Himanshu Sharmash83.himanshu@gmail.com · +91.9599378446Lab 404 · Regional Centre for Biotechnology (RCB) · Faridabad · India 121 001

Current Position

Regional Centre for Biotechnology (RCB) FARIDABAD, INDIA

DBT-Research Associate Jan ’15 – presentWorking on a project as a DBT-research associate (DBT-RA) at RCB, Faridabad, India. Overall themeof the project is to unravel the molecular mechanism of anticancer and immunomodulatory action ofthe bile acids and their drug conjugates.(Details are given in section research experience on a separatesheet)

Previous Position

Centre for Cellular and Molecular Biology (CCMB) HYDERABAD, INDIA

Senior Project Fellow Feb ’14 – Jan ’15Worked on a project as a senior project fellow at CCMB, Hyderabad, India. Overall theme of theproject was to understand molecular basis for antimicrobial action of the variants of natural peptidessuch as defensins.(Details are given in section research experience on a separate sheet)

Education

CCMB HYDERABAD, INDIA

Ph.D. (Life Science) Aug ’07 – Feb ’14Worked as a doctoral fellow with Dr. R. Nagaraj, outstanding scientist, CCMB, Hyderabad, India.

Thesis title: Structure-function relationships in defensins.(Details are given in section research experience on a separate sheet)

Madurai Kamaraj University MADURAI, INDIA

M.Sc. (Biotechnology) July ’05 – May ’07Various courses and practicals were covered on the topics of microbiology, molecular biology, im-munology, cell biology, structural biology and bioinformatics in M.Sc.(Biotechnology) curriculum.Aggregate Percentage 74%.

Thesis title: Protein-protein interaction using yeast two hybrid assay.

Delhi University DELHI, INDIA

B.Sc. (Biochemistry) Jul ’02 – May ’05B.Sc.(Biochemistry) course work included theoretical and practical overview on basic knowledge ofbiochemistry, molecular biology and immunology. Aggregate Percentage 65%.

PublicationsBook Chapter:

Sharma H., and Nagaraj R., Structure-activity relationships in the host-defense antimicrobial peptidesdefensins. Studies in Natural Products Chemistry (Bioactive Natural Products) elsevier science publishers(in Press).

Journal Article:

Sharma H., and Nagaraj R., Antimicrobial activity of human β-defensin 4 analogs: Insights into the roleof disulfide linkages in modulating activity. Peptides, 38, (2012), 255-65.

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Sharma H., and Nagaraj R., Human β-defensin 4 with non-native disulfide bridges exhibit antimicrobialactivity. PLoSOne, DOI: 10.1371/journal.pone.0119525.

Sharma H., Mathew B., and Nagaraj R., Engineering of an inactive linear analog of human β-defensin 4to generate peptides with potent antimicrobial activity. J. Pept. Sci., in press.

SkillsTechnical expertise:

Peptide chemistry Solid phase peptide synthesis using Fmoc chemistry; Multipledisulfide linkage formation using orthogonal cysteine side chainprotections; Modifications such as acylation; Labeling with car-boxyfluorescein

Purification and Characterization Reversed-phase high performance liquid chromatography (RP-HPLC); Mass spectrometry (MALDI-TOF-TOF); Phospholipid andprotein estimations

Bacterial and eukaryotic cell culture Antimicrobial and anticancer activity assays (Broth-dilution as-say and MTT assay); Salt, metabolic inhibitor and serum sensitiv-ity assays

Confocal microscopy Localization studies in bacteria, fungus, eukaryotic cell lines, orsynthetic lipid vesicles (giant unilamellar vesicles (GUVs)); Quan-tification of colocalization

Biophysical techniques Circular dichroism spectroscopy (CD); Fluorescence spectroscopy;Absorption spectroscopy; Ishothermal titration calorimetry (ITC);Surface plasmon resonance (SPR); Langmuir-Blodgett trough(LT); Dynamic Light Scattering (DLS)

Bioinformatics Analytical tools like Peptool; ExPasy

Computer skills:

Operating systems MS Windows XP, 07, 08; Debian based Linux distributions

Business softwares MS office; Adobe Photoshop; OriginLab; gnuplot; GIMP photo editor; LATEXLanguage Elementary knowledge of Perl

ConferencesPoster presentation on topic “Short and linear novel analogs of HBD4 with high salt resistant antimicrobialactivity“ in International Conference on Chemical Biology (ICCB-2014), IICT, Hyderabad, India (Feb ’14).

Poster presentation on topic “Host-defense peptides, role of fatty acid acylation and peptide self-assembly“ inCCMB - 25th foundation day celebrations (Biology 2012 and Beyond), CCMB, Hyderabad, India (Nov ’12).

Poster presentation on topic “Antimicrobial activity of human β-defensin-4 with non-native disulfidebridges: insights into the role of disulfides in modulating activity“ in Gordon Research Conference (GRC) onNew Antibacterial Discovery and Development, Lucca, Italy (Apr ’12).

Poster presentation on topic “Human beta defensin-4 analogs induce apoptosis in A549 and MCF-7cells“ in 3rd Indian peptide symposium, Pune, India (Feb ’11).

Poster presentation on topic “Antimicrobial activity of HBD4 analogs“ in 2nd Indian peptide sympo-sium, 2009, New Delhi, India (Feb ’09).

Participation in 2nd annual meet on cellular and molecular biophysics conducted by Biophysical Soci-ety in CCMB, Hyderabad, India (Jan ’09).

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Supervisory ExperienceAs a Ph.D. and senior project fellow, I have supervised and trained two summer students and three freshlyjoined Ph.D. students in CCMB for their lab rotation. Now summer students are pursuing Ph.D. at differentplaces in United Kingdom (UK) and Ph.D. students are continuing their research work in different laboratoriesof CCMB.

Awards and achievements• Recipient of DBT Research Associateship from DBT, government of India (2015-2017).• Recipient of Nature Publishing Group (NPG) travel grant for GRC meeting in Lucca, Italy (Apr ’12).• Qualified for full travel grant from Council of Scientific and Industrial Research (CSIR), government of India,for GRC meeting in Lucca, Italy (Apr ’12).• Recipient of Senior Research Fellowship from University of Grant commission (UGC), government of India(2009-2012)• Recipient of Junior Research Fellowship from UGC, government of India (2007-2009).• Qualified Junior Research Fellowship from CSIR, government of India, (2008).• Qualified Junior Research Fellowship from Department of Biotechnology, government of India, (2007).• Qualified Graduate Aptitude Test In Engineering (GATE) (2007).• Recipient of fellowship from Department of Biotechnology (DBT), government of India, during post gradua-tion (2005-2007) at Department of Biotechnology, Madurai Kamaraj University, Madurai, INDIA.

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Research Experience

DBT-Research AssociateCurrently working on following project

Unraveling the anticancer therapeutic potential and immune based anticancer mechanism of thebile acids and their drug conjugated variants?

In this project, bile acids such as lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA)carrying varying number of hydroxyl groups will be conjugated with anticancer drugs. Anticancerpotency of the bile acids and their conjugates will be examined against colon cancer cell lines andtumorigenic mice models. Various biophysical and microscopic tools will be used to examine theirmembrane interacting properties. Inflammatory activities will be examined by monitoring the ex-pression of pro- and anti-inflammatory cytokines that could inhibit cell proliferation. Expressionof transcription factors like NF-kB, Stat3 and AP-1 which are involved in tumor progression will beexamined in tumorigenic cells treated with bile acids and their conjugates. This study will introducenovel potential candidates against colon cancer which would not only directly kill cancerous cells butalso inhibit cell proliferation by immune-based mechanism.

Senior Project FellowWorked on following project

Whether acylation can induce salt resistance antimicrobial activity in the linear inactive variants ofhost-defense peptides (HDPs)? and if yes, what would be the mechanism?

In this project, inactive linear peptide corresponding to N-terminal Human β-defensin 4 (HBD4)was acylated by myrstic acid and the antimicrobial activity was examined. Peptide conjugated withmyristic acid acquired the potent activity against various pathogens irrespective of the presence orabsence of salt which is an advantage over the peptides with alternative modifications such as D-Arg orα-aminoisobutyric acid (Aib) substitutions. Various biophysical and microscopic experiments delin-eated the mechanism of action and revealed that these peptides caused membrane permeabilizationand subsequently leads to cell death. This study introduced a novel way of inducing salt tolerancewith robust broad antimicrobial activity in an inactive peptide.

Ph.D.Three inter-related projects were carried out during Ph.D.

Role of disulfide connectivities in antimicrobial activity of HBD4 short analogsIn this project, short peptides corresponding to N-terminal of HBD4 with one, two and three disulfidebridges were synthesized by solid-phase peptide synthesis using Fmoc-chemistry and characterizedby RP-HPLC and mass spectrometry. Antimicrobial properties of the peptides were examined usingstandard microbial cell culture techniques. Mechanistic studies were carried out using combinationof confocal imaging and biophysical techniques such as isothermal titration calorimetry (ITC), CDand fluorescence spectroscopy. This study gave strong evidence that peptides act on the microbesby targeting intracellular components without disrupting membrane integrity unlike other linearantimicrobial peptides.

Significance of cysteine connectivities and positions on antimicrobial action of the full-length HBD4

Three analogs of HBD4 were synthesized containing cysteines at non-native positions. The numberof disulfide bridges are controlled by blocking the side chains of the cysteines. Various microbiology,microscopy and biophysical experiments indicated that peptides are active irrespective of their numberand position of cysteines, however their potency is varied. Conformational flexibility appeared to playan important role as the interaction between cationic residues and anionic membrane seems to bemore pronounced in one and two disulfide constrained peptides indicated by their correlation betweensalt sensitive behavior and secondary structure. This is the first study in which direct role of disulfidebridges in antimicrobial action of defensins has been shown.

How activity can be induced in an inactive linear peptide without introducing disulfide bridges?Three different strategies were acquired to induce the activity: 1. Induction of helicity by introducingα-Aib (aminoisobutyric) acid; 2. Increase in the cationicity; 3. Selective substitution of L-Arg to D-Arg.Peptide with L- to D-Arg substitution was found most active. Interestingly, D-Arg containing peptide

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did not adopt helical structure and as well did not act over the membrane integrity but translocatedacross the membrane and accumulated inside the cytoplasm consequently cell death. In this study, wedid not only induce the activity in the inactive peptide but also delineated its antimicrobial mechanism.

Overall, Ph.D. project significantly contributed to the field of host-defense peptides defensins regardingtheir antimicrobial activity. Moreover, this study gave an alternative to conventional antibiotics byproviding novel strategies for the development of a potential therapeutic agent against pathogenicbacteria and fungus.

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