The National Seminar and Workshop on Research Methods washeld from July 4th to 8thattheAnnaSeminar Hall, University main campus, Maduravoyal, Chennai. The program was convened by Dr.RamaVaidyanathan, Director - Research and Development, Dr.A.P.J. Abdul Kalam Centre of Excellence for Innovation and Entrepreneurship.It aimed to bring students and research fellows together to learn and practise the experimental research methods with hands on experience on biotechnological instruments. In response to the invitation a total of 60 participants attended the plenary sessions and 33registeredparticipants (Appendix 5) attended the workshop. All participants received an agenda and an information sheet (Appendix 1).
This was a 5 days seminar integrated with hands on workshop training to provide an up-to-date experience and exposure to research instruments. The lectures, demonstrationsand hands on sessions provided a comprehensive knowledge and understanding of the ever-changing filed of experimental research.The practical sessions had combine lectures and demonstrations. The participants were given opportunity to practise procedures supported by the guidance of experts in the afternoon. Sumptuous lunch and hi tea was provided to boost energy during mid-sessions.
Dr.HepsibahSharmilcomperedthe event and the program began by welcoming all who attended. Dr.RamaVaidyanathaninvited the chief guest Dr.P.T. Manoharan, Scientist Emeritus, IIT – Madrastoinaugurate the workshop. Acknowledging the tradition, the Kuthuvilaku was lighted by the dignitaries along with Dr.MaliniPande, Dean, SPD and Dr.L.Ramesh, Additional Dean, E&T. Dr.Sreevidya, the Senior Research Scientist assisted the dignitaries to light the lamp.
Dr.RamaVaidyanathan, theconvener of the event gave an overview of the program and extended thanks tothedistinguished guest and participants from and around Chennai. She mentioned the large team of participates who had come on board as part are aiming to make the best use of the week’s seminar and workshop. She highlighted the key area of the Workshop: Bioinformatics, Industry Perspective and Real time PCR, Chemical separations, Search for new antibiotics, Fluorescence spectroscopy and Formulating a Research Project.
Day 1 – Monday 4th July 2016
Scientific Journey - Bioinformatics
Learning of Scientific Pursuit from initiation to continuity
Dr. PT Manoharan, Scientist Emeritus, IIT – Madras
Science in its current level has gone through many phases of development and has also seen diversification and has become the single source of technological development. Just as science has been the creator of new knowledge, technology has become integrated with science to the level of saying that science and technology has become synergic to each other’s continued creation and manifestation. Science can be related to philosophy while technology is related to production of materials for human benefit. But the learning of science depends on the quality of scientific institutions. Though some may believe getting a doctorate degree in science is an achievement by itself it should first have its initiation by self- promoted and devoted interest in the thinking process. But how the scientific pursuit is initiated and nurtured is of paramount importance at the doctoral level; this is influenced by institutional mechanism. Later, however, it is one’s personal efforts, motivation, interactive capability and involvement that matters but a helpful scientific environment is necessary to generate research ideas. The fundamentals of continuation of knowledge creation will depend upon each individuals’ own goals and aspirations in the ambience of other intellectuals.
Dr. Rama Vaidyanathan, Director, Research and Development, Dr.A.P.J. Abdul Kalam Centre of Excellence for Innovation and Entrepreneurship
The materials of bioinformatics are biological data, and its methods are derived from a wide variety of computational techniques. Recent years have seen an explosive growth in biological data, and the development of novel computational methods. These methods have become essential to research progress in structural biology, genomics, structure-based drug design and molecular evolution. The development and maintenance of a robust infrastructure of biological data is of equal importance if biotechnology is to take maximum advantage of research advances in a wide variety of fields. While bioinformatics has already made important contributions, it faces significant challenges as it matures.
Designing Diagnostic PCR Primers for Zika Virus
Dr. Rama Vaidyanathan
Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans. ZIKV infection diagnosis relies on serology–which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. RT-PCR has been successfully used for identification and diagnosis of arboviruses . A rapid, sensitive, and specific RT-PCR was developed and evaluated for the detection of ZIKV in L-15 medium and human serum. The analytical specificity of the assay was evaluated using RNA from 37 ZIKV isolates and from 31 isolates of 19 related flaviviruses. Amplicons of the expected size and sequences were observed only from the ZIKV samples, indicating the specificity of this assay. The detection limit of 337 pfu/ml is similar to that reported for flaviviruses assays and is low enough to detect ZIKV viraemia ranging from 103 pfu/ml to 106 pfu/ml in natural human infection. This detection limit eliminated the need for a nested PCR. In addition, the high repeatability (100%) of the assay in L-15 medium and human serum demonstrated its robustness. Moreover, the one-step RT-PCR assay was easier and faster than virus isolation and serological methods.
Using PCR machine
Mr. Krishnaraju, Research Associate
PCR is an acronym which stands for Polymerase Chain Reaction. Used in many different biological and chemical areas, it produces copies of DNA to generate thousands or even millions of copies of a particular segment. It has so many amazing uses like analyzing genes, phylogeny (studying evolutionary relationships between organisms) and detecting/diagnosing diseases. It is also used to determine genetic fingerprints which are crucial in forensic sciences and paternity testing. PCR is able to copy sections of DNA through a heating cycle. The temperature first rises to about 95oC which melts the DNA strand and splits its two sugar-phosphate backbones apart. The temperature then cools so that primers can bind to the 3’ end of each target sequence. With the assistance of free nucleotides and a DNA polymerase taq, the primers direct the synthesis of the strands. The taq polymerase continues so that at the end of the first cycle, there are two partially double stranded DNA molecules. In the second cycle, the temperature shoots back up to 95oC which melts and splits the strands once again. Like in the first cycle, taq, nucleotides and primers create partially double stranded copies of the DNA molecule: but this time the result is 4 molecules. In the third cycle, this process is once again repeated but the result is 8 molecules: 2 that are just the target sequence (that we were initially trying to copy) and 6 that are longer. The cycles continue to proceed just like this and the target molecules are produced exponentially. Before long, there are thousands of copies of the target sequence.So even though all the amazing functions of a PCR machine may seem like magic, they are purely science!
Day 2 – Tuesday 5th July 2016
Industry Perspective andReal time PCR
The Path to New Drug Discovery and Development - An Industry Perspective
Prof. M.D. Nair, Health Industry Consultant
Ever since the advent of Chemotherapy with the discovery of Salvarsan at the beginning of the last century by Paul Ehrlich, the healthcare sector has depended on the Pharmaceutical industry for the discovery and development of a variety of therapeutic agents to prevent, diagnose or treat a variety of diseases. The modality of this process has changed over time with random screening, rational design based on Structure Activity Relationships , enzyme linked approaches,Genomics ,proteomics etc, all evolving over time . All these were possible due to the continuous and dynamic efforts of the R & D based large Pharmaceutical companies , even though many discoveries were based on basic research carried out in academic laboratories. In spite of dramatic progress in the introduction of new drugs, Drug Research efforts are at the crossroads primarily due to the following reasons.
1) High costs of Research leading to unaffordable treatment costs particularly for patients from the low income countries.
2) Long gestation periods for developing a new drug from concept to market.
3) Emergence of new diseases, drug resistance and Serious Adverse Effects when used in large populations for long periods of time.
4) For economic reasons the slowing down of R& D for discovering drugs for neglected diseases mostly affecting developing countries.
5) Inadequate Understanding of most disease processes , their aetiology and progression.
6) Impact of globally harmonised Patent System on R&D and affordability of Patent Protected Drugs.
Evaluation of Diagnostic assays for mixed Plasmodium infection
Dr. Rama Vaidyanathan
A most commonly used molecular test for Plasmodium infection diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. Three PCR assays (nested, semi-nested multiplex, and one-tube multiplex) were evaluated for the simultaneous detection of human malaria parasites using experimentally mixed cocktails of known quantities of laboratory derived DNA. All three assays detected individual species with high sensitivity and specificity when DNA was from any one single species; however, experimentally mixed DNA cocktails with all four species present were correctly identified most consistently with the nested method. The other two methods failed to consistently identify all four species correctly, especially at lower concentrations of DNA -subclinical levels of malaria (DNA equivalent to or less than 10 parasites per microliter). The nested PCR method remains the method of choice for the detection of mixed malaria infections and especially of sub-clinical infections. Further optimization and/or new molecular gene targets may improve the success rate of detecting multiple parasite species simultaneously using traditional PCR assays.
Expression analysis and Real time PCR
Dr. N. Sudhakar, Associate Professor
Real-time qPCR is one of the most sensitive and reliable method for quantitation of DNA, cDNA and RNA levels in cells or tissues. It is based on detection and quantification of fluorescence emitted from a reporter molecule at real-time. The amplification of target gene during each cycle is monitored and amplification is detected at exponential phase instead of end point analysis in traditional PCR. Two commonly used methods in qPCR are SYBR Green and TaqMan probes.
SYBR Green is a fluorescent dye, which intercalates with double stranded DNA and emits fluorescence upon excitation with Laser. The excitation and emission maxima of SYBR green are at 494 nm and 521nm, respectively. Detection takes place in the extension step of Real-time PCR.
Another method is TaqMan chemistry. TaqMan chemistry uses fluorescently labeledTaqMan probe along with a forward and reverse primer for the target gene. TaqMan probes are sequence specifc oligonucleotides carrying a fluorescent reporter dye at the 5’ end and a quencher at 3’ end. TaqMan probe will be designed between a forward and reverse primer sequence in either + strand or – strand of DNA. When both the reporter and quencher are present at close proximity, there will not be any fluorescence emission. During the extension phase of PCR, Taq Polymerase by the 5’-3’ exonucleaseactivity, cleaves off the 5’ end of reporter dye. That results in detectable fluorescence that is proportional to the accumulated PCR product.
Applications: Real-time PCR is highly suited for a wide range of applications, such as gene expression analysis, SNP genotyping, allelic discrimination, DNA methylation, identification of bacterial DNA, determination of viral load, and determination of genetically modified organisms (GMO).
Day 3 – Wednesday 6th July 2016
Molecular explanation of a few common interesting natural phenomena
Dr. A.G Ramachandran Nair,Former Professor and Head, Dept. of Chemistry, Jawaharlal Institute and Pondicherry Central University
The chemical data obtained in research studies, especially inphytochemical field, have been used in explaining many natural phenomena like Colour changes in flowers, Plant ecology, Self protection of plants, Conversion of bitter orange juice to sweet one, Principle of Chemi-luminescence and Bioluminescence, function of certain Look alike molecules, etc. besides in the well known main Medicinal, Pharmaceutical &Nutraceutical fields. After a short introduction of the prospects and challenges in phytochemical studies, a few cases of some of the not so familiar ones indicated above like molecular basis of: Colour change in Hibiscusmutabilis, Conversion of bitter orange to sweet orange, Self protection by plants, Establishing feeding rights on plants by insects, Accumulation, sequestering and storage of plant poison by insects and their larvae (and even biosynthesis of the protecting molecule, by the insect), Plant interaction with animals, insects and other plants, Use of certain specific plant constituentsto facilitate pollination by insects and biosynthesis and use of the cocktail of attractant pheromones by theBolar spider to trap insects, selection of male by the female for pairing, based on the presence of a protective agent in the male, Applying the principle of Enemy’s enemy is friend by the Zea mays in atritrophic interaction, etc. will be briefly presented.
Phyto Chemical separations
Dr. N. Sreevidya, Senior Research Scientist
Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. Due to an increasing demand for chemical diversity in screening programs, seeking therapeutic drugs from natural products, interest particularly in edible plants has grown throughout the world. Botanicals and herbal preparations for medicinal usage contain various types of bioactive compounds. As extraction is the most important step in the analysis of constituents present in botanicals and herbal preparations, the strengths and weaknesses of different extraction techniques were discussed. The analysis of bioactive compounds present in the plant extracts involves the applications of common phytochemical screening assays, chromatographic techniques such as HPLC and, TLC as well as non-chromatographic techniques such as immunoassay and Fourier Transform InfraRed (FTIR).
Task 3- Microarray Data Analysis
Dr. Venil Sumantran, Adjunct Professor, IIT Madras
For the past 50 years hypothesis driven research has dominated Life Science. Research questions typically addressed problems related to a ‘cause’ and its ‘consequence’, and such studies helped us identify molecular abnormalities underlying specific diseases. Once these molecular abnormalities were understood, specific drugs were designed to attack the ‘molecular culprit/target’ and cure the disease. This line of research is responsible for the large number of drugs we take for granted today! But, this research assumes that a single gene, enzyme, or signaling pathway; causes the cellular/molecular abnormality underlying a specific disease. This assumption is challenged by the discovery that diseases such as obesity, diabetes, heart disease and even cancer; often occur in the same person. Persons with this set of diseases have “Metabolic syndrome” and actually exhibit “shared molecular abnormalities”. Thanks to the OMICS revolution, we have started unravelling the complexities of “shared molecular abnormalities”. With OMICS technologies, the entire set of genes, mRNAs, proteins, and metabolites expressed by a cell/tissue can be captured in real time. Bioinformatic programs for handling and analyzing such big data has shown that a single drug cannot treat a single disease- because each disease has sub-types. For example, different drugs are now used for treatment of different subtypes of breast cancer. This type of medicine is “personalized medicine” and we can now ask entirely different research questions about a given disease.
Lab on chromatography separations
Dr. N. Sreevidya, Senior Research Scientist
Phytochemical separation is essential to recover an isolated single compound as plant materials/crude drugs are complex mixtures of chemicals and have generally higher activity when compared to crude extracts which are the main components for the activity of the extracts. Further more separation is carried out to remove unwanted chemicals like chlorophylls, pigments, fats, waxes, oils, resins, proteins, carbohydrates etc.
Separation techniques can generally be generally carried out either by Distillation or Chromatography. Distillation is a method for the separation of the components in the liquid or for removal of volatile active components present in the crude material. The basis for separation is the different boiling points of the components and can be carried out under low pressure or using steam etc. Chromatography is a preferred method of separation for most of the phytochemicals, in which a mobile phase passes over a stationary phase in such a way that a mixture of substances is separated into its components. Most common chromatographic techniques are Column chromatography, HPLC (High pressure liquid chromatography), thin layer chromatography and Gas Chromatography.
Day 4 – Thursday 7th July 2016
Search for new antibiotics, Fluorescence spectroscopy
Search for New Antibiotics
Dr. Rama Vaidyanathan
Use of antibiotics have saved many lives and has made many medical interventions possible. However, the evolution of antibiotic resistant bacteria is leading to higher mortality and increasing costs of treatment. The discovery of new antibiotics has declined in the pharmaceutical industry. New approaches in antimicrobial discovery such as the discovery of teixobactin from uncultivable microbes, and design of artificial antimicrobial viruses based on lactoferrin will be discussed. Colloaborativeand open access approaches in antimicrobial discovery will be discussed. Finally, some of the promising drugs in the pipeline will be listed.
Methods and Techniques used – AST, MIC, Biofilm, efflux
Dr. H. Magesh, Post Doctorate
1.Antimicrobial Susceptibility Testing
An important task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. Overview of commonly used susceptibility testing method.
a. Broth dilution test
b. Antimicrobial gradient method
c. Disk diffusion test.
Experimental assay – Antibiotic sensitivity test for bacteria & yeast.
2.Screening of K. pneumoniae for biofilm formation
Biofilm - an amorphous and dynamic structure that is not only resistant to antibiotics, but also resistant to host immune clearance. Biofilm formation is a two-stage biological process controlled by surface adhesions and cell-to-cell communication pathways. Aggregated bacterial cells protected and/or coated by extracellular matrix are insensitive to both nutritional stimulation and hostile attacks.
Experimental assay - Biofilm production and quantitative methods
Using a Fluorescence Spectrophotometer
Spectrofluorimetry, as the name suggests takes the advantage of the fluorescent properties. So, before understanding about spectrofluorimetry, it is necessary to know what is fluorescence. When a molecule after absorbing radiations, emits radiation of a longer wavelength, then this phenomenon is referred to as “fluorescence.” Because of this, the compound absorbing in ultraviolet range might emit radiation in visible range. This is called Stoke’s shift wherein the shift is towards a longer wavelength. Fluorescence is an extremely short-lived phenomenon which lasts for about 10-7 seconds or less and thus can provide information about events which take less than 10-7seconds to occur.
Experimental assay – Norfloaxin Standard analysis.
Day 5 – Friday 8th July 2016
Formulating a Research Project
Are you asking the right Research Question ?
Dr. Venil Sumantran, Adjunct Professor, IIT Madras
The most important step in conducting a high-quality research study is to create a study question that will provide the guidance for the planning, analysis, and reporting of your study. The process of generating a novel, answerable study question seems like it should be simple at first blush. Perhaps the keen interest in a particular topic sparks an idea for a study that starts the creative process of hypothesizing and wondering “what if.” It is a wonderful experience to witness or be caught up in the joys of such a process. Finding inspiration for a study may, however, be a challenge, and the study idea emerges, instead, with time after thoughtful consideration of a topic. In either scenario, in order for you to design and execute your study, honing your idea and hypothesis into questions that can be realistically studied is required, adding a level of complexity to what at first seemed simple.Creating the final study question is a formal and iterative process: You create an initial study question by answering questions, defining parameters, getting feedback from colleagues, and conducting a limited literature search. Then you refine your question and define major aspects of your study by using a Patients, Intervention, Comparison, and Outcomes (PICO) table for treatment and diagnostic studies, or a Patients, Prognostic factors, and Outcomes (PPO) table for prognostic studies. By taking the time to complete these steps, you will have a good structure for your research study and will be able to proceed to the next part, a literature review.
Tips on Preparing a Research Project
Dr.Reena Das, Research Scientist
To begin any research project detailed plans are essential. Designing and planning a whole research project involves choosing a researchable, significant topic and preparing a well developed research proposal. Grant writing varies widely across the disciplines, and research intended for epistemological purposes (philosophy or the arts) rests on very different assumptions than research intended for practical applications (medicine or social policy research). It starts with the identification of broad area of interest, evaluating the resources, writing an abstract and discussing and reshaping the idea and finalizing it be narrow enough to address the issue. Collaborators can complement our skills and expertise and can serve as mentors. Finally, it can be summarized that successful grant proposals should have the following:
Reading between the lines
Dr. HepsibahSharmil, Research Scientist
Scientists read a great deal. Reading is central to their professional lives, and the amount read is a decent predictor of their success. Speaking and writing are done in addition to reading. Scientists spend almost two thirds of their informational input time performing reading activities. The time amounts to 553 hours per year or 23.2% of total work time. It is not much of a leap to conclude, time spent was an indicator of the value of the information gained from reading.Evidence show that the amount of reading by scientists has changed little over the past six decades. Further scientists rate reading as essential to their research and it’s the primary source of creative stimulation. Indeed, the award-winning and high-achieving scientists read more than others . It is, therefore, important for science education to know how well the reading of science is taught in schools. More specifically it is important to address the teaching and learning measures on how to read scientific text. To explore the reading strategy here are certain for critical thinking
It could be argued that reading and writing to learn science takes place most effectively in an integrated scientific curriculum. Alternatively, it could be argued that the kind of curriculum is irrelevant. It is important to be knowledgeable and empowered and be familiarized with steps on reading for effective academic writing. One step is to emphasis on reading learn science. Another is to work with publishers to produce flexible guides for using reading and writing activities in the learning of science. Integrated reading and writing activities play a vital role in achieving a minds-on emphasis in the learning of science. Reading and writing activities can support active, constructive learning, inquiry, and problem solving. Such effective reading activity can help individuals to cover science content in greater depth, focusing on related ideas and themes. These activities build upon their prior learning and make real-world connections. In short, the skills of reading for scientific writing serve as dynamic vehicles for learning science meaningfully.
Special Talk -My (and maybe your) Roadmap to Study Abroad
DeepikaAthinarayanan, B.Tech student shared her researchexperience which was fundamental toempower and continueher academic journey to United States tofurther her research prospects. She shared her experience aboutapplying for further studies abroad, specifically USA. She insisted on the main points to be kept in mindwhile applying for studies abroad including the timeline for the start of the process with emphasis on visaprocedures. She also spoke about the pre-preparation steps before leaving the country including bankingprocedures to make life smooth and simple for the parents as well as the students.
A lot of students aspire to study abroad to better their professional credentials and also togain a unique experience in their life. Studying abroad can seem complicated, but the process can be made simpler with good planning. The process can be made more manageable by splitting the whole process into 5 steps:
1. Research your options and determine where, what, and why you want to study abroad.
2. Plan your finances to see how much you can spend, if you need to take any loans, and
also apply for scholarships. Match universities that fit your financial plan.
3. Start preparing for the standardised tests and start filling out the college applications.
Write the statement of purpose and get letters of recommendation from professors.
4. Once you have received admission, start visa procedures and start looking for
accommodation and learning about your city.
5. Shop for clothes and other required essentials, and get ready to fly!
Resource credit: EducationUSA
Round table Discussion on Multi-centric Research Projects – What is the Secret Sauce?
Panelist:Dr.MukeshDoble, Professor, Department of Biotechnology, IIT Madras
Dr.S.Seshadri, Director – Indigenous and Frontier Technology Research Center, Chennai
Dr. Shashank Gupta, VirginiaTech, India centre for Advanced Research and Education, Chennai
Multicentre research project is a great motivator for advanced research.The panellist shared their expertise on organizing the Multicentre research project. The challenges to do collaborative multicentre research are beyond . Small departments, limited budgets, the absence of relevant research centers/programs, and few ongoing sponsored research activities ultimately makes it harder for junior scholars to learn how to organize larger multicentre research projects. They shared five ideas which might help get the ball rolling on collaboration for Multicentre research project:
Seminar and Workshop wrap-up
At the end of the five days program, Dr.Reena Das thanked everyone for their commitment in attending, and stressed that participants’ involvement and enthusiasm in updating skill was much appreciated. It was acknowledged the work of research team is to look after their members as well as looking after other co-researchers and research students. The outstanding research student award was given to DeepikaAthinarayanan and AshifaJafarullafor their project;
Titled: An evaluation of the antibacterial and antiefflux activity of the different parts of Punicagranatumect.
o Research Supervisor:Dr.Sreevidya
Research student appreciation certificate was given to the following candidates
Title:Antibiofilm properties of Natural products against products against Candida Sp.
Title: Isolation and practical characterization of Streptomycetes species with antimicrobial activity against multidrug resistant Gram Positive Bacteria
Title: Smoking behavior of adolescent school children.
o Research Supervisor:Prof.HepsibahSharmil
Dr.RamaVaidyanathan, Director R&D closed the workshop by issuing the participant certificates for those attended. Participants were advised they would all receive a copy of this report.
No Certificate in the Event
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