Aug
12
Mon
2013
Plenary Talk: Watching the network change during the formation of associative memory @ Amriteshwari Hall
Aug 12 @ 9:27 am – 9:58 am

UpinderUpinder S. Bhalla, Ph.D.
Professor & Dean, NCBS, Bengaluru, India


Watching the network change during the formation of associative memory

The process of learning is measured through behavioural changes, but it is of enormous interest to understand its cellular and network basis. We used 2-photon imaging of hippocampal CA1 pyramidal neuron activity in mice to monitor such changes during the acquisition of a trace conditioning task. One of the questions in such learning is how the network retains a trace of a brief conditioned stimulus (a sound), until the arrival of a delayed unconditioned stimulus (a puff of air to the eye). During learning, the mice learn to blink when the tone is presented, well before the arrival of the air puff.

The mice learnt this task in 20-50 trials. We observed that in this time-frame the cells in the network changed the time of their peak activity, such that their firing times tiled the interval between sound and air puff. Thus the cells seem to form a relay of activity. We also observed an evolution in functional connectivity in the network, as measured by groupings of correlated cells. These groupings were stable till the learning protocol commenced, and then changed. Thus we have been able to observe two aspects of network learning: changes in activity (relay firing), and changes in connectivity (correlation groups).

Upi Bhalla Upi

Aug
13
Tue
2013
Invited Talk: A cost-effective approach to Protein Structure-guided Drug Discovery: Aided by Bioinformatics, Chemoinformatics and computational chemistry @ Sathyam Hall
Aug 13 @ 11:15 am – 11:40 am

kalKal 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.

 

Invited Talk: Pertubation of DNA topology in mycobacteria @ Acharya Hall
Aug 13 @ 11:50 am – 12:12 pm

NagarajaV. 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.

Delegate Talk: Designing electrochemical label free immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes
Aug 13 @ 3:53 pm – 4:06 pm
Delegate Talk: Designing electrochemical label free immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes

Pandiaraj Manickam, Niroj Kumar Sethy, Kalpana Bhargava, Vepa Kameswararao and Karunakaran Chandran


Designing electrochemical label free immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes

Release of cytochrome c (cyt c) from mitochondria into cytosol is a hallmark of apoptosis, used as a biomarker of mitochondrial dependent pathway of cell death (Kluck et al. 1997; Green et al. 1998). We have previously reported cytochrome c reductase (CcR) based biosensors for the measurement of mitochondrial cyt c release (Pandiaraj et al. 2013). Here, we describe the development of novel label-free, immunosensor for cyt c utilizing its specific monoclonal antibody. Two types of nanocomposite modified immunosensing platforms were used for the immobilization of anti-cyt c; (i) Self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in conducting polypyrrole (PPy) modified screen printed electrodes (SPE) (ii) Carbon nanotubes (CNT) incorporated PPy on SPE. The nanotopologies of the modified electrodes were confirmed by scanning electron microscopy (SEM). Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were used for probing the electrochemical properties of the nanocomposite modified electrodes. Method for cyt c quantification is based on the direct electron transfer between Fe3+/Fe2+-heme of cyt c selectively bound to anti-cyt c modified electrode. The Faradaic current response of these nanoimmunosensor increases with increase in cyt c concentration. The procedure for cyt c detection was also optimized (pH, incubation times, and characteristics of electrodes) to improve the analytical characteristics of immunosensors. The analytical performance of anti-cyt c biofunctionalized GNP-PPy nanocomposite platform (detection limit 0.5 nM; linear range: 0.5 nM–2 μM) was better than the CNT-PPy (detection limit 2 nM; linear range: 2 nM-500nM). The detection limits were well below the normal physiological concentration range (Karunakaran et al. 2008). The proposed method does not require any signal amplification or labeled secondary antibodies contrast to widespread ELISA and Western blot. The immunosensors results in simple and rapid measurement of cyt c and has great potential to become an inexpensive and portable device for conventional clinical immunoassays.

Aug
14
Wed
2013
Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall
Aug 14 @ 11:35 am – 11:45 am
Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall | Vallikavu | Kerala | India

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.