saurabh vig | IGIB - Academia.edu (original) (raw)

Papers by saurabh vig

Molecular Diagnostics

The polymerase chain reaction (PCR) is a technology used to exponentially amplify a short, specif... more The polymerase chain reaction (PCR) is a technology used to exponentially amplify a short, specific DNA region of interest, generating thousands to millions of copies of this specific region, all with a similar sequence and length. It is one of the few techniques that has undoubtedly revolutionized the field of molecular biology and diagnostics. Since the invention of the classical end-point PCR in 1986, the technique has gone through a revolutionary period, with a boost in 1992 due to the invention of real-time or quantitative PCR (qPCR), which allowed for monitoring the PCR reaction in real-time, thereby making the quantification of nucleic acids much more simple, accurate, and sensitive. It is a key technology that is present on almost every bench in a research or diagnostic laboratory. In this chapter we will give an overview of the qPCR technique, starting from its invention, describing the different methodologies and important factors to be taken into account when optimizing and using this widespread nucleic acid-based technique.

Frontiers in Immunology, 2021

Beta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation a... more Beta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In response to inflammatory signals, beta-cells engage adaptive mechanisms where the endoplasmic reticulum (ER) and mitochondria act in concert to restore cellular homeostasis. In the recent years it has become clear that this adaptive phase may trigger the development of autoimmunity by the generation of autoantigens recognized by autoreactive CD8 T cells. The participation of the ER stress and the unfolded protein response to the increased visibility of beta-cells to the immune system has been largely described. However, the role of the other cellular organelles, and in particular the mitochondria that are central mediator for beta-cell survival and function, remains poorly investigated. In this review we will dissect the crosstalk between the ER and mitochondria in the context of T1D, highlighting the key role played by this interaction in beta-cell dysfunct...

Journal of Proteome Research, 2020

Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, media... more Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, mediated by tissue transglutaminase enzymes, can provoke autoimmunity by generating altered self-epitopes, a process well-known in celiac disease and more recently also described in type 1 diabetes (T1D). To identify deamidated proteins, liquid chromatography-tandem mass spectrometry is the method of choice. However, as nonenzymatic deamidations on asparagine (Asn) and to a minor extent on Gln are frequently induced in vitro during proteomics sample preparation, the accurate detection of in vivo deamidation can be hampered. Here we report on the optimization of a method to reduce in vitro generated deamidation by 70% using improved trypsin digestion conditions (90 min/pH 8). We also point to the critical importance of manual inspection of MS2 spectra, considering that only 55% of the high quality peptides with Gln deamidation were assigned correctly using an automated search algorithm. As proof of principal, using these criteria, we showed a significant increase in levels of both Asn and Gln deamidation in cytokine-exposed murine MIN6 β-cells, paralleled by an increase in tissue transglutaminase activity. These findings add evidence to the hypothesis that deamidation is occurring in stressed β-cell proteins and can be involved in the autoimmune process in T1D.

Cell Death & Disease, 2019

The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum c... more The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum chaperone, with a central role in maintaining protein homeostasis. Recently, an alternative role for GRP78 under stress conditions has been proposed, with stress-induced extracellular secretion and translocation of GRP78 to the cell surface where it acts as a multifunctional signaling receptor. Here we demonstrate translocation of GRP78 to the surface of human EndoC-βH1 cells and primary human islets upon cytokine exposure, in analogy to observations in rodent INS-1E and MIN6 beta cell lines. We show that GRP78 is shuttled via the anterograde secretory pathway, through the Golgi complex and secretory granules, and identify the DNAJ homolog subfamily C member 3 (DNAJC3) as a GRP78-interacting protein that facilitates its membrane translocation. Evaluation of downstream signaling pathways, using N-and C-terminal anti-GRP78 blocking antibodies, demonstrates that both GRP78 signaling domains initiate pro-apoptotic signaling cascades in beta cells. Extracellular GRP78 itself is identified as a ligand for cell surface GRP78 (sGRP78), increasing caspase 3/7 activity and cell death upon binding, which is accompanied by enhanced Chop and Bax mRNA expression. These results suggest that inflammatory cytokines induce a self-destructive pro-apoptotic feedback loop through the secretion and membrane translocation of GRP78. This proapoptotic function distinguishes the role of sGRP78 in beta cells from its reported anti-apoptotic and proliferative role in cancer cells, opening the road for the use of compounds that block sGRP78 as potential beta cell-preserving therapies in type 1 diabetes.

Expert review of proteomics, 2017

Auto-immunity against pancreatic beta-cells leads to an absolute shortage of the hormone insulin,... more Auto-immunity against pancreatic beta-cells leads to an absolute shortage of the hormone insulin, resulting in hyperglycemia and the onset of type 1 diabetes (T1D). Proteomic approaches have been used to elucidate the mechanisms of beta-cell dysfunction and death. Areas covered: In the present review, we discuss discoveries in the beta-cell proteome that have contributed to better insights in the role of the beta-cell in T1D. Techniques, such as 2D-DIGE and MALDI imaging, together with new approaches for sample preparation, including laser capture microdissection and immunopeptidomics, have resulted in novel mechanistic insights in the pathogenesis of T1D. We describe how proteomic studies in beta-cell lines as well as isolated islets from animal models and humans have discovered intracellular signaling pathways leading to beta-cell destruction, the generation of neo-antigens through post-translational modifications of beta-cell antigens as well as better biomarkers of disease progr...

Diabetologia, Mar 1, 2017

Type 1 diabetes is an endocrine disease where a long preclinical phase, characterised by immune c... more Type 1 diabetes is an endocrine disease where a long preclinical phase, characterised by immune cell infiltration in the islets of Langerhans, precedes elevated blood glucose levels and disease onset. Although several studies have investigated the role of the immune system in this process of insulitis, the importance of the beta cells themselves in the initiation of type 1 diabetes is less well understood. The aim of this study was to investigate intrinsic differences present in the islets from diabetes-prone NOD mice before the onset of insulitis. The islet transcriptome and proteome of 2-3-week-old mice was investigated by microarray and 2-dimensional difference gel electrophoresis (2D-DIGE), respectively. Subsequent analyses using sophisticated pathway analysis and ranking of differentially expressed genes and proteins based on their relevance in type 1 diabetes were performed. In the preinsulitic period, alterations in general pathways related to metabolism and cell communicatio...

Calreticulin (CRT), a 46 kDa endoplasmic reticulum chaperone, is critical in the folding and qual... more Calreticulin (CRT), a 46 kDa endoplasmic reticulum chaperone, is critical in the folding and quality control of proteins. However, the mechanisms of its regulation are not fully understood. Our previous study had demonstrated that elevated TNF␣ levels that are hallmarks of diverse metabolic diseases negatively regulate cellular CRT levels. Here, we attempted to study the mode of this regulation of CRT by TNF␣. Using luciferase reporter deletion constructs of the CRT promoter, we demonstrate that while the −2 kb and −1 kb promoter constructs depict comparable activities, the activity of the −0.5 kb region was greatly reduced suggesting the significance of the region between −1.0 kb and −0.5 kb during CRT promoter activity. Of the transcription factors that possess binding elements within this region, C/EBP␣ was prioritized since it was shown to be inhibited by TNF␣ in an earlier report from our laboratory. TNF␣ significantly inhibited the wild-type CRT promoter activity that was attenuated in a C/EBP␣-deleted construct. C/EBP␣ mRNA levels and its nuclear content was also reduced in the presence of TNF␣. This led to reduced C/EBP␣ occupancy on the CRT promoter and a decreased binding of the nuclear protein to the C/EBP␣-consensus sequence. TNF␣ also reduced the nuclear content of C/EBP␤ but it did not bind to the CRT promoter suggesting that it does not contribute to the inhibitory effect of TNF␣. To conclude, our results suggest that C/EBP␣ is critical in mediating the inhibitory effect of TNF␣ on CRT expression that might be crucial in determining the deleterious cellular effects of TNF␣.

Diabetes, 2015

Levels of miR-22-3p, a highly abundant hepatic microRNA, are abnormally increased in mouse models... more Levels of miR-22-3p, a highly abundant hepatic microRNA, are abnormally increased in mouse models of insulin resistance and type 2 diabetes, yet its contribution to deregulated hepatic metabolism under diseased states is not well understood. Here, we unravel a novel link between elevated hepatic miR-22-3p expression and impaired gluconeogenesis in diabetic db/db mice via the regulation of Tcf7 (transcription factor 7). Our data demonstrate that miR-22-3p binds to the 3′ untranslated region of TCF7 and downregulates it, and this microRNA-mediated regulation of TCF7 increases the expression of enzymes of the gluconeogenic pathway in HepG2 cells. Small interfering RNA–mediated knockdown of TCF7 in HepG2 cells also causes similar upregulation of gluconeogenic genes. Furthermore, in vivo silencing of miR-22-3p by antagomiR administration lowered random as well as fasting glucose levels in diabetic mice. miR-22-3p antagonism improved glucose tolerance and insulin sensitivity. Importantly,...

RSC Adv., 2015

Genetic studies pertaining to effector molecules have been pivotal in schizophrenia research.

The International Journal of Biochemistry & Cell Biology, 2012

Journal of Microbiology and Biotechnology, 2010

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phy... more Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-phosphorus (P) available to plants. In the present study, the Citrobacter braakii phytase gene (appA) was overexpressed in rhizobacteria possessing plant growth promoting (PGP) traits, for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broadhost-range expression vector pBBR1MCS-2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains), and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying levels of phytase activity, ranging from 10-folds to 538-folds higher than the corresponding control strains. The transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed the highest expression of phytase (~8 U/mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having the ability to secrete organic acid showed significantly higher release of P from Caphytate in buffered minimal media. AppA overexpressing rhizobacteria showed increased P content, and dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semisolid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of the overexpression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

Genome-wide association studies (GWASs) have discovered association of several loci with Type 2 d... more Genome-wide association studies (GWASs) have discovered association of several loci with Type 2 diabetes (T2D), a common complex disease characterized by impaired insulin secretion by pancreatic b cells and insulin signaling in target tissues. However, effect of genetic risk variants on continuous glycemic measures in nondiabetic subjects mainly elucidates perturbation of insulin secretion. Also, the disease associated genes do not clearly converge on functional categories consistent with the known aspects of T2D pathophysiology. We used a systems biology approach to unravel genome to phenome correlation in T2D. We first examined enrichment of pathways in genes identified in T2D GWASs at genome-wide or lower levels of significance. Genes at lower significance threshold showed enrichment of insulin secretion related pathway. Notably, physical and genetic interaction network of these genes showed robust enrichment of insulin signaling and other T2D pathophysiology related pathways including insulin secretion. The network also overrepresented genes reported to interact with insulin secretion and insulin action targeting antidiabetic drugs. The drug interacting genes themselves showed overrepresentation of insulin signaling and other T2D relevant pathways. Next, we generated genomewide expression profiles of multiple insulin responsive tissues from nondiabetic and diabetic patients. Remarkably, the differentially expressed genes showed significant overlap with the network genes, with the intersection showing enrichment of insulin signaling and other pathways consistent with T2D pathophysiology. Literature search led our genomic, interactomic, transcriptomic and toxicogenomic evidence to converge on TGF-beta signaling, a pathway known to play a crucial role in pancreatic islets development and function, and insulin signaling. Cumulatively, we find that GWAS genes relate directly to insulin secretion and indirectly, through collaborating with other genes, to insulin resistance. This seems to support the epidemiological evidence that environmentally triggered insulin resistance interacts with genetically programmed b cell dysfunction to precipitate diabetes.

Molecular and Cellular …, Jan 1, 2010