Rama Shanker Verma

Bio: Rama Shanker Verma is an academic researcher from Indian Institute of Technology Madras. The author has contributed to research in topics: Mesenchymal stem cell & Stem cell. The author has an hindex of 30, co-authored 159 publications receiving 3160 citations. Previous affiliations of Rama Shanker Verma include University of Pennsylvania & Thapar University.

SNAP-Tag Technology: A Promising Tool for Ex Vivo Immunophenotyping.

Abstract: SNAP-tag, a self-labeling protein tag, is commonly used for in vitro and in vivo analysis of bound target proteins. We report the first evidence that SNAP-tag could be used for ex vivo detection of enriched biological markers. Proof of concept was established for target c-kit receptor, a pathological and diagnostic marker for a variety of cancers. SNAP-tag conjugates with stem-cell factor (SCF) fusion proteins were designed and their binding and specificity was validated in vitro using flow cytometry and immunostaining. Ex vivo diagnostic application of the fusion protein was demonstrated in comparison with anti-c-kit antibody for peripheral blood samples from leukemia patients and colorectal tissue specimens.

Expression Profiling of Differentially Regulated Genes in Fanconi Anemia.

Abstract: Gene expression analysis mainly helps to study gene quantification methods by using various downstream detection approaches like imaging, amplification, probe hybridization, or sequencing. With respect to DNA, which is less static, mRNA levels vary over time, between cell types under divergent conditions. Gene expression analysis is principally focused on determination of mRNA levels transcribed from DNA. DNA microarrays are one of the robust and powerful tools to detect changes in multiple transcripts in larger cohorts in parallel. The basic principle of DNA microarray hybridization is complementary base pairing of single-stranded nucleic-acid sequences. On a microarray platform (also called a chip), known sequences called targets are attached at fixed locations (spots) to a solid surface such as glass using robotic spotting. Since a large number of samples (variables) are used in a typical hybridization experiment, which often leads to impreciseness for example, target mRNA transcribed from the same source should be identical every time. In such cases, developing an optimized protocol for microarray platform to study the expression profiling of differentially regulated genes is a challenging task. Thus genome-wide expression array analysis yields data about candidate genes that may be involved in disease acquisition progression, and helps in better understanding the pathophysiology of the disease. In this chapter we describe in detail the microarray technique, a well-accepted method for understanding the development and progression of Fanconi anemia (FA), a genetic disorder which is characterized by progressive bone marrow failure and a predisposition to cancer.

An Efficient Protocol for Deriving Liver Stem Cells from Neonatal Mice: Validating Its Differentiation Potential

Abstract: The success of liver regeneration depends on the availability of suitable cell types and their potential to differentiate into functional hepatocytes. To identify the stem cells which have the ability to differentiate into hepatocytes, we used neonatal liver as source. However, the current protocol for isolating stem cells from liver involves enzymes like collagenase, hyaluronidase exposed for longer duration which limits the success. This results in the keen interest to develop an easy single step enzyme digestion protocol for isolating stem cells from liver for tissue engineering approaches. Thus, the unlimited availability of cell type favors setting up the functional recovery of the damaged liver, ensuring ahead success towards treating liver diseases. We attempted to isolate liver stem derived cells (LDSCs) from mouse neonatal liver using single step minimal exposure to enzyme followed by in vitro culturing. The cells isolated were characterized for stem cell markers and subjected to lineage differentiation. Further, LDSCs were induced to hepatocyte differentiation and validated with hepatocyte markers. Finally, we developed a reproducible, efficient protocol for isolation of LDSCs with functional hepatocytes differentiation potential, which further can be used as in vitro model system for assessing drug toxicity assays in various preclinical trials.

Osteoblastic cells regulate the haematopoietic stem cell niche

Abstract: Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

Reactive oxygen species in cell signaling

Abstract: Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of “oxidative stress” is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.

G1 events and regulation of cell proliferation.

Abstract: Cells prepare for S phase during the G1 phase of the cell cycle. Cell biological methods have provided knowledge of cycle kinetics and of substages of G1 that are determined by extracellular signals. Through the use of biochemical and molecular biological techniques to study effects of growth factors, oncogenes, and inhibitors, intracellular events during G1 that lead to DNA synthesis are rapidly being discovered. Many cells in vivo are in a quiescent state (G0), with unduplicated DNA. Cells can be activated to reenter the cycle during G1. Similarly, cells in culture can be shifted between G0 and G1. These switches in and out of G1 are the main determinants of post-embryonic cell proliferation rate and are defectively controlled in cancer cells.

Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours

Abstract: Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression.