S. Ramaswamy, Ph.D.
CEO of c-CAMP, Dean, inStem, NCBS, Bangalore, India
Discovery, engineering and applications of Blue Fish Protein with Red Fluorescence
Swagatha Ghosh, Chi-Li Yu, Daniel Ferraro, Sai Sudha, Wayne Schaefer, David T Gibson and S. Ramaswamy
Fluorescent proteins and their applications have revolutionized our understanding of biology significantly. In spite of several years since the discovery of the classic GFP, proteins of this class are used as the standard flag bearers. We have recently discovered a protein from the fish Sanders vitrius that shows interesting fluorescent properties – including a 280 nm stoke shift and infrared emission. The crystal structure of the wild type protein shows that it is a tetramer. We have engineered mutations to make a monomer with very similar fluorescent properties. We have used this protein for tissue imaging as well as for in cell-fluorescence successfully
![Delegate Talk: BrainSurfer- A Novel Neurofeedback Tool for ADHD Training @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/nbcns.jpg)
David Ibanez, Laura Dubreuil and Alejandro Rier
Neurofeedback (NF) is a type of biofeedback that uses real time display of electroencephalography to illustrate brain activity. EEG features are extracted and displayed allowing the user to, with practice, modulate their temporal evolution. Neurofeedback training has many therapeutic applications such as attention deficit hyperactivity disorder (ADHD), migraine, depression or conduct disorders. This document presents NeuroSurfer, a novel general-purpose tool for neurofeedback training with a use case of attention deficit hyperactivity disorder (ADHD) treatment.
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.
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.
Andrey Panteleyev, Ph.D.
Vice Chair, Division of Molecular Biology, NBICS Centre-Kurchatov Institute, Moscow, Russia
The system of PAS proteins (HIF and AhR) as an interface between environment and skin homeostasis
Regulation of normal skin functions as well as etiology of many skin diseases are both tightly linked to the environmental impact. Nevertheless, molecular aspects of skin-environment communication and mechanisms coordinating skin response to a plurality of environmental stressors remain poorly understood.
Our studies along with the work of other groups have identified the family of PAS dimeric transcription factors as an essential sensory and regulatory component of communication between skin and the environment. This protein family comprises a number of hypoxia-induced factors (HIF-alpha proteins), aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), and several proteins implicated in control of rhythmic processes (Clock, Period, and Bmal proteins). Together, various PAS proteins (and first of all ARNT – as the central dimerization partner in the family) control such pivotal aspects of cell physiology as drug/xenobiotic metabolism, hypoxic and UV light response, ROS activity, pathogen defense, overall energy balance and breathing pathways.
In his presentation Dr. Panteleyev will focus on the role of ARNT activity and local hypoxia in control of keratinocyte differentiation and cornification. His recent work revealed that ARNT negatively regulates expression of late differentiation genes through modulation of amphiregulin expression and downstream alterations in activity of EGFR pathway. All these effects are highly dependent on epigenetic mechanisms such as histone deacetylation. Characterisation of hypoxia as a key microenvironmental factor in the skin and the role of HIF pathway in control of dermal vasculature and epidermal functions is another major focus of Dr. Panteleyev’s presentation.
In general, the studies of Dr. Panteleyev’s laboratory provide an insight into the PAS-dependent maintenance of skin homeostasis and point to the potential role of these proteins in pathogenesis of environmentally-modulated skin diseases such as barrier defects, desquamation abnormalities, psoriasis, etc.
![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.