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
Ajith Madhavan
Assistant Professor, School of Biotechnology, Amrita University
Development of a Phototrophic Microbial Fuel Cell with sacrificial electrodes and a novel proton exchange matrix
If micro organisms can solve Sudoku and possibly have feelings, who is to say that they cannot also solve the planet’s energy crisis? Mr. Madhavan employs micro organisms to produce energy using microbial fuel cell (MFC). Micro organisms go through a series of cycles and pathways in order to survive, including the Electron Transport Pathway (ETP) in which bacteria release electrons which can be tapped as energy. In a two-chambered MFC, micro organisms interact with an anode in one chamber and in the presence of an oxidizing agent in the cathodic chamber scavenges electrons from the cathode. The two chambers are connected by an external circuit and connected to a load. In between the two chambers is a proton exchange membrane (PEM) which transports protons from the second chamber to the first and acts as a barrier for electrons. Therefore, a renewable source of energy can be maintained by just providing your bacterial culture with the proper nutrients to thrive and remain happy and satisfied (assuming they have emotions).
Mr. Madhavan has done extensive work on such MFCs and has experimented with various micro organisms and substrates to achieve high energy production. The phototropic MFC Mr. Madhavan designed using Synechococcus elongates using waste water as a substrate was able to generate approximately 10 mȦ and 1 volt of electricity. Other research in this area has even shown that using human urine can be used as a substrate for certain bacteria to produce enough energy to charge a mobile phone.
Although this microbial technology seems to be the “next big thing” (despite their small size) when it comes to renewable energy sources there is still a lot of work to be done before these bacteria batteries hit the market. As of now the MFCs are still much less efficient than solar cells and the search for the perfect bacteria and substrate continues.
![Delegate Talk: Protoplast fusion and transformation: A tool for activation of latent gene clusters @ Sathyam Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/bpbe.jpg)
Abhijeet Kate, Arpana G Panicker, Diana Writer, Giridharan P, Keshav K V Ramamoorthy, Saji George, Shailendra K Sonawane
Protoplast fusion and transformation: A tool for activation of latent gene clusters
In the quest to discover new bioactive leads for unmet medical needs, actinomycetes present a treasure trove of undiscovered molecules. The ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to sparse studies of cryptic gene clusters. These gene clusters can be tapped to explore scaffolds hidden in them. The up-regulation of the dormant genes is one of the most important areas of interest in the bioactive compounds discovery from microbial resources. Genome shuffling is a powerful tool for the activation of such gene clusters. Lei Yu, et al.1, reported enhancement of the lactic acid production in Lactobacillus rhamnosus through genome shuffling brought about by protoplast fusion. D. A. Hopwood et al.2 suggested that an interspecific recombination between strains producing different secondary metabolites, generate producers of ‘hybrid’ antibiotics. They also mentioned that an intraspecific fusion of actinomycetes protoplast bring about random and high frequency recombination. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol (PEG). In our study we had undertaken random genome shuffling by protoplast fusion of two, rather poorly expressed actinomycetes strains A (Figure 1) & B (Figure 2), mediated by PEG; and also by naked DNA transformation of Strain A protoplast with the DNA of Strain B. We generated eight protoplast fusants and seven transformants from parents considering their morphological difference from the two parent strains. These 15 recombinants were checked for their same colony morphologies for five generations to ensure phenotypic stability. Antibiotic resistance pattern was established by using antibiotic octodisc to generate a marker profile of the recombinants and the parent strains. Eight fusants (AP-18, AP-25, AP-2, AP-11, AP-14, AP-19, AP-11 and AP-27) and four transformants (TAP-30, TAP-31, TAP-32 and TAP-33) (Table 1) have shown a different antibiotic sensitivity pattern as compared to the parent strains. We envisage that these recombinants harbor shuffled gene clusters. To support array of conditions to express such shuffled/cryptic genes the recombinants were fermented in 11 different nutrient stress variants. The extracts generated were subjected to metabolite profiling by HPLC-ELSD, bioactivity screening for cytotoxicity and anti-infective capabilities. Two fusants AP-11 (Figure 3) and AP-25; one transformant TAP-32 (in growth media MBA-5 and MBA-7) displayed antifungal activity unlike parent strains (Table 2) Fusant AP-11 (Table 5) exhibited significant cell growth inhibition of five different cancer cell lines. The parents Strain A and Strain B did not exhibit any cell growth inhibition of these cell lines (Table 5). The metabolite profiling of fusant AP-11 and transformant TAP-32 was done by HPLC-ELSD. AP-11 showed the presence of five additional peaks (Figure 5 & Figure 6); TAP-32 extract from medium MBA-5 (Figure 7 & Figure 8) showed the presence of four additional peaks and TAP-32 extract from MBA-7 (Figure 9 & Figure 10) showed 14 additional peaks as compared to parent strains in similar medium and media controls. The study indicated that protoplast fusion and transformation have not only caused morphological changes but also shuffled genes responsible for synthesis of bioactive molecules. Further characterization of these new peaks is warranted.
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.
![Invited Talk: Remote Patient Monitoring – Challenges and Opportunities @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/biomed.jpg)
Jaydeep Unni, Ph.D.
Sr. Project Manager, Robert Bosch Healthcare Systems, Palo Alto, CA
Remote Patient Monitoring – Challenges and Opportunities
Remote Patient Monitoring (RPM) is gaining importance and acceptance with rising number of chronic disease conditions and with increase in the aging population. As instances of Heart diseases, Diabetes etc are increasing the demand for these technologies are increasing. RPM devices typically collect patient vital sign data and in some case also patient responses to health related questions. Thus collected data is then transmitted through various modalities (wireless/Bluetooth/cellular) to Hospitals/Doctor’s office for clinical evaluation. With these solutions Doctors are able to access patient’s vital data ‘any time any where’ thus enabling them to intervene on a timely and effective manner. For older adult population chronic disease management, post-acute care management and safety monitoring are areas were RPM finds application. That said, there are significant challenges in adoption of Remote Patient Monitoring including patient willingness and compliance for adoption, affordability, availability of simpler/smarter technology to mention a few. But experts contend that if implemented correctly Remote Patient Monitoring can contain healthcare expenditure by reducing avoidable hospitalization while greatly improving quality of care.
Lalitha 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: Development of Supercritical Fluid Chromatography methods for the replacement of existing USP Normal phase liquid chromatography methods @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/bioanalt.jpg)
Syed Salman Lateef and Vinayak A K
Development of Supercritical Fluid Chromatography methods for the replacement of existing USP Normal phase liquid chromatography methods
Normal phase liquid chromatography methods often have long run times and involve environmentally toxic/costly solvents. Supercritical chromatography methods on the other hand are faster, inexpensive, and eco-friendly. The low viscous supercritical carbon dioxide operates at high flow rates compared to LC without losing separation efficiency. In this work, SFC methods are developed to replace three United States Pharmacopeial (USP) normal phase achiral methods – prednisolone, tolazamide and cholecalciferol. System suitability parameters of the normal phase method are compared against the SFC method. Precision, linearity and robustness of the new SFC methods are demonstrated. SFC methods were found to be cost effective in terms of analysis time and solvent savings. The SFC method does not require purchase and disposal of expensive environmentally hazardous chemicals. Hence, the newly developed SFC method provides a faster and safer solution.