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Curriculum Vitae

Prashant Kumar Singh


Senior Research Fellow (S.R.F.), Division of Parasitology, Central Drug Research Institute (CDRI), Chhattar Manzil Palace, M.G. Road, Lucknow (U.P.), I DIA 226 001. Email: prashantcdri@gmail.com Contact o.: +91-9795998745

Research Experience: 1. April, 2009 April, 2102 : Senior Research Fellow at Division of Parasitology, Central Drug Research Institute, Lucknow, India. 2. April 2007 April 2009 : Junior Research Fellow at Division of Parasitology, Central Drug Research Institute, Lucknow, India.

About doctoral research work: Ph.D. in Biotechnology (Thesis submitted) Institution: Central Drug Research Institute, Lucknow (U.P.), India. University: Jawaharlal Nehru University, New Delhi, India. Supervisor: Dr. (Mrs.) Shailja Bhattacharya, Scientist G, CDRI, Lucknow.

Thesis Title:
Molecular cloning and characterization of functional protein(s) of human lymphatic filariid Brugia malayi

Thesis work:
My doctoral work is aimed at characterization of Brugia malayi recombinant proteins immunologically as vaccine candidates, and parasite specific and vital enzymes as a drug targets. The objective precisely emphasized on independent Phosphoglycerate mutase. The recombinant protein was immuno-characterized in BALB/c mouse model and thereafter its prophylactic efficacy was explored in Mastomys coucha after challenge infection with parasite. The vaccine efficacy has also been analyzed using different human administrable adjuvants. In vivo and in vitro RNAi technique was utilized to validate Bm-iPGM as a potential antifilarial drug target. To fulfill the aims and objectives of the above work, I had an opportunity to learn and use the following techniques in BALB/c, B. malayi M. coucha and Meriones unguiculatus models. i) Animal handling (Rabbit, mice, rodent).

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Rearing and breeding of Aedes aegypti mosquitoes in temperature and humidity controlled insectarium and cyclically transmission and maintenance of B. malayi infection in susceptible hosts (M. coucha and jirds). iii) Database search viz. NCBI, PubMed, Use of online tools. iv) Cloning, Expression and Purification of recombinant proteins. v) Circular Dichroism and Fluorescense spectroscopy. vi) ELISA vii) Vaccination procedures and protocols. viii) Immune characterization of the recombinant protein in animal models. ix) Challenge of animals with infective larvae of the parasite. x) Lymphocyte transformation test (3H-Thymidine uptake assay). xi) Antibody determination by indirect ELISA (IgG and its subclasses, IgA, IgM and IgE). xii) Estimation of CD molecules (CD4, CD8 and CD19) and intracellular cytokines (IL-2, IL4, IL-10 and IFN-) by Flow cytometry. xiii) Estimation of nitric oxide (Griess method) and ROS (using DCF-DA through FACS). xiv) Antibody dependent cell cytotoxicity assays. xv) Data compilation, statistical analyses using scientific software Graphpad Prism and other parts of manuscript preparation like graphical representations etc. xvi) RNA interference Besides my doctoral work I have also gained experience in immunoscreening of filarial infective stage cDNA library and identified three immunodominant proteins that can be potent vaccine candidates.

ii)

Research Publi ca tio ns: (Publi shed/ Co mmunicated )

1.Shilpy Shakya, Prashant Kumar Singh, Susheela Kushwaha and Shailja MisraBhattacharya. Adult Brugia malayi ~34kDa (BMT-5) antigen offers Th1 mediated significant protection against infective larval challenge in Mastomys coucha. Parasitology international. 2009, 58 (4); 346-53. 2. Prashant Kumar Singh, Arya Ajay, Susheela Kushwaha, Rami Pati Tripathi & Shailja Misra-Bhattacharya. Towards Novel Antifilarial Drugs: Challenges and Recent Developments. Future Medicinal Chemistry, February 2010, 2 (2); 251-83. 3. Meghna Singh, Prashant Kumar Singh and Shailja Misra-Bhattacharya. Functional characterization of DEAD Box RNA Helicase of human Lymphatic Filarial Parasite Brugia malayi using RNA Interferene. Journal of Biotechnology. (2011), doi:10.1016/j.jbiotec. 2011.12.003 4. Prashant Kumar Singh, Susheela Kushwaha, Ajay K. Rana and Shailja MisraBhattacharya. Cofactor independent Phosphoglycerate Mutase of Brugia malayi: structural and immunological characterization. (mss communicated to Vaccine). 5. Prashant Kumar Singh, Susheela Kushwaha and Shailja Misra-Bhattacharya. Cofactor independent Phosphoglycerate Mutase of Brugia malayi Protects Mastomys coucha Against Larval Challenge through Mixed Th1/Th2 Milieu. (mss communicated PLoS Neglected Tropical Diseases) 6. Susheela Kushwaha, Prashant Kumar Singh, Ajay Rana and Shailja MisraBhattacharya. Cloning, expression, purification and kinetics of Trehalose 6 phosphate phosphatase of filaria parasite Brugia malayi. Acta Tropica, June 2011, (119), 151159.

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7. Susheela Kushwaha, Vishal Kumar Soni, Prashant Kumar Singh, Nasreen Bano, Anil Kumar, Rajendra Singh Sangwan and Shailja-Misra Bhattacharya.Withania somnifera chemotypes NMITLI 101R, NMITLI 118R, NMITLI 128R and Withaferin A protect Mastomys coucha from Brugia malayi infection. Parasite Immunology. , 2012, 34, 111. 8. Susheela Kushwaha, Saptarshi Roy, Rita Maity, Asish Mallick, Vishal K. Soni, Prashant K. Singh, Narayan D. Chaurasiya, Rajender S. Sangwan, Shailja-Misra Bhattacharya and Chitra Mandal. Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice. Vaccine (2011), doi:10.1016/j.vaccine.2011.12.031 9. Susheela Kushwaha, Prashant Kumar Singh, Mohd. Shahab, Manisha Pathak and Shailja Misra Bhattacharya. In vitro silencing of Brugia malayi Trehalose-6-phosphate phosphatase impairs embryogenesis and in vivo development of infective larvae in jirds. (mss accepted PLoS Neglected Tropical Diseases) 10. Prashant Kumar Singh, Susheela Kushwaha, Mohd. Shahab, Manisha Pathak and Shailja Misra-Bhattacharya. In vitro gene silencing of independent phosphoglycerate mutase (iPGM) in filarial parasite Brugia malayi weakens parasite motility, impairs microfilaria release, promotes infective larval mortality and retards their in vivo development. (mss communicated to Journal of Biotechnology) 11. Susheela Kushwaha, Prashant Kumar Singh, Jyoti Gupta, Vishal Kumar Soni and Shailja Misra-Bhattacharya. Recombinant trehalose-6-phosphate phosphatase of Brugia malayi cross reacts with human bancroftian antibodies and engenders a robust protective immune outcome in mice. (mss communicated to Microbes and Infection). Papers Presented in ational/International Conferences/Symposia

1. Cofactor independent phosphoglycerate mutase of human lymphatic filariid Brugia malayi: Cloning, Expression and immune characterization Prashant K Singh, Susheela Kushwaha, Vishal K. Soni, Jeetendra K. Nag and Shailja MisraBhattacharya* 21st National congress of Indian Society of Parasitology, November 14-16, 2009, Panjab University, Chandigarh. 2. Cloning and Expression of trehalose -6-phosphate synthase gene of human lymphatic filariid Brugia malayi: Purification and characterization of the recombinant protein. Susheela Kushwaha, Prashant K Singh, Vishal K. Soni, Nidhi Srivastava and Shailja MisraBhattacharya* 21st National congress of Indian Society of Parasitology, November 14-16, 2009, Panjab University, Chandigarh. 3. Vaccination with recombinant independent phosphoglycerate mutase against challenge infection with Brugia malayi confers protective immunity in rodent. Prashant Kumar Singh, Susheela Kushwaha and Shailja Misra-Bhattacharya*

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4th International Symposium on Current trends in drug discovery research- 2010, 17th-21st February, 2010, Central Drug Research Institute, Lucknow. Published in Medicinal Chemistry Research, Volume 19, Supplement 1, 2010 4. Cloning, Expression and characterization of repetitive infective larval antigen BML3 R15 of Lymphatic filaria Brugia malayi. Vishal K. Soni, Prashant K. Singh, Susheela Kushwaha and Shailja Misra-Bhattacharya* 4th International Symposium on Current trends in drug discovery research- 2010, 17th-21st February, 2010, Central Drug Research Institute, Lucknow. Published in Medicinal Chemistry Research, Volume 19, Supplement 1, 2010. 5. The recombinant Trehalose phosphate phosphatase (TPP) of Brugia malayi provides protection against infective larval challenge in rodent via mixed Th1/Th2 response. Susheela Kushwaha, Prashant K Singh and Shailja Misra-Bhattacharya* 14th International Conference of Indian Society of Chemists & Biologists, January 15 18, 2010, Central Drug Research Institute (CSIR), Lucknow. 6. Functional characterization of ATPase R A Helicase of human Lymphatic filarial parasite Brugia malayi using R A interference. Meghna Singh, Prashant Kumar Singh and Shailja Misra-Bhattacharya. 37th Annual Conference of Indian Immunology Society (Immunocon 2010). Sher-i-Kashmir Institute of Medical Sciences, Srinagar, India. 7. Expression, purification and characterization of trehalose-6- phosphate phosphatase from human lymphatic filarial parasite Brugia malayi. Susheela Kushwaha, Prashant Kumar Singh, Ajay Kumar Rana and Shailja Misra-Bhattacharya*. 37th Annual Conference of Indian Immunology Society (Immunocon 2010), 7th-9th Feb, 2011, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, India. 8. Cocktail vaccination with Brugia malayi recombinant proteins confer effective protection against infective larval challenge in Mastomys coucha. Nidhi Shrivastava, Jeetendra Kumar Nag, Susheela Kushwaha, Prashant Kumar Singh and Shailja Misra-Bhattacharya. 37th Annual Conference of Indian Immunology Society (Immunocon 2010), 7th-9th Feb, 2011, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, India. 9. DAM methylase of endosymbiotic Wolbachia from lymphatic filarial Brugia malayi, an ideal drug target causes immunosuppression in mice. Ajay Rana, Vishal Kumar Soni, Susheela Kushwaha, Prashant Kumar Singh and Shailja Bhattacharya. 37th Annual Conference of Indian Immunology Society (Immunocon 2010), 7th-9th Feb, 2011, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, India.

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10. Silencing of Brugia malayi trehalsoe-6-phosphate phosphatase gene by R A interference impairs female worm embryogenesis and parasite survival. Susheela Kushwaha, Prashant K Singh, Mohd. Shahab and Shailja Misra Bhattacharya 80th Annual meeting of Society of biological Chemists. (SBC-2011), 12-15, November, 2011, Lucknow, India. 11. Identification and purification of potentially protective cD A clones of Brugia malayi by screening of L3 cD A library with irradiated L3 protected Mastomys coucha serum. Prashant K Singh, Susheela Kushwaha, Jyoti Gupta and Shailja Misra Bhattacharya 80th Annual meeting of Society of biological Chemists. (SBC-2011), 12-15, November, 2011, Lucknow, India. 12. Brugia malayi recombinant protein cocktail exhibit enhanced protection in experimental host mastomys coucha by eliciting augmented immune activation. Nidhi Shrivastava, Jeetendra K Nag, Prashant K Singh, Susheela Kushwaha and Shailja MisraBhattacharya 80th Annual meeting of Society of biological Chemists. (SBC-2011), 12-15, November, 2011, Lucknow, India. Sequences Submitted EU519463 EU668900 EU684476.1 Training Programs Attended 1. Participated in CSIRs HRDG 5th Technology Led Entrepreneurship Programme held at IICT, Hyderabad, India (June 2 27, 2008). 2. Participated in Workshop on Bioinformatics Tools and Techniques for Gene and Protein Analysis held at SGPGI, Lucknow, India (Aug. 10-12, 2008). 3. Participated in Workshop on IPR Awareness held at CDRI, Lucknow, India (Apr. 26, 2009). 4. Undertook two months research training on Maintenance and propagation of Human MCF-7 cell line and associated biochemical techniques at Tissue Culture Unit, CDRI, Lucknow, India (Jun.16- Aug.16, 2004). 5. Undertook one month practical training at Mohan Meakins Ltd, Mohannagar, India (Jul. 4 - Aug. 4, 2003). Affiliation in Academic/Scientific Societies 1. The Society of Biological Chemists, India: Life member (No. 2666)

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2. The Indian Society of Parasitology: Life member (No. LM 776)

Aca de mi c Pro g re s s i o n
Ph.D. Biotechnology (Thesis to be submitted) Institution - CDRI, Lucknow, India. University Jawaharlal Nehru University, New Delhi, India. Supervisor - Dr. (Mrs.) S. Bhattacharya, Chief Scientist, CDRI, Lucknow, India. Area of research - Parasite immunology. Topic: Molecular cloning and characterization of functional protein(s) of human lymphatic filariid Brugia malayi Master of Science (M.Sc.), 2004 (Ist Div.) Department of Botany, Ch. Charan Singh University, Meerut (U.P.), India. This course covered principle and application of Biotechnology in the field of Industrial biotechnology, Biochemistry, Microbiology, Microbial Metabolism, Microbial genetics, Plant tissue culture, Immunology, Molecular and cell biology, Environmental Biotechnology, and Food and feed technology. Bachelor of Science (B.Sc.), 2002 (Ist Div.) Ch. Charan Singh University, Meerut (U.P.), India. In addition to a comprehensive study program this course included all aspects of Chemistry, Zoology and Botany that are of importance to all fields. Intermediate in Biology, 1999 (Ist Div.) Meerut Public School (CBSE), Meerut (U.P), India. Physics, Chemistry, Biology, English and Physical Education. High school in Science, 1997 (Ist Div.) St. Thomas English Medium School (ICSE), Meerut (U.P), India. Science, Mathematics, Biology, Hindi, Computers and Social Sciences. Honours and Award 1. Dec. 2005: 2. March 2006: 3. ovember 2009: Qualified Indian Council of Scientific and Industrial Research National Eligibility Test ( ET) for JRF. Qualified Graduate Aptitude Test in Engineering (GATE) - 2006 in Life Sciences. Best Poster Award at 21st National congress of Indian Society of Parasitology, November 14-16, 2009, Panjab University, Chandigarh. Techn iques Known

Basic techniques
1. Microscopy, Spectrophotometry. 2. Gel electrophoresis, PCR, Real Time PCR, Western blotting.

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Culture Techniques
1. Macrophage cell culture and cell line culture. 2. Culture of splenocytes and lymphocyte from rodent animal models.

Immunological
1. Estimation of cytokines in cell and cell culture supernatants by FACS or Sandwich ELISA/ cytokine bead array method. 2. Estimation of intracellular cytokine and cell surface receptors using flouresecent dye conjugated antibodies by flow cytometry. 3. Antibody (IgG & subclasses, IgM and IgE) detections by ELISA. 4. Cell mediated immune responses by thymidine (H3) (Lymphocyte transformation test) uptake. 5. Nitric oxide and ROS estimation. 6. Antibody dependent cell cytotoxicity assay.

Molecular Biology
1. Polyacrylamide gel electrophoresis (Native and reduced) 2. Identification and characterization of parasite proteins against different test sera and cell lysate by Western blotting 3. Electro-elution of protein from acrylamide gels. 4. Plasmid and genomic DNA isolation. 5. PCR and Real Time PCR. 6. Cloning, expression and affinity purification of proteins.

Analytical Sciences
1. 2. Protein sample (soluble and total) preparation of parasite and cells. Purification of soluble homogenate of parasite by Sephadex G-200 column chromatography and estimation of protein.

Animal handling
1. Handling of M. coucha, jird, rabbit, rat and mice. 2. Rearing, breeding of mosquitoes and maintenance of insectarium. Co mpu t er Prof i ci en cy 1. Word Processor (Word), Spreadsheet (Excel) and PowerPoint in Microsoft Windows 2. Experience in database search, online tools like BLAST, FASTA etc. Personal Details Date of Birth Fathers ame Mothers ame ationality Languages known Marital Status Sex Address (Permanent/ Correspondence) : 16 January, 1982 : Dr. Surendra Singh : Mrs. Rajnee Singh : Indian : English, Hindi : Unmarried : Male : C/o Sh. K. Singh,

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414, Brahmpuri, Meerut (U.P.), INDIA 250002 Mobile o. E-mail Id Passport no. : +91-9795998745 : prashantcdri@gmail.com : J1074177

RE FE RE CES

Dr. (Smt.) Shailja Misra-Bhattacharya Chief Scientist, Division of Parasitology, Central Drug Research Institute, M.G. Marg, Lucknow-226 001, India #: 00+91-522-2212411-18 Ext.4221 Fax 00+91-522-2223405 Email: shailja_cdri@rediffmail.com Mob: 9415010721 Dr. Sunil Kumar Puri Chief Scientist, Head, Division of Parasitology, Central Drug Research Institute, M.G. Marg, Lucknow-226 001, India #: 00+91-522-2212411-18 Ext.4357 Fax 00+91-522-2223405 Email: skpuricdri@yahoo.co.in Mob: 9919566792 Dr. Anuradha Dube Chief Scientist, Division of Parasitology, Central Drug Research Institute, M.G. Marg, Lucknow-226 001, India #: 00+91-522-2212411-18 Fax 00+91-522-2223405 Email: anuradhadube@gmail.com Mob: 9450931332

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