Vural Özdemir Ph.D.
Sanjeeva Srivastava Ph.D.
Rajgopal Srinivasan, Ph.D.
Principal Scientist & Head Bio IT R&D, TCS Innovation Labs, India
Interpretation of Genomic Variation – Identifying Rare Variations Leading to Disease
Genome sequencing technologies are generating an abundance of data on human genetic variations. A big challenge lies in interpreting the functional relevance of such variations, especially in clinical settings. A first step in understanding the clinical relevance of genetic variations is to annotate the variants for region of occurrence, degree of conservation both within and across species, pattern of variation across related individuals, novelty of the variation and know effects of related variations. Several tools already exist for this purpose. However, these tools have their strengths and weaknesses. A second issue is the development of algorithms, which, given a rich annotation of variants are able to prioritize the variants as being relevant to the phenotype under investigation.
In my talk I will detail work that has been done in our labs to address both of the above problems. I will also illustrate the application of these tools that helped identify a rare mutation in the ATM gene leading to a diagnosis of AT in two infants.
D. Narasimha Rao, Ph.D.
Professor, Dept of Biochemistry, Indian Institute of Science, Bangalore, India
Genomics of Restriction-Modification Systems
Restriction endonucleases occur ubiquitously among procaryotic organisms. Up to 1% of the genome of procaryotic organisms is taken up by the genes for these enzymes. Their principal biological function is the protection of the host genome against foreign DNA, in particular bacteriophage DNA. Restriction-modification (R-M) systems are composed of pairs of opposing enzyme activities: an endonuclease and a DNA methyltransferase (MTase). The endonucleases recognise specific sequences and catalyse cleavage of double-stranded DNA. The modification MTases catalyse the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, R-M systems are generally classified into three groups. In general R-M systems restrict unmodified DNA, but there are other systems that specifically recognise and cut modified DNA. More than 3500 restriction enzymes have been discovered so far. With the identification and sequencing of a number of R-M systems from bacterial genomes, an increasing number of these have been found that do not seem to fit into the conventional classification.
It is well documented that restriction enzyme genes always lie close to their cognate methyltransferase genes. Analysis of the bacterial and archaeal genome sequences shows that MTase genes are more common than one would have expected on the basis of previous biochemical screening. Frequently, they clearly form part of a R-M system, because the adjacent open reading frames (ORFs) show similarity to known restriction enzyme genes. Very often, though, the adjacent ORFs have no homologs in the GenBank and become candidates either for restriction enzymes with novel specificities or for new examples of previously uncloned specificities. Sequence-dependent modification and restriction forms the foundation of defense against foreign DNAs and thus RM systems may serve as a tool of defense for bacterial cells. RM systems however, sometimes behave as discrete units of life, and any threat to their maintenance, such as a challenge by a competing genetic element can lead to cell death through restriction breakage in the genome, thus providing these systems with a competitive advantage. The RM systems can behave as mobile-genetic elements and have undergone extensive horizontal transfer between genomes causing genome rearrangements. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.
The similarities and differences in the different mechanisms used by restriction enzymes will be discussed. Although it is not clear whether the majority of R-M systems are required for the maintenance of the integrity of the genome or whether they are spreading as selfish genetic elements, they are key players in the “genomic metabolism” of procaryotic organisms. As such they deserve the attention of biologists in general. Finally, restriction enzymes are the work horses of molecular biology. Understanding their enzymology will be advantageous to those who use these enzymes, and essential for those who are devoted to the ambitious goal of changing the properties of these enzymes, and thereby make them even more useful.
R. Manjunath, Ph.D.
Associate Professor, Dept of Biochemistry, Indian Institute of Science, Bengaluru, India
REGULATION OF THE MHC COMPLEX AND HLA SOLUBILISATION BY THE FLAVIVIRUS, JAPANESE ENCEPHALITIS VIRUS
Viral encephalitis caused by Japanese encephalitis virus (JEV) and West Nile Virus (WNV) is a mosquito-borne disease that is prevalent in different parts of India and other parts of South East Asia. JEV is a positive single stranded RNA virus that belongs to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. It is a neurotropic virus which infects the central nervous system (CNS) and causes death predominantly in newborn children and young adults. JEV follows a zoonotic life-cycle involving mosquitoes and vertebrate, chiefly pigs and ardeid birds, as amplifying hosts. Humans are infected when bitten by an infected mosquito and are dead end hosts. Its structural, pathological, immunological and epidemiological aspects have been well studied. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral neutrophil leucocytosis in the brain and leads to elevated levels of type I interferon, macrophage-derived chemotactic factor, RANTES,TNF-α and IL-8 in the serum and cerebrospinal fluid.
Major Histocompatibility Complex (MHC) molecules play a very important role in adaptive immune responses. Along with various classical MHC class I molecules, other non-classical MHC class I molecules play an important role in modulating innate immune responses. Our lab has shown the activation of cytotoxic T-cells (CTLs) during JEV infection and CTLs recognize non-self peptides presented on MHC molecules and provide protection by eliminating infected cells. However, along with proinflammatory cytokines such as TNFα, they may also cause immunopathology within the JEV infected brain. Both JEV and WNV, another related flavivirus have been shown to increase MHC class I expression. Infection of human foreskin fibroblast cells (HFF) by WNV results in upregulation of HLA expression. Data from our lab has also shown that JEV infection upregulates classical as well as nonclassical (class Ib) MHC antigen expression on the surface of primary mouse brain astrocytes and mouse embryonic fibroblasts.
There are no reports that have discussed the expression of these molecules on other cells like endothelial and astrocyte that play an important role in viral invasion in humans. We have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecules, HLA-E as well as HLA-F in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial cell line (ECV304), human glioblastoma cell line (U87MG) and human foreskin fibroblast cells (HFF). Nonclassical MHC molecules such as mouse Qa-1b and its human homologue, HLA-E have been shown to be the ligand for the inhibitory NK receptor, NKG2A/CD94 and may bridge innate and adaptive immune responses. We show that JEV infection of HBMEC and ECV 304 cells upregulates the expression of HLA-A, and –B antigens as well as HLA-E and HLA-F. Increased expression of total HLA-E upon JEV infection was also observed in other human cell lines as well like, human amniotic epithelial cells, AV-3, FL and WISH cells. Further, we show for the first time that soluble HLA-E (sHLA-E) was released from infected ECV and HBMECs. In contrast, HFF cells showed only upregulation of cell-surface HLA-E expression while U87MG, a human glioblastoma cell line neither showed any cell-surface induction nor its solubilization. This shedding of sHLA-E was found to be dependent on matrix metalloproteinase (MMP) and an important MMP, MMP-9 was upregulated during JEV infection. Treatment with IFNγ resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Also, sHLA-E was shed upon treatment with IFNβ and both IFNβ and TNFα, when present together caused an additive increase in the shedding of sHLA-E. HLA-E is an inhibitory ligand for CD94/NKG2A receptor of Natural Killer cells. Thus, MMP mediated solubilization of HLA-E from infected endothelial cells may have important implications in JEV pathogenesis including its ability to compromise the blood brain barrier.
Vural Özdemir, MD, Ph.D., DABCP
Co-Founder, DELSA Global, Seattle, WA, USA
Crowd-Funded Micro-Grants to Link Biotechnology and “Big Data” R&D to Life Sciences Innovation in India
Vural Özdemir, MD, PhD, DABCP1,2*
- Data-Enabled Life Sciences Alliance International (DELSA Global), Seattle, WA 98101, USA;
- Faculty of Management and Medicine, McGill University, Canada;
ABSTRACT
Aims: This presentation proposes two innovative funding solutions for linking biotechnology and “Big Data” R&D in India with artisan small scale discovery science, and ultimately, with knowledge-based innovation:
- crowd-funded micro-grants, and
- citizen philanthropy
These two concepts are new, and inter-related, and can be game changing to achieve the vision of biotechnology innovation in India, and help bridge local innovation with global science.
Background and Context: Biomedical science in the 21(st) century is embedded in, and draws from, a digital commons and “Big Data” created by high-throughput Omics technologies such as genomics. Classic Edisonian metaphors of science and scientists (i.e., “the lone genius” or other narrow definitions of expertise) are ill equipped to harness the vast promises of the 21(st) century digital commons. Moreover, in medicine and life sciences, experts often under-appreciate the important contributions made by citizen scholars and lead users of innovations to design innovative products and co-create new knowledge. We believe there are a large number of users waiting to be mobilized so as to engage with Big Data as citizen scientists-only if some funding were available. Yet many of these scholars may not meet the meta-criteria used to judge expertise, such as a track record in obtaining large research grants or a traditional academic curriculum vitae. This presentation will describe a novel idea and action framework: micro-grants, each worth $1000, for genomics and Big Data. Though a relatively small amount at first glance, this far exceeds the annual income of the “bottom one billion” – the 1.4 billion people living below the extreme poverty level defined by the World Bank ($1.25/day).
We will present two types of micro-grants. Type 1 micro-grants can be awarded through established funding agencies and philanthropies that create micro-granting programs to fund a broad and highly diverse array of small artisan labs and citizen scholars to connect genomics and Big Data with new models of discovery such as open user innovation. Type 2 micro-grants can be funded by existing or new science observatories and citizen think tanks through crowd-funding mechanisms described herein. Type 2 micro-grants would also facilitate global health diplomacy by co-creating crowd-funded micro-granting programs across nation-states in regions facing political and financial instability, while sharing similar disease burdens, therapeutics, and diagnostic needs. We report the creation of ten Type 2 micro-grants for citizen science and artisan labs to be administered by the nonprofit Data-Enabled Life Sciences Alliance International (DELSA Global, Seattle: http://www.delsaglobal.org). Our hope is that these micro-grants will spur novel forms of disruptive innovation and life sciences translation by artisan scientists and citizen scholars alike.
Address Correspondence to:
Vural Özdemir, MD, PhD, DABCP
Senior Scholar and Associate Professor
Faculty of Management and Medicine, McGill University
1001 Sherbrooke Street West
Montreal, Canada H3A 1G5