Microbiology World Nov - Dec 2013 ISSN 2350 - 8774

Microbiology World
Nov Dec 2013
ISSN 2350 - 8774
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Microbiology World
Nov Dec 2013
ISSN 2350 - 8774
President
Mobeen Syed, M.D. King Endward Medical University Lahore MSc. from ASD, BSc. from Punjab University D-Lab from MIT MA USA
Vice-President
Sudheer Kumar Aluru, Ph.D Human Genetics, Sri Venkateswara University, India HOD of Biology Department (Narayana Institutions)
Managing Director
Dr. D K Acharya, Ph.D Asst Prof., Biotech Dept. A. M. Collage of Science, Management and Computer Technology, India
Chief Editor
Mr. Sagar Aryal Medical Microbiology (M.Sc), Nepal
Reviewers
Mr. Samir Aga Department of Immunological Diseases Medical Technologist, Iraq Mr. Saumyadip Sarkar, Ph.D ELSEVIER Student Ambassador South Asia, Reed Elsevier (UK) M.Sc., Research Scholar (Human Genetics), India
Editors
Dr. Sao Bang Hanoi Medical University Dean of Microbiology Department (Provincial Hospital) Microbiology Specialist, Vietnam Mr. Tankeshwar Acharya Lecturer: Patan Academy of Health Sciences (PAHS) Medical Microbiology, Nepal Mr. Avishekh Gautam Lecturer: St. Xaviers College Medical Microbiology, Nepal Mr. Manish Thapaliya Lecturer: St. Xaviers College Food Microbiology, Nepal
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Table of Content
Page No. Nobel prize winners in Microbiology, Immunology and Genetics Genetic Background of Alzheimers Disease Interview with Dr. Ayush Kumar Microbiology and art: A comfortable combination? Research Process Non Treponemal Tests Cultivation of viruses: Cell culture systems Immunoglobulins in Periodontitis Online Microbiology Course 4-7 8-10 11-13
14-17 18-21 22-25 26-29 30-33 34-36
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NOBEL PRIZE WINNERS IN MICROBIOLOGY, IMMUNOLOGY AND GENETICS

2011: Bruce A. Beutler Jules A. Hoffmann Ralph M. Steinman Discoveries concerning the activation of innate immunity Discovered the role of dendritic cell in adaptive immunity 2008: the Nobel Prize was shared between Harald zur Hausen, for his discovery that human papillomaviruses can cause cervical cancer, and Franoise Barr-Sinoussi and Luc Montagnier, for their discovery of HIV 2005: Barry Marshall and Robin Warren: For the identification of Helicobacter pylori and its role in gastritis and peptic ulcer disease 2001: Leland Hartwell Paul Nurse Tim Hunt Identified key molecular steps in the cell cycle using yeast as a model organism 1997: STANLEY B. PRUSINER Discovered and characterized prions as a new biological infectious agent containing only protein and no nucleic acid 1996: The prize was awarded jointly to PETER C. DOHERTY and ROLF M. ZINKERNAGEL for their discoveries concerning the specificity of the cell mediated immune defense. 1990: The prize was awarded jointly to JOSEPH E. MURRAY and E. DONNALL THOMAS for their discoveries concerning organ and cell transplantation in the treatment of human disease. 1989: The prize was awarded jointly to J. MICHAEL BISHOP and HAROLD E. VARMUS for their discovery of the cellular origin of retroviral oncogenes.
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1987: SUSUMU TONEGAWA for his discovery of the genetic principle for generation of antibody diversity. 1984: The prize was awarded jointly to NIELS K. JERNE, GEORGES J.F. KHLER and CSAR MILSTEIN for theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies. 1983: BARBARA MC CLINTOCK for her discovery of mobile genetic elements (transposons). 1980: The prize was awarded jointly to BARUJ BENACERRAF, JEAN DAUSSET and GEORGE D. SNELL for their discoveries concerning genetically determined structures on the cell surface that regulate immunological reactions. 1978: The prize was awarded jointly to WERNER ARBER, DANIEL NATHANS and HAMILTON O. SMITH for the discovery of restriction enzymes and their application to problems of molecular genetics. 1975: The prize was awarded jointly to DAVID BALTIMORE, RENATO DULBECCO and HOWARD MARTIN TEMIN for their discoveries concerning the interaction between tumor viruses and the genetic material of the cell. 1972: The prize was awarded jointly to GERALD M. EDELMAN and RODNEY R. PORTER for their discoveries concerning the chemical structure of antibodies 1969: The prize was awarded jointly to MAX DELBRCK, ALFRED D. HERSHEY and SALVADOR E. LURIA for their discoveries concerning the replication mechanism and the genetic structure of viruses.
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1968: The prize was awarded jointly to ROBERT W. HOLLEY, HAR GOBIND KHORANA and MARSHALL W. NIRENBERG for their interpretation of the genetic code and its function in protein synthesis. 1966: The prize was awarded to PEYTON ROUS for his discovery of tumor inducing viruses 1965: The prize was awarded jointly to FRANOIS JACOB, ANDR LWOFF and JACOUES MONOD for their discoveries concerning genetic control of enzyme and virus synthesis. 1960: The prize was awarded jointly to SIR FRANK MACFARLANE BURNET and SIR PETER BRIAN MEDAWAR for discovery of acquired immunological tolerance. 1959: The prize was awarded jointly to SEVERO OCHOA and ARTHUR KORNBERG for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid. 1958: The prize was divided equally, one half awarded jointly to GEORGE WELLS BEADLE and EDWARD LAWRIE TATUM for their discovery that genes act by regulating definite chemical events and the other half to JOSHUA LEDERBERG for his discoveries concerning genetic recombination and the organization of the genetic material of bacteria. 1954: The prize was awarded jointly to JOHN FRANKLIN ENDERS, THOMAS HUCKLE WELLER and FREDERICK CHAPMAN ROBBINS for their discovery of the ability of poliomyelitis viruses to grow in cultures of various types of tissue. 1952: SELMAN ABRAHAM WAKSMAN for his discovery of streptomycin, the first antibiotic effective against tuberculosis.
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1951: MAX THEILER for his discoveries concerning yellow fever and how to combat it. 1946: HERMANN JOSEPH MULLER for the discovery of the production of mutations by means of X-ray irradiation. 1945: The prize was awarded jointly to SIR ALEXANDER FLEMING, SIR ERNST BORIS CHAIN and LORD HOWARD WALTER FLOREY for the discovery of penicillin and its curative effect in various infectious diseases. 1930: KARL LANDSTEINER for his discovery of human blood groups. 1928: CHARLES JULES HENRI NICOLLE for his work on typhus. 1919: JULES BORDET for his discoveries relating to immunity. 1913: CHARLES ROBERT RICHET in recognition of his work on anaphylaxis 1908: The prize was awarded jointly to ILYA ILYICH METCHNIKOV and PAUL EHRLICH in recognition of their work on immunity. 1907: CHARLES LOUIS ALPHONSE LAVERAN in recognition of discovery of malarial parasite. 1905: ROBERT KOCH for his investigations and discoveries in relation to tuberculosis. 1902: SIR RONALD ROSS for his work on malaria, where he discovered the life cycle of Plasmodium in mosquito. 1901: EMIL ADOLF VON BEHRING for his work on serum therapy, especially its application against diphtheria
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Genetic Background of Alzheimers Disease

Alzheimers disease, the well-known progressive neurologic disease of the brain, leading to irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. There are multiple researchers and also it has carried an importance of current research which carried an important understanding about the genetical background of Alzherimers. A research published in Journal of Neuroscience where the UK researchers, University of Kentucky identified several variations in DNA sequence, where each can modify Alzheimers risk. They identified a potential genetic variation near the gene CD33, which is thought to inhibit clearance of amyloid beta The results obtained, indicating the inhibition of CD33 may reduce Alzheimers disease. A drug was tested for acute myeloid leukemia targets CD33, suggesting the potential for treatments based on CD33 to mitigate the risk for Alzheimer's disease. Although they suggested that further studies must be conducted before this treatment approach could be tested in humans. Another research where four new genes have been identified where researchers from a consortium of 44 universities and research institutions in the United States, including Rush University Medical Center, identified four new genes linked to Alzheimer's disease. The research was published in the journal Nature Genetics.
Source: http://images.sciencedaily.com/2011/04/110403141329-large.jpg
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The genetic analysis was performed with more than 11,000 people with Alzheimers disease, along with the same number of elderly people with no symptoms of dementia. Earlier a gene of apolipoprotein E-e4, APOE-e4, identified over 15 years ago, has the largest effect on risk. Then more additional genes was identified, CR1, CLU, and BIN1. But in through this research adds more four genes MS4A, CD2AP, CD33, and EPHA1 -- and contributes to identifying and confirming two other genes, BIN1 and ABCA7, thereby doubling the number of genes known to play a role in Alzheimer's disease. The above two research investigates that CD33 p lays major role in Alzheimers disease. Hence it allows easy focus that some cell signaling ability takes up a major role behind the unrevealed story behind this disease. In the next recent research in 2013, researchers from Washington University School of Medicine in St. Louis identified several genes linked to the tau protein, which is found in the tangles that develop in the brain as Alzheimer's progresses and patients develop dementia. APOE, had been identified as a risk factor for Alzheimer's appears to be connected to elevated levels of tau. Researchers in an article said Some of the effects are mediated through amyloid -beta and others by tau. That suggests there are at least two ways in which the gene can influence our risk for Alzheimer's disease. Finally in addition to their research along with APOE, the researchers found that a gene called GLIS3, and the genes TREM2 and TREML2 also affect both tau levels and Alzheimer's risk. Regarding tau protein early research was carried out in 2009, published in The American Journal of Pathology based on the hypothesis that an unfolded protein response contributed to neurodegeneration in Alzheimer's disease partially though its effects on the accumulation of hyperphosphorylated tau, a major component of tangles in Alzheimer's disease patients. They identified that markers of the unfolded protein response were expressed in areas of tau accumulation in patients with Alzheimer's disease.
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So in conclusion of this report, it is obvious to understand that for any genetic analysis signaling pathway and protein modification towards a disease is important. Alzheimers disease which allows researchers to reach a proper research finding that Alzheimers disease is caused due to multiple gene involvement. Initially CD33 was identified which holds a major role on amyloid beta, followed by other genes too. tau protein also been identified to have so me major impact on Alzheimers, hence amyloid beta alone does not always play the role. Even in this story I pushed back a little from the solutions identified from the genetic analysis but the better understanding of correlation highlights major importance toward the disease. References:
M. Malik, J. F. Simpson, I. Parikh, B. R. Wilfred, D. W. Fardo, P. T. Nelson, S. Estus. CD33 Alzheimer's Risk-Altering Polymorphism, CD33 Expression, and Exon 2 Splicing. Journal of Neuroscience, 2013; 33 (33): 13320 Naj et al. Common variants at MS4A4/MS4A6E, CD2AP, CD33 and EPHA1 are associated with late-onset Alzheimer's disease. Nature Genetics, 2011 Carlos Cruchaga, John S.K. Kauwe, Oscar Harari, Sheng Chih Jin, Yefei Cai, Celeste M. Karch, Bruno A. Benitez, Amanda T. Jeng, Tara Skorupa, David Carrell, Sarah Bertelsen, Matthew Bailey, David McKean, Joshua M. Shulman, Philip L. De Jager, Lori Chibnik, David A. Bennett, Steve E. Arnold, Denise Harold, Rebecca Sims, Amy Gerrish, Julie Williams, Vivianna M. Van Deerlin, Virginia M.-Y. Lee, Leslie M. Shaw, John Q. Trojanowski, Jonathan L. Haines, Richard Mayeux, Margaret A. Pericak-Vance, Lindsay A. Farrer, Gerard D. Schellenberg, Elaine R. Peskind, Douglas Galasko, Anne M. Fagan, David M. Holtzman, John C. Morris, Alison M. Goate. GWAS of Cerebrospinal Fluid Tau Levels Identifies Risk Variants for Alzheimers Disease. Neuron, 2013 Hoozemans JJM, van Haastert ES, Nijholt DAT, Rozemuller AJM, Eikelenboom P, Scheper W. The Unfolded Protein Response Is Activated in Pretangle Neurons in Alzheimer's Disease Hippocampus. American Journal Of Pathology, 2009
- Mr. Saumyadip Sarkar

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Interview with Dr. Ayush Kumar
Q) Dr. Ayush Kumar Assistant Professor, Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. Starting off with your early life, how you had been keen in science during your school life in India and who had been influencing you while you were a kid? Comments: My parents had the biggest influence on me when I was growing up. Studies were a very important of my childhood primarily because my parents always kept me reminding of the important of education in life. As for the interest in Science, I think I was lucky to have some very good teachers who played a big role in piquing my interest in science.
Q) You have received both bachelors and master's degrees in Microbiology from Awadh University in Faizabad, UP, India. So how was this travel as a research scholar, studied in India and continuing research in Canada and US? Comments: The transition was surprisingly quite easy. I think it is the strength of our education system in India that allows us to flourish in any research environment.
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Q) You have done your PhD from Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada and then Post-Doctoral research at Department of Microbiology, Immunology, and Pathology, Colorado State University, Ft. Collins, CO, USA. So how was the diversity of the research background you feel in Countries Like US and Canada? Comments: It varies from institution to institution. Canada and US in general are quite similar when it comes to research, however you can see a huge difference from one institute to another within the same country.
Q) Microbes are prevailing everywhere, not only inside body but places where people might have not been reached (like on bed rocks of sea as example). Some microbes develop resistance and sometimes some are pathogenic. How would you explain in simple terms how these microbes develop resistance? Comments: The biggest reason why we are seeing more and more resistance in bacterial pathogens is irresponsible use of antibiotics. While it is becoming increasingly clear that a number of antibiotic resistance mechanisms are ancient, it is also true that improper use of antibiotics has resulted in a massive rise in antibiotic resistant organisms in recent year. So much so that a number of bacterial pathogens are now becoming untreatable with our current repertoire of antibiotics. Antibiotic resistant mechanisms in bacteria can be broadly classified into intrinsic and acquired. Intrinsic mechanisms include impermeability of the outer membrane in Gram-negative bacteria. Acquired mechanisms include are clinically very important as these can be transferred from organism to organism on mobile DNA elements, plasmids etc.
Q) Membranous proteins play an important role while understanding the mechanisms of function and regulatory pathways in developing pathogenicity. Will you please explain us through your research?
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Comments: The proteins we work on are called multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) family. While homologs of these proteins are found every organism, in Gram-negative bacteria they form a tripartite complex with two other proteins, an outer membrane protein and a periplasmic protein. Together these three proteins form a continuous channel traversing the inner membrane, periplasm, and the outer membrane of Gram-negative bacteria. The RND protein acts as an antiporter pumping out the antibiotic molecule through this tripartite structure into the external medium. The fascinating feature of these proteins is their broad substrate specificity as they are able to pump out a wide variety of antibiotics with no structural similarity.
Q) Your Lab renown preferably as Kumar Lab. So will you Introduce with your co-researchers as well as provide some information highlighting the development of your lab? Comments: I currently have one PhD student, one Masters student, and an undergraduate researcher in my lab.
Q) What would be your personal message towards young researchers and students of Microbiology? Comments: Microbiology is a very vast field and it continues to grow. My advice would be find your passion and follow it with all your heart.
Q) Finally concluding with the questions, would like to express your life apart from your work? Comments: I love to cook and watch cricket.
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Microbiology and art: a comfortable combination?

Art provides an opportunity for visualisation and communication of science, and I have found that in this context and others, microbiology and art link very well. Over the past few years, I have collected examples of this interdisciplinarity, and produced a lecture which I give to first year Biology undergraduates. The lecture is one of a series given during a module entitled Frontiers in Biology. In the module, a lecture is presented by each specialist subject area within our School, and students sign up to an Figure 1: Model of influenza virus and accompanying PowerPoint presentation assignment in any of the six subjects. For all other subjects, the accompanying assignment is a poster plan. For microbiology, students have the opportunity to produce, alone or in groups, an item to illustrate some link between art and microbiology. There is no upper limit to the number of students who can take this option; students can work alone or in groups. Ideas are discussed during a tutorial, and assessment criteria are negotiated between myself and the student(s). The outcomes are diverse, often creative and inspiring, and several have been used to illustrate this article.
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The topics covered in the lecture, entitled Microbiology and Art: the Final Frontier are: Deterioration of Art Microbially-induced spoilage of art and heritage material is perhaps the most obvious link between the subjects, but I also describe aspects of prevention and control of such deterioration, and give examples of microbiologically-induced remediation. Students were especially interested to hear of this unusual example of applied microbiology, and several opted to do assignment work on, for example, spoilage of film, ancient Egyptian art and stone monuments, and on some of the EU funded conservation projects. Their work was primarily presented in poster format. Beauty of microorganisms The excitement generated when a good specimen is found down the microscope can be infectious: it is not only the success of finding the microorganism sometimes they look really nice! Indeed, sophisticated imaging techniques enable differentiation of components of microbial communities, or individual cells, Figure 2: Tuberculosis and mid-20th century graphics, accompanied and such images are common in by text describing links between the disease and the arts textbooks and in lecture presentations. Images can be enhanced and modified, and find a diverse range of uses: calendars (e.g. www.veeco.com), websites, graphic art posters, clothing (www.iawareables.com), toys (www.giantmicrobes.com) collage and so on. Student products included three-dimensional models; customised lab coats; silk paintings; and designer jewellery!
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Microbiology World Microorganisms in art
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Surprisingly, microorganisms themselves may provide material as well as inspiration for art: pigmented bacteria can be used to paint images on agar plates; pictures made on microscope slides from diatoms can be purchased for educational use (www.diatoms.co.uk); models of toadstools can be found in many a craft shop, with more scientifically accurate models also available for purchase, or viewing (www.hps.cam.ac.uk/whipple/explore/models/glassfungi; www.britmycolsoc.org.uk/resources.asp). The 20-sided polygon, the icosahedron, provides the maximum volume for the minimum building material, thus is an ideal structure for both viruses and geodesic buildings such as the Eden project. During the assignment, some students produced 3-D models of viruses (Figure 1): one group ingeniously used the clear balls used for guinea pig exercise, as the transparent envelope! The consequences of bacterial and viral diseases, rather than the microorganisms themselves, provide ideal subjects for visualising the destruction wreaked by plagues through history (e.g. www.wellcome.ac.uk), and upon individuals (e.g. The Inheritance by Edvard Munch). Students created PowerPoint presentations outlining the influence of plague on contemporary art; produced conceptual images using FISH technology; painted a representation of the history of science; produced a collage in 1930s style of the importance of tuberculosis in literature (Figure 2); designed panels for the AIDS quilt; profiled artists who interpret science through the medium of paint, constructed a large model of influenza virus, accompanied by PowerPoint lecture notes and smaller cutaway models, for use in lectures. Of course, not all products are aesthetically pleasing, nor do they meet the negotiated assessment criteria (Figure 3).
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Microbiology World Combining Microbiology and Art
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Previously, projects in poster and leaflet design (Verran, 1992, 1993) have incorporated consideration of communication, cost and co-operation with peers. More ambitious cross disciplinary projects involved undergraduates in art and biology working together to Figure 3: Fairly anaesthetic and poorly conceived collage to demonstrate use of design an artwork for the fungi (wrappers, labels etc): unfortunately the real mushrooms which were also part of the collage went mouldy, necessitating the disposal of the piece! foyer of a new science building. Although not successful due to the cost implications, one proposed installation considered the importance of repeating, yet evolving structures in all aspects of science (fractals, DNA, evolution, polymers). Discussions with artists at the University have led to projects where the outcome has been artwork, exhibition, or installation, rather than scientific paper, report or presentation. Postgraduate art students have also used microorganisms and/or principles of microbiology as part of their project work. Such SciArt partnerships are not uncommon, and the Wellcome Institute is particularly interested in this type of development. Joanna Verran School of Biology, Chemistry and Health Science, Manchester Metropolitan University, Manchester M1 5GD
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Research Process
In research process, the following order concerning various steps provides a useful procedural guideline regarding the research process: (1) formulating the research problem; (2) extensive literature survey; (3) developing the hypothesis; (4) preparing the research design; (5) determining sample design; (6) collecting the data; (7) analysis of data; (8) hypothesis testing; (9) generalisations and interpretation, and (10) preparation of the report or presentation of the results, i.e., formal write-up of conclusions reached. 1. Formulating the research problem: There are two types of research problems, viz., those which relate to states of nature and those which relate to relationships between variables. At the very outset the researcher must single out the problem he wants to study, i.e., he must decide the general area of interest or aspect of a subject-matter that he would like to inquire into. Initially the problem may be stated in a broad general way and then the ambiguities, if any, relating to the problem be resolved. Then, the feasibility of a particular solution has to be considered before a working formulation of the problem can be set up. The formulation of a general topic into a specific research problem, thus, constitutes the first step in a scientific enquiry. Essentially two steps are involved in formulating the research problem, viz., understanding the problem thoroughly, and rephrasing the same into meaningful terms from an analytical point of view. 2 Literature review: Once the problem is formulated, a brief summary of it should be written down. It is compulsory for a research worker writing a thesis for a Ph.D. degree to write a synopsis of the topic and submit it to the necessary Committee or the Research Board for approval. At this juncture the researcher should undertake
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extensive literature survey connected with the problem. For this purpose, the abstracting and indexing journals and published or unpublished bibliographies are the first place to go to. Academic journals, conference proceedings, government reports, books etc., must be tapped depending on the nature of the problem. In this process, it should be remembered that one source will lead to another. The earlier studies, if any, which are similar to the study in hand should be carefully studied. A good library will be a great help to the researcher at this stage. 3. Development of working hypotheses: After extensive literature survey, researcher should state in clear terms the working hypothesis or hypotheses. Working hypothesis is tentative assumption made in order to draw out and test its logical or empirical consequences. As such the manner in which research hypotheses are developed is particularly important since they provide the focal point for research. They also affect the manner in which tests must be conducted in the analysis of data and indirectly the quality of data which is required for the analysis. In most types of research, the development of working hypothesis plays an important role. Hypothesis should be very specific and limited to the piece of research in hand because it has to be tested. The role of the hypothesis is to guide the researcher by delimiting the area of research and to keep him on the right track. It sharpens his thinking and focuses attention on the more important facets of the problem. It also indicates the type of data required and the type of methods of data analysis to be used. 4. Preparing the research design: The research problem having been formulated in clear cut terms, the researcher will be required to prepare a research design, i.e., he will have to state the conceptual structure within which research would be conducted. The preparation of such a design facilitates research to be as efficient as possible yielding maximal information. In other words, the function of research design is to provide for the collection of relevant evidence with minimal expenditure of effort, time and money. But how all these can be achieved depends mainly on the research purpose. Research purposes may be grouped into four categories, viz., (i) Exploration, (ii) Description, (iii) Diagnosis, and (iv) Experimentation. A flexible research design which provides opportunity for considering many different aspects of a problem is considered appropriate if the purpose of the research study is that of exploration. But when the purpose happens
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to be an accurate description of a situation or of an association between variables, the suitable design will be one that minimizes bias and maximizes the reliability of the data collected and analyzed. 5. Determining sample design: All the items under consideration in any field of inquiry constitute a universe or population. A complete enumeration of all the items in the population is known as a census inquiry. It can be presumed that in such an inquiry when all the items are covered no element of chance is left and highest accuracy is obtained. But in practice this may not be true. Even the slightest element of bias in such an inquiry will get larger and larger as the number of observations increases. Moreover, there is no way of checking the element of bias or its extent except through a resurvey or use of sample checks. Besides, this type of inquiry involves a great deal of time, money and energy. Not only this, census inquiry is not possible in practice under many circumstances. For instance, blood testing is done only on sample basis. Hence, quite often we select only a few items from the universe for our study purposes. The items so selected constitute what is technically called a sample. 6. Collecting the data: In dealing with any real life problem it is often found that data at hand are inadequate, and hence, it becomes necessary to collect data that are appropriate. There are several ways of collecting the appropriate data which differ considerably in context of money costs, time and other resources at the disposal of the researcher. Primary data can be collected either through experiment or through survey. If the researcher conducts an experiment, he observes some quantitative measurements, or the data, with the help of which he examines the truth contained in his hypothesis. But in the case of a survey, data can be collected by any one or more ways. 7. Analysis of data: After the data have been collected, the researcher turns to the task of analyzing them. The analysis of data requires a number of closely related operations such as establishment of categories, the application of these categories to raw data through coding, tabulation and then drawing statistical inferences. The unwieldy data should necessarily be condensed into a few manageable groups and tables for further analysis. Thus, researcher should classify the raw data into some
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purposeful and usable categories. Coding operation is usually done at this stage through which the categories of data are transformed into symbols that may be tabulated and counted. Editing is the procedure that improves the quality of the data for coding. With coding the stage is ready for tabulation. Tabulation is a part of the technical procedure wherein the classified data are put in the form of tables. The mechanical devices can be made use of at this juncture. A great deal of data, especially in large inquiries, is tabulated by computers. Computers not only save time but also make it possible to study large number of variables affecting a problem simultaneously. 8. Hypothesis-testing: After analyzing the data as stated above, the researcher is in a position to test the hypotheses, if any, he had formulated earlier. Do the facts support the hypotheses or they happen to be contrary? This is the usual question which should be answered while testing hypotheses. Various tests, such as Chi square test, t-test, F-test, have been developed by statisticians for the purpose. The hypotheses may be tested through the use of one or more of such tests, depending upon the nature and object of research inquiry. Hypothesis-testing will result in either accepting the hypothesis or in rejecting it. 9. Generalizations and interpretation: If a hypothesis is tested and upheld several times, it may be possible for the researcher to arrive at generalization, i.e., to build a theory. As a matter of fact, the real value of research lies in its ability to arrive at certain generalizations. If the researcher had no hypothesis to start with, he might seek to explain his findings on the basis of some theory. It is known as interpretation. The process of interpretation may quite often trigger off new questions which in turn may lead to further researches. 10. Preparation of the report or the thesis: Finally, the researcher has to prepare the report of what has been done by him. Writing of report must be done with great care keeping in view the following: i. Draft report ii. Final report - Posh Raj Khanal St. Xaviers College, Nepal
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Non Treponemal Tests

Syphilis Syphilis is a sexually transmitted infection caused by the spirochete bacterium Treponema pallidum subsp. pallidum. The primary route of transmission is through sexual contact It may also be transmitted from mother to foetus during pregnancy or at birth resulting in congenital syphilis Syphilis is difficult to diagnose clinically in early stage Confirmation is either via. Serological tests or direct microscopic examination. Serological test is divided into non-treponemal and treponemal test
Non Treponemal Tests After infection, host forms treponemal antibodies to T.pallidum ; in addition, the host also forms non treponemal anti-lipoidal antibodies in response to lipoidal materials released from the damaged host cells and to lipid from the cell surface of Treponema itself. These antibodies are, traditionally, referred to as Reagins. NTT refers to a type of test that looks for non-specific (non-treponemal) antibodies in the blood of patient that indicates that the organism (T.pallidum)that caused syphilis is present NTTs are widely used for qualitative syphilis screening. These tests are based on Ag-Ab reaction,ie, flocculation test and complement fixation test. Reagin antibodies in the patient serum is detected by cardiolipin antigen. Cardiolipin antigen is an alcoholic extract of bovine heart muscle to which lecithin and cholesterol are added. Cardiolipin reacts with non-specific reagin.
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Flocculation test a)VDRL b)RPR c)TRUST d)KT VDRL (Venereal Disease Research Laboratory) test It is the most widely used simple and rapid test. It was developed by Harris, Rosenberg and Riedel in 1946. It is a type of slide flocculation test. It can be done on serum sample or csf. It can also be used to detect congenital syphilis. It becomes positive 1-2 weeks after appearance of primary lesion (chancre). Test becomes reactive(50-75%) in late phase of primary syphilis, highly reactive(100%) in secondary syphilis & reactivity decreases (75%) thereafter.
Qualitative test atient serum is inactivated by heating at C for 30 mins to destroy the complements. Freshly prepared VDRL antigen is used which is a alcoholic solution of 0.03% cardiolipin, 0.21% lecithin and 0.9% cholesterol. Add exactly 1 free-falling drop (17 l) of antigen suspension in each circle. Place the card on the rotator and rotate the card at 140 rpm for 4 mins. The results are then checked under the microscope. The highest dilution showing flocculation is considered as reactive titre.
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RPR (Rapid Plasma Reagin) test It is a macroscopic non-treponemal floculation card test to screen syphilis. Also called RPR 18mm circle card test. Antigen used is VDRL antigen which is coated to charcoal or carbon particles. Addition of charcoal enables the result to be read visually (by eye) instead of microscopically. Unheated serum is used. If Abs are present, Ab combines with lipid particles of Ag & show up as black clumps against white card. If Ab is absent, the test mixture is uniformly gray. TRUST (Toludine Red Unheated Serum Test) It is a modification of RPR card test It has got an additional advantage in hot climates as the antigen is more stable than RPR antigen and can be stored at RT. The TRUST antigen suspension is based on the VDRL antigen but contains a toluidine red pigment. When a specimen contains antibody, the TRUST antigen agglutinates and the reaction appears as red clumps against the white background of the test cards. The test is read macroscopically using a high intensity incandescent lamp. If antibody is not present, the test mixture remains a faint red color and no agglutination occurs.
WR (Wassermann Reaction) It is a type of complement fixation test. named after the bacteriologist August Paul Von Wassermann, in 1906. Test serum is inactivated by heating at C for half an hour before the test to destroy the complement activity of test serum. Antibody coated red cells is used as indicator system.
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Complement lyses antibody coated red cells and hemolysis ocurs. A fourfold or greater rise in reagin titre usually occurs during evolution of primary syphilis. A titre of 32 or higher is ususlly seen in secondary syphilis. An increase in reagin titre with time may be used to confirm a diagnosis of congenital syphilis. A persistant fall in reagin titre following treatment of early syphilis can be used to monitor efficacy of antibacterial therapy.
Limitations of NTTs Their usefulness is limited by decreased sensitivity in early primary syphilis and during late syphilis. With NTTs, false positive rxns can occur in diseases such as leprosy, malaria, toxoplasmosis, tuberculosis, infectious mononucleosis, SLE, hyperglobulinemia, viral infections(HIV, measles, vaccinia, varicella, hepatitis, epstein barr virus infection). IV drug users, pregnant woman & elderly may have false positive rxns. They may also show false negative rxn when patients Ab titre is very high(prozone effect). Because of issues with false positives, a reactive nontreponemal test does not confirm T. pallidum infection without some other evidence for the diagnosis of syphilis . If positive results are obtained, the more specific treponemal testing (FTAABS, MHA-TP, etc.) should be performed. - Angelica Rajbhandari St. Xaviers College, Nepal
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Cultivation of viruses
Viruses can replicate only in living cells. Different methods are used: Cell culture Tissue culture Organ culture Chick Embryo culture Animal Inoculation
Cell culture systems

Cell cultures are prepared either as single layers (monolayers), on a glass or plastic surface, or as suspensions, the choice depending on their proposed used. Cell monolayers are most commonly used for the culture of viruses. There are three categories, namely a) primary, b) semi-continuous, and c) continuous cell cultures a) Primary cultures:
Viable cell suspensions may be obtained by dissociaating tissues or organs, e.g. human amnion, with trypsin, collagenases or other enzymes such as versene. Tissues from young or embyonic animals give better results than those from adults. When placed in culture vessels with nutrient media, viable cells adhere to the wall of the vessel and begin to multiply. Multiplication ceases when neighbouring cells touch (contact inhibition), resulting in the formation of a sheet of cells, one cell deep (a monolayer). Because of metabolic activity in such a culture is low, the accumulation of acid in the medium is low, which in turn means that the cells are easily maintained. The cells in primary cultures generally possess a diploid complement of chromosomes characteristic of the tissue cells of the donor animal. Although particularly useful for the isolation of viruses such as the echoviruses or orthomyxoviruses, primary culture techniques have several practical disadvantages. Cultures have to be prepared de novo from fresh tissue samples,
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which may either be difficult to obtain or may require special care in preparation. The presence of contaminating or enodgenous viruses, latent in the animal host, could be hazardous and may give difficulties with cell growth and virus isolation. Primary cultures established from the organs of different individuals of the same animal species may vary in their ability to support replication of the same virus. Primary cell cultures cannot be subcultured repeatedly. b) Semi-continuous cell cultures (cell strains):
Semi-continuous cell cultures are established with the successful subculture of primary cell monolayers. With methods currently available to the diagnostic laboratory, these cultures consist mostly of spindle shaped fibroblastoid cells (Euploid cells). When established from human embryonic tissue or neonatal foreskin, these semi-continuous cell cultures may undergo up to 50-100 population doublings before senescence and death of the culture. Although semi-continuous cultures area easily eastablished and provide abundant source of cells, they may not be as susceptible to some viruses as true primary cultures; also at higher passage levels, the cells may vary in their ability to support virus replication. Examples of fibrolast strains are rhesus fetal lung diploid cells or chicken fibroblasts. MRC-5 and WI-38. MRC-5 human diploid fibroblasts have been used for the viral vaccine preparation for killed HepatitisA, killed Rabies, live Varicella and WI-38 has been used for the vaccine preparation of Live Adenovirus, Live Rubella. Rhesus fetal lung diploid cells or chicken fibroblasts are used for the killed viral vaccine of Rabies virus. c) Continuous cell cultures (cell lines):
Cell lines are also derived from primary cells, but can be repeatedly subcultured. These continuous cell lines are aneuploid cells scuh as those from many human and animal tumours and these cells can be subcultured indefinitely. Aneuploid cell lines have a chromosome complement between that of diploid and tetraploid cells. Indefinite subculture can be done on glass or plastic sufaces. Some lines will also grow in suspension culture, unlike primary or semi-continuous cell cultures which require an anchorage on glass or plastic. Continuous cultures are produced either
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by transformation (spontaneous or engineered) of cell strains in vitro, or by culture of cells taken form tumours. The number of cells required to initiate a culture on glass or plastic is low compared with that for primary cells or semi-continuous cell strains (i.e. high plating efficiency). Relative to the latter, requirements for serum or other nutrients are less, growth rate is faster and contact inhibition is lost. Consequently the cells have a tendency to overgrow, and often require more frequent attention than those in primary cultures. Although the cells of continuous lines are not necessarily tumorogenic in vivo, they are effectively immortal in vitro and their chromosome complement is heteroploid (aneuploid). Patterns of respiratory metabolism are altered with increased production of acid from glucose. The ease of propagation of continuous cell lines is to some extent offset by the need to maintain the pH is of the culture around 7.0 by addition of buffer, or changing the medium, or both. Without this attention cells die or become detached (metabolic degeneration). Control of pH is effected by the addition of sodium bicarbonate to the medium, and incubation of the cells in a carbon dioxideenriched atmosphere. Usually some phosphate buffer is also present. The zwitterionic buffer N-2-hydroxyethylpiperazine-N-3-ethanesulphonic acid (HEPES) is useful because it overrides all other buffers present and obviates the need, in many instances, for a CO2- enriched atmonsphere. This allows incubation of open cell cultures (e.g. in multi-well plates) in a humidified incubator. Following are the continuous cell lines commonly used for cultivation of different viruses: Cell lines Viruses cultivated HeLa (Human epithelial cell line of Respiratory syncytial virus, cervical carcinoma) Adenovirus, HSV, Poliovirus and some Coxsackie viruses HEp-2 (Human epithelial type 2 Adenovirus, HSV, Poliovirus and cells) some Coxsackie viruses BHK 21( Baby Hamster Kidney Lymphocytic choriomeningitis cells) (LCM) virus, Lassa virus, Rubella virus MDCK (Dog kidney cell line) Influenza virus, Oral and killed
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polio vaccine production PBC (peripheral blood cells) HIV AGMK ( African green monkey Rubella Kidney) Vero (monkey kidney cell line) HSV, Lassa, LCM, Dengue, yellow fever virus, bunya virus, Rift valley fever virus, Colorado Tick Fever virus and other hemorrhagic fever viruses and arboviruses HEK (human embyonic Kidney) VZV, Measles, Adenovirus, PMK(Primary monkey Kidney) Mumps, Enterovirus, Influenza virus Cell Culture Viruses cultivated HFF (human foreskin fibroblasts) CMV semi continuous cell line culture RD (Rhamdomyosarcoma cell line) CoxsackieA virus LLC-MK2 (Rhesus monkey kidney) Parainfluenza virus
Insect cell lines are continuous cultures derived from, for example, the SF9 ovary (fall army worm, Spodoptera frugiperda) and from mosquitoes (e.g. Aedes aegypti and Ae. albopictus). The SF9 line, which is grown in special media, is highly susceptible to infection with baculoviruses and can be used for baculovirus expression vectors and other needs. Mosquito lines are used as suspension cultures for the replication of many mosquito-borne viruses; they have special media and subculturing requirements. - Avishekh Gautam St. Xaviers College, Nepal
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Immunoglobulins in Periodontitis
Studies on the immunological diagnosis are gaining importance due to their specificity and reliability. So an effort is constantly made in this direction globally to identify periodontal diseases at a most early stage possible. Periodontal disease is considered to be a mixed infection wherein the pathogens act directly or indirectly in the destruction of the tooth-supporting tissues. The host reacts to this bacterial challenge by activating its defense mechanisms in an attempt to localize and eventually eliminate the pathogens. The immune responses can be mediated either by antibodies (humoral) or by sensitized lymphocytes (cellular). Our understanding of dental plaque biofilm has evolved since the non-specific plaque hypothesis that considered plaque as a nonspecific mass of native microorganisms that, because of lack of oral hygiene, builds up in great Source: 1 http://www.sageproducts.com/prod proportions to overcome the host resistance limit and ucts/oral-hygiene/biofilms.cfm thereby affecting the tooth structure and tooth supporting tissues. A great diversity of microorganisms, over 700 species was detected in the oral cavity and evidence shows that the investigation of specific microorganisms as etiological agents for periodontal diseases and caries is not a simplistic approach. Although oral mechanical hygiene is fundamental to control caries and prevent periodontal disease, it is important to highlight that optimal control is not achieved by most individuals. Thus, a complementary use of chemotherapeutic agents has been investigated to overcome the deficiencies of mechanical oral hygiene habits, insofar as they reduce both plaque formation and gingival inflammation, and
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represent a valid strategy to change the biofilm and maintain dental and periodontal health. Antibodies belong to the third fastest migrating group of serum globulins, the gamma globulins. The term Immunoglobulin (Ig) refers to the immunityconferring portion of the gamma globulin fraction. Based on physicochemical and antigenic differences, five classes of immunoglobulins have been recognized IgG, IgA, IgM, IgD and IgE. These immunoglobulins contribute to the inhibition of bacterial adherence and colonization, enhance bacterial phagocytosis, and help detoxify bacterial toxins and thus play a major role in the defense against bacterial infections. The inflammatory and immune responses clearly contribute to the maintenance of homeostasis between the host and the microbial biofilm of the periodontium. For the host to maintain homeostasis within the oral cavity, three distinct but interrelated immune responses contribute to controlling the microbial challenge. These are the salivary and gingival tissue (local) and the serum (systemic) immune systems. According to Lehner, immunological responses (through local secretory and systemic serum antibodies) can be mediated by three related fluid compartments: Saliva, crevicular fluid and blood. Hence, immunoglobulins if present, should be detected in these fluid compartments Studies with evaluation of either serum or salivary quantitation of immunoglobulins have provided varying results. Some studies revealed increased serum IgG, IgA and IgM in patients with periodontitis, while others showed no significant differences in serum Ig levels between periodontitis patients and healthy individuals. A study conducted by Kaslick et al. revealed increased levels of serum IgA, IgG and IgM in periodontosis patients, but paradoxically 41% of patients had no increase in IgG, IgA or IgM levels. Studies revealed increased salivary IgA in periodontitis patients, elevated salivary IgG and A levels in severe periodontitis patient, and another study showed salivary IgG and IgA to be elevated in juvenile periodontitis patients. Contradicting these studies, study by Basu MK et al. revealed decrease in salivary IgA in periodontitis patients compared to healthy individuals. Study by Bratthal GT and Ellen RP revealed
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elevated salivary and crevicular antibodies to periodontal pathogens after conventional gingivitis treatment. Reiff RL stated that levels of salivary and serum Ig G and A declined after Phase I therapy, but in the same study, some study subjects revealed elevations in the immunoglobulin levels after therapy. Basu MK et al. observed higher salivary IgG and lower salivary IgA levels in periodontitis patients before oral hygiene therapy. The concentrations of these immunoglobulins after periodontal therapy was comparable with those found in clinically normal individuals. Discussion It is pretty much agreed that the immune system is involved in the pathogenesis of periodontal disease. The literature is replete with studies involving the immunoglobulin levels in different forms of periodontal diseases and these studies have yielded varying results. In few studies, individual variations have been observed with regard to the immunoglobulin levels, i.e., although the serum and salivary levels are elevated in most of the cases, there are some exceptions where the levels were lesser than the controls, but still within the normal range. After therapy, in some cases there was a decline in the levels while in some others there was an increase. One of the possible causes for this may be the individual patient variation with respect to the oral microflora present at the time of sampling, the varying degrees of periodontal pathology and varying degrees of inflammation present at the site. The role of immunoglobulins in the pathogenesis of periodontitis is still not clear. Several unanswered questions still remain. What stage of infection will be able to elucidate or detect the antibody? i.e., it is not clear at what point in the infection and subsequent disease process the initial seroconversion occurs. Once detected should it be considered as a sign of improvement in the condition or decline in the condition? How is the antibody associated with active disease? and can early immune responses be detected prior to gross infection to enable early institution of therapeutic modalities? Also, the effects of therapy on the levels of
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immunoglobulins are not clear. Whether the raise in the immunoglobulin levels after Phase I therapy induced by the scaling procedure is beneficial is unanswered. The long-term study of the disease process from its inception and in its various stages may provide answers to the questions raised. Further long-term studies with a larger sample population and with advanced immunological techniques have to be undertaken to understand the role played by the immunoglobulins in the pathogenesis of periodontitis, to define at-risk population, which may help to use immunological data for diagnosis, classification and monitoring of periodontal diseases. Long-term follow-up studies shall shed light on the changes in the immunoglobulin levels following various treatment modalities employed for treatment of periodontal diseases. Sources: 1. Srinivasan PC (2012). Immunoglobulin Levels and Periodontal Diseases-A Clinical Immunological Study. 1: 254. doi:10.4172/scientificreports.254 2. Landi L, Amar S, Polins AS, Van Dyke TE (1997) Host mechanisms in the pathogenesis of periodontal disease. Curr Opin Periodontol 4: 3-10. 3. Sahingur SE, Cohen RE (2004) Analysis of host responses and risk for disease progression. Periodontol 2000 34: 57-83. 4. Ananthanarayan R, Paniker CKJ (1990) Antibodies-Immunoglobulins. In: Textbook of Microbiology. (4th edn), Orient Longman Ltd, Madras, PP: 84-91. 5. Rode Sde M, Gimenez X, Montoya VC, Gmez M, Blanc SL, Medina M, Salinas E, Pedroza J, Zaldivar-Chiapa RM, Pannuti CM, Cortelli JR, Oppermann RV. Braz Oral Res. 2012;26 Suppl 1:133-43. Daily biofilm control and oral health: consensus on the epidemiological challenge--Latin American Advisory Panel.
- Sudheer Aluru
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Online Microbiology Course

Course Name: DNA - From Structure to Therapy Dr. rer. nat. habil. Susanne Illenberger and Prof. DPhil. Sebastian Springer Jacobs University Bremen About this course This course explains one of the key molecules in life: Deoxyribonucleic Acid (DNA). DNA stores the genetic information in all living cells. The sequence of its building blocks defines both individual identity and species diversity. Changes in DNA can lead to cancer and other diseases. DNA-based technology is now used to detect and treat diseases. Our Journey In this course, we will take you on a journey from the DNA molecule to the development of novel therapies. We will first look at some historical aspects and key experiments such as how DNA was identified and proven to be the keeper of genetic information. We will then describe how DNA becomes duplicated (when cells divide) and how the genetic information stored on DNA is organized into genes. Next, we will explain how genes become transcribed into a messenger molecule (mRNA) and eventually translated into proteins that carry out the actual cellular functions. With this basic knowledge, we will then look at simple DNAbased techniques and how they are employed not just in basic research but also in everyday life: for the analysis of food, in crime scene investigations and forensics, for the diagnosis of genetic diseases, and for therapy development in modern molecular medicine. When students have completed the course, they will know a lot about structure, function, and uses of DNA, and they will understand the DNArelated words that are often used in the news (for example "gene", "mutation", "DNA fingerprint").
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Course Structure The course will be organized into 14 units of approx. 15 min each. The units will follow a common scheme with a short introductory sequence that discusses the importance of the topic covered. The main part of the lecture will have molecular models, power point animations, live drawings, and short lecture video sequences. At the end of each unit, we will provide a question catalogue and links to supporting material. Immediate self testing will be possible through multiple choice questions and interactive tasks. As homework, students will work on "open questions" in student-centered discussion groups. Open questions are based on but not restricted to the material covered in the course, and students will be encouraged to do their own reading to answer them. The fourteen units comprise the following topics: 1. How DNA is present in everyday life (DNA History I) 2. DNA History II 3. The structure and properties of DNA DNA fingerprint 4. Replication the copying of DNA 5. From DNA to Protein I - Transcription of Genes 6. From DNA to Protein II - RNA processing 7. From DNA to Protein III translating the genetic code 8. Methods I making Genes visible 9. Methods II amplifying Genes 10. Introduction to Genetic Diseases - Cancer 11. Consequences of Mutations 12. How to repair mutations? 13. Muscular Dystrophies - when muscles die 14. Stem Cell Therapies replacing defective cells
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Learning Outcomes Participants will be able to answer the following questions after completing the course: 1. Why and how has DNA become one of the most powerful tools in research? 2. What exactly does gene therapy mean? 3. Why is it so hard to find a cure for cancer? 4. For which diseases may DNA-based technology help developing a therapy? 5. What are potentials and risks in gene-based medicine? Prior Knowledge Participants that possess an interest in modern biomedical research will be able to grasp the key concepts without strong background knowledge. In order to pass the final exam, though, high school level knowledge in chemistry and biology will be advantageous when we draw chemical structures or address cell biological questions.
Enroll Free From: https://iversity.org/c/10?r=43d1f
This is the Free Online Course offered by: iversity https://iversity.org
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You can also send your articles to info@microbiologyworld.com or broneps1@gmail.com Selected ones will be published in our next issue of Jan-Feb. Thanks, Microbiology World Team
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Cell culture systems