👤 Amarjit Mishra

Pancreatic ductal adenocarcinoma (PDAC) is the most common and among the deadliest of pancreatic cancers. Its 5-year survival is only ∼8%. Pancreatic cancers are a heterogeneous group of diseases, of which PDAC is particularly aggressive. Like many other cancers, PDAC also starts as a pre-invasive precursor lesion (known as pancreatic intraepithelial neoplasia, PanIN), which offers an opportunity for both early detection and early treatment. Even advanced PDAC can benefit from prognostic biomarkers. However, reliable biomarkers for early diagnosis or those for prognosis of therapy remain an unfulfilled goal for PDAC. In this study, we selected 153 PDAC patients from the TCGA database and used their clinical, DNA methylation, gene expression, and micro-RNA (miRNA) and long non-coding RNA (lncRNA) expression data for multi-omics analysis. Differential methylations at about 12,000 CpG sites were observed in PDAC tumor genomes, with about 61% of them hypermethylated, predominantly in the promoter regions and in CpG-islands. We correlated promoter methylation and gene expression for mRNAs and identified 17 genes that were previously recognized as PDAC biomarkers. Similarly, several genes (B3GNT3, DMBT1, DEPDC1B) and lncRNAs (PVT1, and GATA6-AS) are strongly correlated with survival, which have not been reported in PDAC before. Other genes such as EFR3B, whose biological roles are not well known in mammals are also found to strongly associated with survival. We further identified 406 promoter methylation target loci associated with patients survival, including known esophageal squamous cell carcinoma biomarkers, cg03234186 (ZNF154), and cg02587316, cg18630667, and cg05020604 (ZNF382). Overall, this is one of the first studies that identified survival associated genes using multi-omics data from PDAC patients. Show less

Ventricular Septal Defect (VSD), the most common congenital heart defect, is characterized by a hole in the septum between the right and left ventricles. The pathogenesis of VSD is unknown in most clinical cases. There is a paucity of data relevant to epigenetic changes in VSD. The placenta is a fetal tissue crucial in cardiac development and a potentially useful surrogate for evaluating the development of heart tissue. To understand epigenetic mechanisms that may play a role in the development of VSD, genome-wide DNA methylation assay on placentas of 8 term subjects with isolated VSD and no known or suspected genetic syndromes and 10 unaffected controls was performed using the Illumina HumanMethylation450 BeadChip assay. We identified a total of 80 highly accurate potential CpGs in 80 genes for detection of VSD; area under the receiver operating characteristic curve (AUC ROC) 1.0 with significant 95% CI (FDR) p-values < 0.05 for each individual locus. The biological processes and functions for many of these differentially methylated genes are previously known to be associated with heart development or disease, including cardiac ventricle development (HEY2, ISL1), heart looping (SRF), cardiac muscle cell differentiation (ACTC1, HEY2), cardiac septum development (ISL1), heart morphogenesis (SRF, HEY2, ISL1, HEYL), Notch signaling pathway (HEY2, HEYL), cardiac chamber development (ISL1), and cardiac muscle tissue development (ACTC1, ISL1). In addition, we identified 8 microRNAs that have the potential to be biomarkers for the detection of VSD including: miR-191, miR-548F1, miR-148A, miR-423, miR-92B, miR-611, miR-2110, and miR-548H4. To our knowledge this is the first report in which placental analysis has been used for determining the pathogenesis of and predicting VSD. Show less

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA - promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment. 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

In the present study, expression level of various ATP-binding cassette (ABC) viz., ABCA1, ABCA7, ABCG1, ABCG2, and ABCG5; associated transcription factors viz., SREBF1, LXRα (NR1H3), PPARA, and Solute Carriers (SLC); or Glucose transporters (GLUT) viz., SLC2A1(GLUT1), SLC2A4 (GLUT4), SLC2A8 (GLUT8), and SLC2A12 (GLUT12) superfamily of transporters were compared across physiological stages of buffalo mammary gland. The relative expression of ABCA1, and ABCG1 was significantly (p < 0.05) higher in mammary gland of heifer followed by involution and lactation stages. Similarly, ABCA7 gene expression was highest in heifer mammary gland followed by lactation and involution stages. ABCG2 gene expression was significantly (p < 0.05) high in lactating mammary gland in comparison to involution and heifer stages. On the other hand, ABCG5 gene expression was highest in involuting mammary gland followed by lactation and involution stages. Additionally, the expression of LXRα SREBF1, and PPARA which are known to regulate some of the ABC tranporters were also analyzed. The expression of LXRα gene was high in involuting as compared to lactating mammary gland. In contrast, SREBF1 and PPARA expression was significantly (p < 0.05) high in lactating mammary gland. Among the several SLC transporters studied, SLC2A1, SLC2A4, and SLC2A8 showed significant (p < 0.05) higher expression during lactation stage, whereas SLC2A12 expression was greater during heifer stage suggesting SLC2A1, SLC2A4, and SLC2A8 to be the major transporters associated with glucose uptake in buffalo mammary gland. The expression profile of (lactoferrin) LTF, known to be expressed at high level in mammary gland during involution was also studied. As expected, its expression was significantly (p < 0.05) higher during involution in comparison to lactating mammary gland.in buffaloes as well. The inclusion of LTF as a control gene further provided the confidence in the buffalo mammary gland expression data generated in the present study. This study thus helped to provide information about the distinct expression pattern of various transporters and their regulators in buffalo mammary gland during different physiological states. Show less

Neuronal regeneration and axonal re-growth in the injured mammalian central nervous system remains an unsolved field. To date, three myelin-associated proteins [Nogo or reticulon 4 (RTN4), myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMG)] are known to inhibit axonal regeneration via activation of the neuronal glycosylphosphatidylinositol-anchored Nogo receptor [NgR, together with p75 neurotrophin receptor (p75NTR) and Lingo-1]. In the present study we describe the novel protein MANI (myelin-associated neurite-outgrowth inhibitor) that localizes to neural membranes. Functional characterization of MANI overexpressing neural stem cells (NSCs) revealed that the protein promotes differentiation into catecholaminergic neurons. Yeast two-hybrid screening and co-immunoprecipitation experiments confirmed the cell division cycle protein 27 (Cdc27) as an interacting partner of Mani. The analyses of Mani-overexpressing PC12 cells demonstrated that Mani retards neuronal axonal growth as a positive effector of Cdc27 expression and activity. We show that knockdown of Cdc27, a component of the anaphase-promoting complex (APC), leads to enhanced neurite outgrowth. Our finding describes the novel MANI-Cdc27-APC pathway as an important cascade that prevents neurons from extending axons, thus providing implications for the potential treatment of neurodegenerative diseases. Show less

The metazoan nuclear pore complex (NPC) disassembles during mitosis, and many of its constituents distribute onto spindles and kinetochores, including the Nup107-160 sub-complex. We have found that Nup107-160 interacts with the gamma-tubulin ring complex (gamma-TuRC), an essential and conserved microtubule nucleator, and recruits gamma-TuRC to unattached kinetochores. The unattached kinetochores nucleate microtubules in a manner that is regulated by Ran GTPase; such microtubules contribute to the formation of kinetochore fibres (k-fibres), microtubule bundles connecting kinetochores to spindle poles. Our data indicate that Nup107-160 and gamma-TuRC act cooperatively to promote spindle assembly through microtubule nucleation at kinetochores: HeLa cells lacking Nup107-160 or gamma-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Moreover, co-precipitated Nup107-160- gamma-TuRC complexes nucleated microtubule formation in assays using purified tubulin. Although Ran did not regulate microtubule nucleation by gamma-TuRC alone, Nup107-160-gamma-TuRC complexes required Ran-GTP for microtubule nucleation. Collectively, our observations show that Nup107-160 promotes spindle assembly through Ran-GTP-regulated nucleation of microtubules by gamma-TuRC at kinetochores, and reveal a relationship between nucleoporins and the microtubule cytoskeleton. Show less

Eukaryotic genomes encode a zinc finger protein (ZPR1) with tandem ZPR1 domains. In response to growth stimuli, ZPR1 assembles into complexes with eukaryotic translation elongation factor 1A (eEF1A) and the survival motor neurons protein. To gain insight into the structural mechanisms underlying the essential function of ZPR1 in diverse organisms, we determined the crystal structure of a ZPR1 domain tandem and characterized the interaction with eEF1A. The ZPR1 domain consists of an elongation initiation factor 2-like zinc finger and a double-stranded beta helix with a helical hairpin insertion. ZPR1 binds preferentially to GDP-bound eEF1A but does not directly influence the kinetics of nucleotide exchange or GTP hydrolysis. However, ZPR1 efficiently displaces the exchange factor eEF1Balpha from preformed nucleotide-free complexes, suggesting that it may function as a negative regulator of eEF1A activation. Structure-based mutational and complementation analyses reveal a conserved binding epitope for eEF1A that is required for normal cell growth, proliferation, and cell cycle progression. Structural differences between the ZPR1 domains contribute to the observed functional divergence and provide evidence for distinct modalities of interaction with eEF1A and survival motor neuron complexes. Show less