Dr. KSHITISH ACHARYA

IBAB-Shodhaka joint research work wins the best poster award (shared by 3 posters) at Bangalore Bio 2009!

He has more than one specific research interest and enjoys the flexibility in technical approaches. The variety across his current research interests illustrates the same.

1. Analysis of mechanisms of regulation of transcription:

A. Database of co-regulated genes and development of a mammalian promoter prediction tool:
A database of transcriptional elements of co-regulated genes, with manual curation of the data is being created (MGEXdb). This database would then be used to create a more reliable promoter prediction tool. The current focus is on the genes expressed in mammalian testis and prostate tissues. MGEXdb is currently available on our website with limited public access. The database can provide easy information on the expression pattern of genes in different cell types within testis. More data will be included and user-friendly features completed before completing the database for the selected tissues.

B. Studies on RNA-binding proteins (RBPs) in the context of transcription regulation:

RNA-binding motifs are present in many proteins that are involved in regulation of transcription. However, a generalized role of such proteins in some of the transcription phases has not been paid its due attention. Similarly several other aspects of the mechanisms of interactions of RBPs with the nucleic acid molecules have not been assessed to obtain a holistic picture. Studies and databases related to RBPs are being reviewed.  

2. Cloning and expressing genetically engineered peptides with potential epitopic regions of chosen diagnostic antigens:

Genetically engineered peptides that represent epitopic regions of certain antigens can be useful components of diagnostic kits.

Some of the bioinformatics strategies are used to predict putative epitopic regions of specific antigens (e.g., PR3, MPO and PSA). Primers are then designed to amplify regions that express putative epitopic regions. A single peptide would be designed and expressed in bacterial and/or mammalian cells. The expressed peptides would then be tested for their ability to raise antibodies which would also identify the antigen in its native form.

PR3, a potential tool for diagnosis for Wegener's granulomatosis, a systemic autoimmune disorder, is one of the target antigens being considered. PR 3 is a 26kDa neutral serine proteinase, involved in immune defense reactions of neutrophils and monocytes. The anti-neutrophil cytoplasmic antibodies (PR3-ANCA) in patients with Wegener's granulomatosis (WG) are highly specific for Proteinase 3 (PR3) only. The presence of autoantibodies to neutrophil cytoplasmic proteinase-3, usually reported as c-ANCA, is virtually diagnostic for Wegener's granulomatosis and related conditions.

A 250bp region of the PR3 gene is identified. This would code for one of the potentially epitopic regions of the PR3 protein. Primers designed for amplifying this region have worked (Fig. 2). This fragment is being cloned using plasmid vectors. Other important PR3 gene fragments will be integrated with this cloned sequence to generate the genetically engineered gene.

Fig 1: The genomic DNA was subjected to PCR using the specific primers designed. The PCR product was then analyzed by electrophoresis. The left lane has molecular weight markers (250bp ladder) and the right line shows the amplimer of ~250bp size.

3.  Programs for molecular biology

A. Primer designing software (Exprimer and primer junction):

a) ExPrimer: A web based application to design primers from exon junctions.
Identification of appropriate primers is crucial for several techniques that involve DNA hybridization and amplification. The following important features are rarely combined in a single primer designing program: (1) Determining sequence specificity of the primer with minimum user-effort (2) Selecting a primer from an exon junction (3) Automated determination of primer characteristics like temperature of melting, self annealing etc. A program that integrates these features and provides other unique advantages has been almost completed in our laboratory by Mr. Kuljeet Singh, a second batch student of our PG Diploma course in Bio-informatics. The program, upon receiving information of specific organism and gene, interfaces with the NCBI BLASTn site and scan the nr-nucleotide database. Possible sequences of user-specified primer length, which represent a small region of exon junction, are considered. The program can perform a comparative analysis of these potential primers, with minimum or no user intervention. The crucial parameters considered for analysis include sequence specificity, GC content, self annealing temperature, etc. A primer with optimum combination of such characters is chosen and suggested. A summary of results of all the analysis for primers from different exon-junctions of the gene is also given. Further, one can view the detailed alignment patterns that are significant in the context of primer-dimer formation. The dynamic programming is done using the PERL script. The primer scoring is done by reference point approximation method.

Advantages: 1) The primers obtained through this program can reduce chances of junk products in RT-PCR. Primers spanning exon-junctions will not bind to genomic DNA and/or hn-RNA present in the sample for RT-PCR experiment. 2) Expression profiling of splice variants of a gene: Splice variant-specific primers from exon junctions can be obtained. 3) Identification and characterization of novel splice variants of a gene can be aided by this program. The user can edit the probable exon-junctions in the genbank file and can validate the existence of the same by in vitro experiments.

Validation of the tool: About 100 randomly taken test genbank files were used for evaluation. The results were satisfactory. The program’s ability to design exon-junction primers for splice variants was tested using BRCA1 (Breast Cancer gene) gene.

b) Primer junction: Currently the Ex-primer has been improved and this new comprehensive tool is under test. This program is available on request for academic purposes (please send in your request to info@ibab.ac.in with your name, designation and affiliation; also add in the subject: Attention to Dr. Kshitish).

c) Experimental evaluation of the efficiency of primers generated by oligo-designing tools. The Ex-ex Primer is currently being validated using PCR and reverse-transcription PCRs. Studies are also being done to compare the reliability of Tm calculation methods.
 
B. Mot-det: A software to detect presence of patterns of short nucleotide sequences or motifs within one or more sequences submitted. The program can identify a) specific pattern, b) variants of a consensus pattern or c) most common patterns in the given sequence or a set of sequences. The program can also identify the patterns within different blocks of the sequences.

C. PERLit: A literature search tool developed, using PERL language, to facilitate the PubMed usage. The tool also aids creation of a quick database which can be further searched using the program.

D. A few other small programs developed include:

a) Oligo-tracer (can detect even part of the short query sequence in a bigger sequence submitted)

b) Oligo-number (numbers the nucleotides if a sequence is submitted)

c) Gen-sum (Gene summary extractor: With any gene name as input, the tool retrieves basic information, including the synonyms, sequence, potential promoter, TSS etc)

d) List matcher (with 2 or more lists of any terms, gene names - for example, the tool classifies the terms into those which are unique to each list vs. those common to multiple lists)

Publications:

1. Kshitish K. Acharya, Chhabi K. Govind, Amy N. Shore, Mark H. Stoler, Prabhakara P. Reddi  (2006) cis-Requirement for the Maintenance of Round Spermatid-Specific Dev Biol. Jul 15;295(2):781-90.

2. Kshitish Acharya K. (2006) The biotech-bioinfo interface in the context of education and growth of the biotechnology industry in India today. Biovistas 1(1): 7-13. Click here to access the full article.

3. Sandhu, K S and Acharya, K, ExPrimer: To design primers from exon-exon junctions
Bioinformatics, 21: 2091 (2005)

4. Sreekumar A, Acharya K K, Lalitha H S, India S S, and Seshagiri P B
Germ cell-specific localization of immunoreactive riboflavin carrier protein in the male golden hamster: Appearance during spermatogenesis and role in sperm function. Reproduction - accepted for publication (2004)

5. Reddi P P, Shore A, Shapiro J A, Anderson A, Stoler M H and Acharya K K and Herr J C Spermatid-specific promoter of the SP-10 gene functions as an insulator in somatic cells. Developmental Biology 262: 173-182 (2003)

6. Reddi, P P, Shore A, Acharya K K and Herr J C
Transcriptional regulation of spermiogenesis: Insights from the study of SP-10 gene, which codes for an acrosomal protein. Journal of Reproductive Immunology, 53:25-36 (2002)

7. Seshagiri P B, Acharya, K K, Jayaprakash, D, Satish, K S and Shetty, G
Ovarian hyperstimulation in bonnet monkeys using gonadotrophins. In: Follicular Growth, Ovulation and Fertilization: Molecular and Clinical Basis. A. Kumar and A.K. Mukhopadhyay (Eds.). Narosa Publishing House. New Delhi (2002)

8.Acharya K K, Roy A and Amitabh K
Relative role of olfactory cues and certain non-olfactory factors in foraging of fruit-eating bats. Behavioural Processes 44(1): 59-64 (1998)

9. Acharya K K and Dominic C J
Duration of the luteotrophic memory of the stud male odours formed in female mouse.
J. Exp. Zool. 279: 626-632 (1997)

10. Acharya K K and Dominic C J
Male-induced post-implantation pregnancy failure in the laboratory mouse: A re-investigation. J. Reprod. Biol. Comp. Endocrinol. 6: 69-75 (1994)

11. Acharya KK and Dominic C J
Male-induced acceleration of sexual maturation in female mice: Absence in the Parkes (P) strain. Indian
J. Exp. Biol. 32: 906-907(1994)

CONSULTANCY:

Dr. Kshitish is interested in being a subject matter expert (SME) in teams providing consultancy or an independent consultant for

(a) specific research projects in biotechnology or bioinformatics organizations,

(b) product quality or stability assessments,

(c) strategies in biotechnology companies,

(d) training employees in specific biotechnology/basic bioinformatics areas as well as general professional skills, and

(e) improving the efficiency and economy of peripheral activities across departments in research and/or production units.

Apart from research and teaching, he also has experience in certain aspects of management and organizational activities. Following is a summary of his experiences:

Experience (15 years of research, including Ph.D.):

Consultancy/research collaborations: Has been a consultant/subject matter expert (SME) to a few biotech companies, including the /Wipro/Health Sciences (in the area of microarray data analysis). He is currently working in collaboration with a/ leading research group at an American University/. Dr. Acharya is also playing a pivotal role, an adviser, in a special research enhancement program for medical researchers at a research center; he has designed a unique program for training in molecular biology methods for clinical research.

General experience and domain expertise:
Teaching: 4 years of teaching in

• molecular biology

• bioinformatics and

• some of the general professional skills.

He has also been invited for several lectures on various topics in the above-mentioned areas.

Organization and management:

• The industrial exposure as the head of the research and production unit of a biotech company has been a good primer to his skills in these areas.

• Dr. Kshitish has organized several short term programs, including custom-made corporate courses, at IBAB.

• He has also conceived and initiated a novel 6-12 months program in the laboratory bio-techniques, to cater to the needs of Indian biotech-industry and

• has been the convener of this program since the beginning

Research areas: Molecular biology, bio-techniques and bioinformatics

Overall experience:

Academic, India: About 17 years of research; teaching in IBAB for 4 years.

Academic, USA: 3.5 years of patent-oriented research (molecular biology).

Industry, India: An year as head of the research and production unit in a growing Indian Biotechnology company.

Research assistants in various projects with Dr. Kshitish Acharya
Name	Qualification (degree or diploma) details
	Qualification	Year	Institute
Akhilesh K.B.	MSc	2006	Presidency college, Bangalore
Archana K.	BE	2006	Vemana Institute of Technology, Bangalore
Ashrefunisa S.	Bioinformatics diploma after MSc	2006	IBAB, Bangalore
Darshan S. C.	MSc	2007	Kuvempu University, Shivamogga
Greta K.	Biotechniques diploma after BTech	2007	IBAB, Bangalore
Harishanker K.	MSc	2006	FMKMC (Mangalore University), Madikeri,
Haritha H.	Biotechniques diploma after  MSc	2004	IBAB, Bangalore
Hima K.	MSc	2006	Vydehi Inst. Biotech Sc., Bangalore
Himadri S.	MSc	2006	Sri Ramachandra University, Chennai
Mohammed F.	MSc	2006	Sri Ramachandra University, Chennai
Nagaraja D.	MSc	2007	Kuvempu University, Shivamogga
Neelima C.	MSc	2006	SRM Arts and Science college, University of Madras
Praveena B.K.G.	BTech	2006	MITS, Madanapalle
Rama Kumar P.	MSc	2007	IASE university, Rajasthan
Shamshad  K.B.	MSc	2006	St Aloysius College, Mangalore
Shreemathi V.	MSc	2007	City College, Bangalore
Suhail M.K.	MSc	2007	DGRD college, Coimbatore
Sundar S.K.	MTech	2005	Anna University, Chennai
Sunil kumar R.	MSc	2007	Kuvempu University, Shivamogga
Vanitha M.	MTech	2006	SITST, Chennai