Nader Pourmand, Ph.D.
Director, UCSC Genome Technology Center,University of California, Santa Cruz
Biosensor and Single Cell Manipulation using Nanopipettes
Approaching sub-cellular biological problems from an engineering perspective begs for the incorporation of electronic readouts. With their high sensitivity and low invasiveness, nanotechnology-based tools hold great promise for biochemical sensing and single-cell manipulation. During my talk I will discuss the incorporation of electrical measurements into nanopipette technology and present results showing the rapid and reversible response of these subcellular sensors to different analytes such as antigens, ions and carbohydrates. In addition, I will present the development of a single-cell manipulation platform that uses a nanopipette in a scanning ion-conductive microscopy technique. We use this newly developed technology to position the nanopipette with nanoscale precision, and to inject and/or aspirate a minute amount of material to and from individual cells or organelle without comprising cell viability. Furthermore, if time permits, I will show our strategy for a new, single-cell DNA/ RNA sequencing technology that will potentially use nanopipette technology to analyze the minute amount of aspirated cellular material.
Michelle Hermiston, MD, Ph.D.
Assistant Professor, Department of Pediatrics University of California San Francisco, USA
Interrogating Signaling Networks at the Single Cell Level In Primary Human Patient Samples
Multiparameter phosphoflow cytometry is a highly sensitive proteomic approach that enables monitoring of biochemical perturbations at the single cell level. By combining antisera to cell surface markers and key intracellular proteins, perturbations in signaling networks, cell survival and apoptosis mediators, cell cycle regulators, and/or modulators of other cellular processes can be analyzed in a highly reproducible and sensitive manner in the basal state and in response to stimulation or drug treatment. Advantages of this approach include the ability to identify the biochemical consequences of genetic and/or epigenetic changes in small numbers of cells, to map potential interplay between various signaling networks simultaneously in a single cell, and to interrogate potential mechanisms of drug resistance or response in a primary patient sample. Application of this technology to patients with acute lymphoblastic leukemia or the autoimmune disease systemic lupus erythematosus (SLE) will be discussed.
Kal Ramnarayan, Ph.D.
Co-founder President & Chief Scientific Officer, Sapient Discovery, San Diego, CA, USA
A cost-effective approach to Protein Structure-guided Drug Discovery: Aided by Bioinformatics, Chemoinformatics and computational chemistry
With the mapping of the human genome completed almost a decade ago, efforts are still underway to understand the gene products (i.e., proteins) in the human biological and disease pathways. Deciphering such information is very important for the discovery and development of small molecule drugs as well as protein therapeutics for various human diseases for which no cure exists. As an example, with more than 500 members, the kinase family of protein targets continues to be an important and attractive class for drug discovery. While how many of the members in this family are actually druggable is still to be established, there are several ongoing efforts on this class of proteins across a broad spectrum of disease categories. Even though in general the protein structural topology might looks similar, there are issues with respect selectivity of identified small molecule inhibitors when, the lead molecule discovery is carried out at the ATP binding site. As an added complexity, allosteric modulators are needed for some of the members, but the actual site for such modulation on the protein target can not resolved with uncertainty. In this presentation we will describe a bioinformatics and computational based platform for small molecule discovery for protein targets that are involved in protein-protein interactions as well as targets like kinases and phosphatases. We will describe a computational approach in which we have used an informatics based platform with several hundred kinases to sort through in silico and identify inhibitors that are likely to be highly selective in the lead generation phase. We will discuss the implication of this approach on the drug discovery of the kinase and phosphatase classes in general and independent of the disease category.
V. Nagaraja Ph.D.
Professor, Indian Institute of Science, Bengaluru, India
Perturbation of DNA topology in mycobacteria
To maintain the topological homeostasis of the genome in the cell, DNA topoisomerases catalyse DNA cleavage, strand passage and rejoining of the ends. Thus, although they are essential house- keeping enzymes, they are the most vulnerable targets; arrest of the reaction after the first trans-esterification step leads to breaks in DNA and cell death. Some of the successful antibacterial or anticancer drugs target the step ie arrest the reaction or stabilize the topo -DNA covalent complex. I will describe our efforts in this direction – to target DNA gyrase and also topoisomerase1 from mycobacteria. The latter, although essential, has no inhibitors described so far. The new inhibitors being characterized are also used to probe topoisomerase control of gene expression.
In the biological warfare between the organisms, a diverse set of molecules encoded by invading genomes target the above mentioned most vulnerable step of topoisomerase reaction, leading to the accumulation of double strand breaks. Bacteria, on their part appear to have developed defense strategies to protect the cells from genomic double strand breaks. I will describe a mechanism involving three distinct gyrase interacting proteins which inhibit the enzyme in vitro. However, in vivo all these topology modulators protect DNA gyrase from poisoning effect by sequestering the enzyme away from DNA.
Next, we have targeted a topology modulator protein, a nucleoid associated protein(NAP) from Mycobacterium tuberculosis to develop small molecule inhibitors by structure based design. Over expression of HU leads to alteration in the nucleoid architecture. The crystal structure of the N-terminal half of HU reveals a cleft that accommodates duplex DNA. Based on the structural feature, we have designed inhibitors which bind to the protein and affect its interaction with DNA, de-compact the nucleoid and inhibit cell growth. Chemical probing with the inhibitors reveal the importance of HU regulon in M.tuberculosis.
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.
Karmeshu, Ph.D.
Dean & Professor, School of Computer & Systems Sciences & School of Computational & Integrative Sciences, Jawaharlal Nehru University, India.
Interspike Interval Distribution of Neuronal Model with distributed delay: Emergence of unimodal, bimodal and Power law
The study of interspike interval distribution of spiking neurons is a key issue in the field of computational neuroscience. A wide range of spiking patterns display unimodal, bimodal ISI patterns including power law behavior. A challenging problem is to understand the biophysical mechanism which can generate the empirically observed patterns. A neuronal model with distributed delay (NMDD) is proposed and is formulated as an integro-stochastic differential equation which corresponds to a non-markovian process. The widely studied IF and LIF models become special cases of this model. The NMDD brings out some interesting features when excitatory rates are close to inhibitory rates rendering the drift close to zero. It is interesting that NMDD model with gamma type memory kernel can also account for bimodal ISI pattern. The mean delay of the memory kernels plays a significant role in bringing out the transition from unimodal to bimodal ISI distribution. It is interesting to note that when a collection of neurons group together and fire together, the ISI distribution exhibits power law.
Seeram Ramakrishna, Ph.D.
Director, Center for Nanofibers & Nanotechnology, National University of Singapore
Biomaterials: Future Perspectives
From the perspective of thousands of years of history, the role of biomaterials in healthcare and wellbeing of humans is at best accidental. However, since 1970s with the introduction of national regulatory frameworks for medical devices, the biomaterials field evolved and reinforced with strong science and engineering understandings. The biomaterials field also flourished on the backdrop of growing need for better medical devices and medical treatments, and sustained investments in research and development. It is estimated that the world market size for medical devices is ~300 billion dollars and for biomaterials it is ~30 billion dollars. Healthcare is now one of the fastest growing sectors worldwide. Legions of scientists, engineers, and clinicians worldwide are attempting to design and develop newer medical treatments involving tissue engineering, regenerative medicine, nanotech enabled drug delivery, and stem cells. They are also engineering ex-vivo tissues and disease models to evaluate therapeutic drugs, biomolecules, and medical treatments. Engineered nanoparticles and nanofiber scaffolds have emerged as important class of biomaterials as many see them as necessary in creating suitable biomimetic micro-environment for engineering and regeneration of various tissues, expansion & differentiation of stem cells, site specific controlled delivery of biomolecules & drugs, and faster & accurate diagnostics. This lecture will capture the progress made thus far in pre-clinical and clinical studies. Further this lecture will discuss the way forward for translation of bench side research into the bed side practice. This lecture also seeks to identify newer opportunities for biomaterials beyond the medical devices.
![Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/btindia.jpg)
Sukhithasri V, Nisha N, Vivek V and Raja Biswas
The host innate immune system acts as the first line of defense against invading pathogens. During an infection, the host innate immune cells recognize unique conserved molecules on the pathogen known as Pathogen Associated Molecular Patterns (PAMPs). This recognition of PAMPs helps the host mount an innate immune response leading to the production of cytokines (Akira et al. 2006). Peptidoglycan, one of the most conserved and essential component of the bacterial cell wall is one such PAMP. Peptidoglycan is known to have potent proinflammatory properties (Gust et al. 2007). Host recognize peptidoglycan using Nucleotide oligomerization domain proteins (NODs). This recognition of peptidoglycan activates the NODs and triggers downstream signaling leading to the nuclear translocation of NF-κB and production of cytokines (McDonald et al. 2005). Pathogenic bacteria modify their peptidoglycan as a strategy to evade innate immune recognition, which helps it to establish infection in the host. These peptidoglycan modifications include O-acetylation and N-glycolylation of muramic acid and N-deacetylation of N-acetylglucosamine (Davis et al. 2011). Modification of mycobacterial peptidoglycan by N-glycolylation prevents the catalytic activity of lysozyme (Raymond et al. 2005). Additionally, mycobacterial peptidoglycan is modified by amidation for unknown reasons.
Here, we have investigated the role of amidated peptidoglycan in Mycobacterium sp in modulating the innate immune response. We isolated amidated peptidoglycan from Mycobacterium sp and non-amidated peptidoglycan from Escherichia coli. We made a comparative analysis of the cytokine response produced on stimulation of innate immune cells by peptidoglycan from E. Coli and Mycobacterium sp. Macrophages and whole blood were treated with peptidoglycan and the cytokines secreted into spent medium and plasma respectively were analyzed using ELISA. Our results show that peptidoglycan from Mycobacterium sp is less effective in stimulating innate immune cells to produce cytokines. This intrinsic modulation of the cytokine response suggests that mycobacteria modify their peptidoglycan by amidation to evade innate immune response.