Aug
12
Mon
2013
Invited Talk: Biology of plant infection by Magnaporthe oryzae @ Sathyam Hall
Aug 12 @ 12:10 pm – 12:30 pm

bharatBharat B. Chattoo, Ph.D.
Professor, Faculty of Science M.S.University of Baroda, India


Biology of plant infection by Magnaporthe oryzae

The rice blast disease caused by the ascomycetous  fungus  Magnaporthe oryzae is a major constraint in rice production. Rice-M.oryzae is also emerging as a good model patho-system to investigate how the fungus invades and propagates within the host. Identification and characterisation of genes critical for fungal pathogenesis provides opportunities to explore their use as possible targets for development of strategies for combating fungal infection and to better understand the complex process of host-pathogen interaction.

We have used insertional mutagenesis and RNAi based approaches to identify pathogenesis related genes in this fungus. A large number of mutants were isolated using Agrobacterium tumefaciens mediated transformation (ATMT). Characterisation of several interesting mutants is in progress. We have identified a novel gene, MGA1, required for the development of appressoria. The mutant mga1 is unable to infect and is impaired in glycogen and lipid mobilization required for appressorium development. The glycerol content in the mycelia of the mutant was significantly lower as compared to wild type and it was unable to tolerate hyperosmotic stress. A novel ABC transporter was identified in this fungus. The abc4 mutant did not form functional appressoria, was non-pathogenic and showed increased sensitivity to certain antifungal molecules implying the role of ABC4 in multidrug resistance (MDR). Another mutant MoSUMO (MGG_05737) was isolated  using a Split Marker technique; the mutant showed defects in growth, germination and infection. Immuno-fluorescence microscopy revealed that MoSumo is localized to septa in mycelia and nucleus as well as septa in spores. Two Dimensional Gel Electrophoresis showed differences in patterns of protein expression between Wild Type B157 and MoΔSumo mutant.  We also isolated and charaterised mutants in MoALR2 (MGG_08843) and MoMNR2 (MGG_09884). Our results indicate that both MoALR2 and MoMNR2 are Mg2+ transporters, and the reduction in the levels of CorA transporters caused defects in surface hydrophobicity, cell wall stress tolerance, sporulation, appressorium formation and infection are mediated through changes in the key signaling cascades in the knock-down transformants. (Work supported by the Department of Biotechnology, Government of India)

 

 

Aug
13
Tue
2013
Plenary Talk: Biosensor and Single Cell Manipulation using Nanopipettes @ Amriteshwari Hall
Aug 13 @ 10:06 am – 10:49 am

NaderNader 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.

Invited Talk: Nanoscale Simulations – Tackling Form and Formulation Challenges in Drug Development and Drug Delivery @ Sathyam Hall
Aug 13 @ 2:15 pm – 2:40 pm

lalithaLalitha Subramanian, Ph.D.
Chief Scientific Officer & VP, Services at Scienomics, USA


Nanoscale Simulations – Tackling Form and Formulation Challenges in Drug Development and Drug Delivery

Lalitha Subramanian, Dora Spyriouni, Andreas Bick, Sabine Schweizer, and Xenophon Krokidis Scienomics

The discovery of a compound which is potent in activity against a target is a major milestone in Pharmaceutical and Biotech industry. However, a potent compound is only effective as a therapeutic agent when it can be administered such that the optimal quantity is transported to the site of action at an optimal rate. The active pharmaceutical ingredient (API) has to be tested for its physicochemical properties before the appropriate dosage form and formulation can be designed. Some of the commonly evaluated parameters are crystal forms and polymorphs, solubility, dissolution behavior, stability, partition coefficient, water sorption behavior, surface properties, particle size and shape, etc. Pharmaceutical development teams face the challenge of quickly and efficiently determining a number of properties with small quantities of the expensive candidate compounds. Recently the trend has been to screen these properties as early as possible and often the candidate compounds are not available in sufficient quantities. Increasingly, these teams are leveraging nanoscale simulations similar to those employed by drug discovery teams for several decades. Nanoscale simulations are used to predict the behavior using very little experimental data and only if this is promising further experiments are done. Another aspect where nanoscale simulations are being used in drug development and drug delivery is to get insights into the behavior of the system so that process failures can be remediated and formulation performance can be improved. Thus, the predictive screening and the in-depth understanding leads to experimental efficiency resulting in far-reaching business impacts.

With specific examples, this talk will focus on the different types of nanoscale simulations used to predict properties of the API in excipients and also provide insight into system behavior as a function of shelf life, temperature, mechanical stress, etc.

Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall
Aug 13 @ 6:39 pm – 6:49 pm
Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall | Vallikavu | Kerala | India

Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi


Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.

Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.

Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.

Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.