C, suggesting that factors beyond HDL concentration may be responsible for the increased risks of CVD. Therefore, recent studies have focused more on HDL function than on HDL levels. The metabolic environment in diabetes mellitus condition such as hyperglycemia-induced advanced glycation end products, oxidative stress, and inflammation promote HDL dysfunction leading to greater risks of CVD. This review discusses dysfunctional HDL as one of the mechanisms of increased CVD risks in diabetes mellitus through adversely affecting components that support HDL function in cholesterol efflux and LDL oxidation. The dampening of reverse cholesterol transport, a key process that removes cholesterol from lipid-laden macrophages in the arterial wall, leads to increased risks of CVD in diabetic patients. Therapeutic approaches to keep diabetes under control may benefit patients from developing CVD. Show less

Coronary artery disease (CAD) occurring in less than 45 years of age is termed as young CAD. Recent studies show a prevalence of 1.2% of CAD cases in this age group. Ethnic wise south Asians especially Indians are more vulnerable to have CAD in young age group with a prevalence of 5% to 10%. Conventional risk factors such as smoking, diabetes, hypertension, obesity and family history seems to be as important as in older CAD subjects. But the prevalence of these risk factors seems to vary in younger subjects. By far the most commonly associated risk factor is smoking in young CAD. Several genes associated with lipoprotein metabolism are now found to be associated with young CAD like cholesterol ester transfer protein ( Show less

Mutations in sarcomeric genes are common genetic cause of cardiomyopathies. An intronic 25-bp deletion in cardiac myosin binding protein C (MYBPC3) at 3' region is associated with dilated and hypertrophic cardiomyopathies in Southeast Asia. However, the frequency of sarcomeric gene polymorphisms and associated clinical presentation have not been established with left ventricular dysfunction (LVD). Therefore, the aim of the present study was to explore the association of MYBPC3 25-bp deletion, titin (TTN) 18 bp I/D, troponin T type 2 (TNNT2) 5 bp I/D and myospryn K2906N polymorphisms with LVD. This study includes 988 consecutive patients with angiographically confirmed coronary artery disease (CAD) and 300 healthy controls. Among the 988 CAD patients, 253 with reduced left ventricle ejection fraction (LVEF≤45%) were categorized as LVD. MYBPC3 25-bp deletion, TTN 18 bp I/D and TNNT2 5 bp I/D polymorphisms were determined by direct polymerase chain reaction method, while myospryn K2906N polymorphism by TaqMan assay. Our results showed that MYBPC3 25-bp deletion polymorphism was significantly associated with elevated risk of LVD (LVEF <45) (healthy controls versus LVD: OR=3.85, P <0.001; and nonLVD versus LVD: OR=1.65, P = 0.035), while TTN 18 bp I/D, TNNT2 5 bp I/D and myospryn K2906N polymorphisms did not show any significant association with LVD. The results also showed that MYBPC3 25-bp deletion polymorphism was significantly associated with other parameters of LV remodelling, i.e. LV dimensions (LV end diastole dimension, LVEDD: P = 0.037 and LV end systolic dimension, LVESD: P = 0.032). Our data suggests that MYBPC3 25-bp deletion may play significant role in conferring LVD as well as CAD risk in north Indian population. Show less

Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor-clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures. Show less

Gallbladder cancer (GBC) is a violent neoplasm associated with late diagnosis, unsatisfactory treatment, and poor prognosis. The disease shows complex interplay between multiple genetic variants. We analyzed 15 polymorphisms in nine genes involved in various pathways to find out combinations of genetic variants contributing to GBC risk. The genes included in the study were matrix metalloproteinases (MMP-2, MMP-7, and MMP-9), tissue inhibitor of metalloproteinases (TIMP-2), cytochrome P450 (CYP)1A1, CYP1B1, phospholipase C epsilon 1 (PLCE1), liver X receptor (LXR)-alpha, and LXR-beta. Genotypes were determined by PCR-RFLP and TaqMan probes. Statistical analysis was done by SPSS version 16. Multilocus analysis was performed by Classification and Regression Tree (CART) analysis and multifactor dimensionality reduction (MDR) to gene-gene interactions in modifying GBC risk. In silico analysis was done using various bioinformatics tools (F-SNP, FAST-SNP). Single locus analysis showed association of MMP-2 (-735 C > T, -1306 C > T), MMP-7 - 181 A > G, MMP-9 (P574R, R668Q), TIMP-2 - 418 G > C, CYP1A1-MspI, CYP1A1-Ile462Val, PLCE1 (rs2274223 A > G, rs7922612 T > C) and LXR-beta T > C (rs3546355 G > A, rs2695121 T > C) polymorphisms with GBC risk (p < 0.05) whereas CYP1B1 and LXR-α variants were not associated with GBC risk. Multidimensional reduction analysis revealed LXR-β (rs3546355 G > A, rs2695121 T > C), MMP-2 (-1306 C > T), MMP-9 (R668Q), and PLCE1 rs2274223 A > G to be key players in GBC causation (p < 0.001, CVC = 7/10). The results were further supported by independent CART analysis (p < 0.001). In silico analysis of associated variants suggested change in splicing or transcriptional regulation. Interactome and STRING analysis showed network of associated genes. The study found PLCE1 and LXR-β network interactions as important contributory factors for genetic predisposition in gallbladder cancer. Show less

Leptospirosis is a zoonotic infectious disease of tropical, subtropical and temperate zones, which is caused by the pathogenic spirochetes of genus Leptospira. Although this zoonosis is generally not considered as fatal, the pathogen can eventually cause severe infection with septic shock, multi-organ failure and lethal pulmonary hemorrhages leading to mortality. In this study, we have performed a proteomic analysis of serum samples from leptospirosis patients (n=6), febrile controls (falciparum malaria) (n=8) and healthy subjects (n=18) to obtain an insight about disease pathogenesis and host immune responses in leptospiral infections. 2DE and 2D-DIGE analysis in combination with MALDI-TOF/TOF MS revealed differential expression of 22 serum proteins in leptospirosis patients compared to the healthy controls. Among the identified differentially expressed proteins, 8 candidates exhibited different trends compared to the febrile controls. Functional analysis suggested the involvement of differentially expressed proteins in vital physiological pathways, including acute phase response, complement and coagulation cascades and hemostasis. This is the first report of analysis of human serum proteome alterations in leptospirosis patients, which revealed several differentially expressed proteins, including α-1-antitrypsin, vitronectin, ceruloplasmin, G-protein signaling regulator, apolipoprotein A-IV, which have not been reported in context of leptospirosis previously. This study will enhance our understanding about leptospirosis pathogenesis and provide a glimpse of host immunological responses. Additionally, a few differentially expressed proteins identified in this study may further be investigated as diagnostic or prognostic serum biomarkers for leptospirosis. This article is part of a Special Issue entitled: Integrated omics. Show less

Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC. Show less

The purpose of the study was to identify epithelial and stromal proteins that exhibit up- or down-regulation in keratoconus (KC) vs. normal human corneas. Because previous proteomic studies utilized whole human corneas or epithelium alone, thereby diluted the specificity of the proteome of each tissue, we selectively analyzed the epithelium and stromal proteins. Individual preparations of epithelial and stromal proteins from KC and age-matched normal corneas were analyzed by two independent methods, i.e., a shotgun proteomic using a Nano-Electrospray Ionization Liquid Chromatography Tandem Mass Spectrometry [Nano-ESI-LC-MS (MS)(2)] and two-dimensional-difference gel electrophoresis (2D-DIGE) coupled with mass spectrometric methods. The label-free Nano-ESI-LC-MS (MS)(2) method identified 104 epithelial and 44 stromal proteins from both normal and KC corneas, and also quantified relative changes in levels of selected proteins, in both the tissues using spectral counts in a proteomic dataset. Relative to normal corneal epithelial proteins, six KC epithelial proteins (lamin-A/C, keratin type I cytoskeletal 14, tubulin beta chain, heat shock cognate 71 kDa protein, keratin type I cytoskeletal 16 and protein S100-A4) exhibited up-regulation and five proteins (transketolase, pyruvate kinase, 14-3-3 sigma isoform, phosphoglycerate kinase 1, and NADPH dehydrogenase (quinone) 1) showed down-regulation. A similar relative analysis showed that three KC stromal proteins (decorin, vimentin and keratocan) were up-regulated and five stromal proteins (TGF-betaig h3 (Bigh3), serotransferrin, MAM domain-containing protein 2 and isoforms 2C2A of collagen alpha-2[VI] chain) were down-regulated. The 2D-DIGE-mass spectrometry followed by Decyder software analysis showed that relative to normal corneas, the KC corneal epithelium exhibited up-regulation of four proteins (serum albumin, keratin 5, L-lactate dehydrogenase and annexin A8) and down-regulation of four proteins (FTH1 [Ferritin heavy chain protein 1], calpain small subunit 1, heat shock protein beta 1 and annexin A2). A similar relative analysis of stroma by this method also showed up-regulation of aldehyde dehydrogenase 3A1 (ALDH3A1), keratin 12, apolipoprotein A-IV precursor, haptoglobin precursor, prolipoprotein and lipoprotein Gln in KC corneas. Together, the results suggested that the Nano-ESI-LC-MS(MS)(2) method was superior than the 2D-DIGE method as it identified a greater number of proteins with altered levels in KC corneas. Further, the epithelial and stromal structural proteins of KC corneas exhibited altered levels compared to normal corneas, suggesting that they are affected due to structural remodeling during KC development and progression. Additionally, because several epithelial and stromal enzymes exhibited up- or down-regulation in the KC corneas relative to normal corneas, the two layers of KC corneas were under metabolic stress to adjust their remodeling. Show less

Mutations in MYBPC3 encoding cardiac myosin binding protein C are common genetic cause of hereditary cardiac myopathies. An intronic 25-bp deletion in MYBPC3 at 3' region is associated with dilated (DCM) and hypertrophic (HCM) cardiomyopathies in Southeast Asia. However, the frequency of MYBPC3 25 bp deletion and associated clinical presentation has not been established in an unrelated cohort of left ventricular dysfunction (LVD) secondary to coronary artery disease (CAD) patients. We sought to determine the role of MYBPC3 25 bp polymorphism on LVD in two cohorts of CAD patients. The study included 265 consecutive patients with angiographically confirmed CAD and 220 controls. MYBPC3 25 bp polymorphism was determined by polymerase chain reaction. Our results showed that carrier status of MYBPC3 25 bp deletion was associated with significant compromised left ventricle ejection fraction (LVEF ≤45) in CAD patients (p value  =  <0.001; OR = 4.49). To validate our results, we performed a replication study in additional 140 cases with similar clinical characteristics and results again confirmed consistent findings (p = 0.029; OR = 3.3). Also, presence of the gene deletion did not have significant association in CAD patients with preserved ejection fraction (LVEF>45) (p value  = 0.1; OR  = 2.3). The frequency of MYBPC3 DW genotype and D allele was associated with compromised LVEF implying that genetic variants of MYBPC3 encoding mutant structural sarcomere protein could increase susceptibility to left ventricular dysfunction. Therefore, 25 bp deletion in MYBPC3 may represent a genetic marker for cardiac failure in CAD patients from Southeast Asia. Show less

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide, or GIP), a 42-amino acid incretin hormone, modulates insulin secretion in a glucose-concentration-dependent manner. Its insulinotropic action is highly dependent on glucose concentration that surmounts the hypoglycemia side effects associated with current therapy. In order to develop a GIP-based anti-diabetic therapy, it is essential to establish the 3D structure of the peptide and study its interaction with the GIP receptor (GIPR) in detail. This will give an insight into the GIP-mediated insulin release process. In this article, we report the solution structure of GIP(1-42, human)NH(2) deduced by NMR and the interaction of the peptide with the N-terminus of GIPR using molecular modelling methods. The structure of GIP(1-42, human)NH(2) in H(2)O has been investigated using 2D-NMR (DQF-COSY, TOCSY, NOESY, (1)H-(13)C HSQC) experiments, and its conformation was built by constrained MD simulations with the NMR data as constraints. The peptide in H(2)O exhibits an alpha-helical structure between residues Ser8 and Asn39 with some discontinuity at residues Gln29 to Asp35; the helix is bent at Gln29. This bent gives the peptide an 'L' shape that becomes more pronounced upon binding to the receptor. The interaction of GIP with the N-terminus of GIPR was modelled by allowing GIP to interact with the N-terminus of GIPR under a series of decreasing constraints in a molecular dynamics simulation, culminating with energy minimization without application of any constraints on the system. The canonical ensemble obtained from the simulation was subjected to a detailed energy analysis to identify the peptide-protein interaction patterns at the individual residue level. These interaction energies shed some light on the binding of GIP with the GIPR N-terminus in a quantitative manner. Show less

Gastric inhibitory polypeptide (GIP, or glucose-dependent insulinotropic polypeptide) is a 42-amino acid incretin hormone moderating glucose-induced insulin secretion. Antidiabetic therapy based on GIP holds great promise because of the fact that its insulinotropic action is highly dependent on the level of glucose, overcoming the sideeffects of hypoglycemia associated with the current therapy of Type 2 diabetes. The truncated peptide, GIP(1-30)NH2, has the same activity as the full length native peptide. We have studied the structure of GIP(1-30)NH2 and built a model of its G-protein coupled receptor (GPCR). The structure of GIP(1-30)NH2 in DMSO-d6 and H2O has been studied using 2D NMR (total correlation spectroscopy (TOCSY), nuclear overhauser effect spectroscopy (NOESY), double quantum filtered-COSY (DQF-COSY), 13C-heteronuclear single quantum correlation (HSQC) experiments, and its conformation built by MD simulations with the NMR data as constraints. The peptide in DMSO-d6 exhibits an alpha-helix between residues Ile12 and Lys30 with a discontinuity at residues Gln19 and Gln20. In H2O, the alpha-helix starts at Ile7, breaks off at Gln19, and then continues right through to Lys30. GIP(1-30)NH2 has all the structural features of peptides belonging to family B1 GPCRs, which are characterized by a coil at the N-terminal and a long C-terminal alpha-helix with or without a break. A model of the seven transmembrane (TM) helices of the GIP receptor (GIPR) has been built on the principles of comparative protein modeling, using the crystal structure of bovine rhodopsin as a template. The N-terminal domain of GIPR has been constructed from the NMR structure of the N-terminal of corticoptropin releasing factor receptor (CRFR), a family B1 GCPR. The intra and extra cellular loops and the C-terminal have been modeled from fragments retrieved from the PDB. On the basis of the experimental data available for some members of family B1 GPCRs, four pairs of constraints between GIP(1-30)NH2 and its receptor were used in the FTDOCK program, to build the complete model of the GIP(1-30)NH2:GIPR complex. The model can rationalize the various experimental observations including the potency of the truncated GIP peptide. This work is the first complete model at the atomic level of GIP(1-30)NH2 and of the complex with its GPCR. Show less

Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (Grl), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in grl cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression. Show less

The basic helix-loop-helix transcriptional repressor Hairy-related transcription factor 2 (Hrt2) is expressed in ventricular, but not atrial, cardiomyocytes, and in endothelial and vascular smooth muscle cells. Mice homozygous for a null mutation of Hrt2 die perinatally from a spectrum of cardiac abnormalities, raising questions about the specific functions of this transcriptional regulator in individual cardiac cell lineages. Using a conditional Hrt2 null allele, we show that cardiomyocyte-specific deletion of Hrt2 in mice results in ectopic activation of atrial genes in ventricular myocardium with an associated impairment of cardiac contractility and a unique distortion in morphology of the right ventricular chamber. Consistent with the atrialization of ventricular gene expression in Hrt2 mutant mice, forced expression of Hrt2 in atrial cardiomyocytes is sufficient to repress atrial cardiac genes. These findings reveal a ventricular myocardial cell-autonomous function for Hrt2 in the suppression of atrial cell identity and the maintenance of postnatal cardiac function. Show less

Various population studies have reported the association of rare S2 allele of apolipoprotein C3 (APOC3) SstI polymorphism with hypertriglyceridemia (HTG) and coronary artery disease (CAD). We were the first to report an association of S2 allele with high triglyceride (TG) levels in healthy volunteers from Northern India. Since HTG is suggested to be a predominant risk factor for CAD among Indians, we have elucidated the relationship of APOC3 SstI polymorphism with the lipid profile and CAD. A total of 158 patients with > or = 70% stenosis in one or more coronary artery (angiographically proven CAD patients), 35 subjects with < 70% stenosis (NCAD) and 151 normal controls (free of heart disease) from Northern plains of India were recruited in the study. DNA samples were analyzed by polymerase chain reaction (PCR) followed by SstI digestion. Lipid profile was estimated by enzymatic kit. We found a strong association of S2 allele with high TG levels, which was more significant in patients. Prevalence of S2 allele in normal controls and CAD patients were comparable, despite the fact that mean TG level was significantly higher in patients. A greater insight into this observation revealed that the prevalence of high TG, if not coupled with other risk factors (like high total cholesterol, low HDL), was comparable in patients and controls. Thus, our study reveals that rare S2 allele may be employed as a susceptibility marker for high TG. However, high TG or S2 allele alone may not contribute to the etiology of CAD. Show less

Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase B alpha relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner. Show less

Several studies including a small case-control (hypertriglyceridemic/normotriglyceridemic individuals) study by us revealed close association between rare S2 allele ofAPOC3 Sstl polymorphism and hypertriglyceridemia. With the understanding that Asian Indians are highly vulnerable to the adverse effects of hypertriglyceridemia, we extended the investigation and studied the frequency distribution of this polymorphism in 216 healthy volunteers from Northern plains of India. We found that more than 50% of the study population had one or two S2 allele. This may suggest that a larger fraction of this population is genetically predisposed to hypertriglyceridemia. Show less

A close association between Sst I polymorphism in the 3' untranslated region of the apolipoproteinC3 (APOC3) gene and levels of plasma triglycerides (TG) had been reported by different investigators. Hypertriglyceridemia(HTG) is a known risk factor for coronary artery disease (CAD) in the context of Asian Indians. We conducted a study on the relationship between APOC3 SstI polymorphism (S1S1, S1S2 and S2S2 genotypes) and plasma TG levels in a group of 139 male healthy volunteers from Northern India. DNA samples were analyzed by polymerase chain reaction (PCR) followed by SstI digestion. Digested PCR products were run on 3% agarose gel and visualized by ethidium bromide staining. Rare S2 allele was highly prevalent in our study population (0.313) as compared to the Caucasians (0.00-0.11). The genotypic distribution was in agreement with Hardy-Weinberg equilibrium. S2 allele was almost two times more prevalent in the HTG group (N = 34) as compared to NTG group (N = 105) (p = 0.001). Multiple logistic regression revealed S1S2 individuals had age-adjusted odds ratio of 2.43 (95%CI = 0.99-6.01, p = 0.054) and S2S2 had 9.9 (95%CI = 2.66-37.29, p = 0.0006) for developing HTG in comparison to S1S1 genotype. Our study shows a significant association between rare S2 allele and HTG in Asian Indians. Show less