DEPARTMENT OF BIOTECHNOLOGY,MINISTRY OF SCIENCE & TECHNOLOGY

GOVERNMENT OF INDIA, NEW DELHI

PROFORMA FOR SUBMISSION OF COMPLETION REPORT (2012-17)(Infrastructure and DBT-BUILDER Programme)

A. PROJECT SUMMARY

1. Project Title: DBT-Maharshi Dayanand University, Interdisciplinary Life Science Programme for Advance Research and Education, BT/PR4329/INF/22/144/2011

2. Date of Start of the project (as per sanction order): 12th March, 2012

1. Completion Report Duration (12.3.2012 to 11.3.2017):

2. Total cost of the project (including revision): Rs 2.81 crores

3. Coordinator’s detail: Prof SK Gakhar/Prof Promod Mehta Director, Centre for Medical

Biotechnology/Centre for Biotechnology, MD University, Rohtak-124001

4. Sanctioned objectives and objectives achieved (Tabular format):

Objectives sanctioned Objectives achieved1. Proteomics and Genomic analysis of malaria

parasite and its vector under different physiological conditions

Proteome analysis of infected and uninfected vector (midgut, salivary glands, haemolymph and ovary).

Proteome analysis of different development stages of parasite in mosquitoes.

Proteome analysis of blood stages parasites under various stress conditions(temperature/drugs/blomolecules).

Bioinformatics-based analysis of chromatin remodeling proteins and /or for transmission blocking activity will be analyzed.

Cloning and expression of chromatin remodeling proteins and /or transmission blocking protein.

Purification of the recombinant proteins and generation of antibodies.

Characterization of the proteins (localization studies, oligomeric status, immunoprecipitations).

Attempts will be made to analyze the expression at mRNA.

Analysis of data.

1. Proteomics and Genomic analysis of malarial parasite and its vector under different physiological conditions

Proteome analysis of uninfected vector (salivary gland and ovary).

Bioinformatics-based analysis of chromatin remodeling proteins

Transmission blocking activity was analyzed with drug resistance.

Cloning and expression of chromatin remodeling proteins and /or transmission blocking protein.

Purification of the recombinant proteins and generation of antibodies.

Characterization of the proteins was done. The mRNA expression of chromatin assembly

factor 1 and zuotin related factor 1 Localization of chromatin assembly factor 1 in

asexual stage

2. Detection of candidate antigens and their antibodies or the diagnosis of tuberculosis by Immuno-PCR and for human viruses using Real-time PCR

Diagnosis of Tuberculosis

2. Detection of candidate antigens and their antibodies for the diagnosis of tuberculosis by Immuno-PCR and for human viruses using Real Time PCR.Diagnosis of tuberculosis

Development of Immuno-PCR (I-PCR) with recombinant antigens and antibodies.

Spiking of biological fluids with known concentration of recombinant antigens to mimic the clinical TB samples and perform I-PCR.

Detection of antigens and antibodies in clinical samples of pulmonary TB patients.

Detection of antigen antibodies in clinical samples of extra pulmonary TB and HIV-TB coinfected patients.

Quantification of antigens in clinical samples of TB patients by real-time PCR/I-PCR.Dengue and Chikungunya

Collection of mosquitoes to establish the role of various species in the Transmission of Dengue and Chikungunya in different geographical regions.

Standardization of Conventional Monoplex and Multiplex RT-PCR for Dengue and Chikungunya viuruses.

Compare different method of virus diagnosis. Development of district-wise risk maps for

prediction of epidemics of Dengue and Chikungunya.Rotavirus

Sample collection and screening by ELISA. Electropherotypic analysis of collected samples

RNA. RNA will be subjected to reverse transcription. G and P genotyping by multiplex PCR using

genotype specific primers. Designing of qRT-PCR primers and probe. Standardization of qRT-PCR protocol for various

dilutions of faecal like ELISA and electropherotyping etc.

3. Isolation and characterization of bioactive molecules form Microbes and PlantsTo explore the plant diversity for novel bioactive compounds. To prepare of various plant extracts according to

polarity. To screen of various plant extracts to evaluate

their antimicrobial potential using various assays. To study the toxicity of the most active extract.To explore the different micro-biomes for novel bioactive compounds. To isolate and characterize of microbial isolates

form different ecosystems. To screen of microbial isolates for their

antibacterial and antifungal properties. To purify of antifungal/antibacterial molecules.

Development of Immuno-PCR (I-PCR) with recombinant antigens and antibodies.

Spiking of biological fluids with known concentration of recombinant antigens to mimic the clinical TB samples and perform I-PCR.

Detection of antigens in clinical samples of pulmonary TB patients.

Detection of antigens in clinical samples of extrapulmonary TB patients.

Quantification of antigen in clinical samples of TB patients by real-time I-PCR (optimized the method and could optimize with known concentration of protein and glycolipid antigens

Analysis of body fluids of TB samples based on the detection of PstS1 (Rv0934) by real-time immuno-PCRDengue and Chikungunya

Standardization of Conventional Monoplex and MultiplexRT_PCR for Dengue and Chikungunya viruses.

Multiplex RT-PCR Assay for Detection and Typing Dengue and Chikungunya viruses in clinical samples.

Multiplex RT-PCR assay for surveillance of Dengue and Chikungunya viruses in mosquitoes of Rohtak district.

Rotavirus Designing of qRT-PCR primers and probe. Standardization of qRT-PCR protocol for various

dilutions of fecal samples. G and P genotyping of Rotavirus samples

positive through RNA-PAGE

3. Isolation and characterization of bioactive molecules from microbes and plants

To explore the plant diversity for novel bioactive compounds

To screen various plant extracts to evaluate their antimicrobial potential using various assays

Antibacterial potential of Cassia fistula and Cordia dichotoma

Purification and characterization of α-amylase/ laccase from Bacillus subtilis and B. pumilus.To explore the different micro-biomes for novel bioactive compounds.

To isolate and characterize of microbial isolates form different ecosystems.

To screen of microbial isolates for their antibacterial and antifungal properties.

To purify of antifungal/antibacterial molecules. To characterize antifungal/antibacterial

compounds. Antifungal activity of Tamoxifen against

Filamentous Fungi.

To characterize antifungal/antibacterial compounds.

5. Reason for non-achievement of objectives (Bullet points only):Reason for non-achievement of objectives:

Proteome analysis of different development stages of parasite in mosquitoes and proteome analysis of blood stages parasites under various stress conditions. We could not finish this task due to lack of funds at appropriate time.

Though we have collected mosquitoes in different geographical regions of Haryana and also Standardized Conventional Monoplex and Multiplex RT-PCR for the detection of Dengue viruses, we could only analyze ~ 60 samples as the money was lapsing to buy appropriate kits and chemicals at appropriate time..

6. Infrastructure established (as per sanction order): M.Sc Medical Biotech and Biotech labs are provided with laminar flow and BSL-2 facilities. The students are well aware of working with pathogenic bacteria such as E.coli, S. typhi, M. smegmatis, M. marinum, etc. at biosafety level -1 and biosafety level-2 facilities, cell culture, sterilizing of heat-labile compounds such as serum, cell culture media etc in laminar flow. All the basic chemicals in the labs of several Depts of Life Sciences such as Biotech, Medical Biotech, Botany, Microbiology, Biochemistry, Genetics, etc. are provided so that the M.Sc. students can comfortably work for practicals and dissertation of their research topics. The basic equipments such as cold centrifuge, cold microfuge, PCR, Gel documentation system, SDS-PAGE electrophoresis, ELISA readeretc could facilitate good infrastructure to do good science among the students of several Departments of Life Sciences.

7. Manpower trained (as per sanction order, plz include the break up with figures only e.g. number of M.Sc. Students/JRF/SRF/RA and Assistant Professor, etc.): MSc students ~ 160, JRF 5 SRF 3 RA 3

8. Number of SC/ST/OBC category people trained (Plz provide figures separately for each category):i)Hitesh Singh joined as JRF since May 2016 to March 11, 2017with Dr Neelam Shrawat, Astt Prof Dept of

Genetics ii) Ms Renu Dharra, joined as SRF since 30 th Sept, 2016 to March 11, 2017with Prof Promod Mehta, CBT9. Research outcome:

- Publications (figures only; kindly include only those publications having DBT’s acknowledgement as per the sanction order and comes out of this project ONLY) - 12

- Patents (filed and granted separately; kindly also include the patent numbers/reference number) NA- Technology developed, transferred or commercialized (Kindly include the name of the company to which

the technology has been commercialized) A robust and accurate immuno-PCR test has been developed for an early diagnosis of both pulmonary and extrapulmonary TB patients, which showed superiority over ELISA though it needs further validation in a large number of samples and commercialized.

- Status of commercialized technology (whether in market or not; if yes the name of the country) NA10. Grant remaining unspent along with justification: Rs 1.47 lakhs (DD prepared and will be sent by courier).11. Justification for non-submission of yearly financial documents NAB. PROGRESS REPORT IN DETAIL 1. Status of the Civil Works/Building Construction (kindly provide justification, if required): NA2. Facilities Established (including the up-gradation of existing laboratories and facilities): Biosafety level-2 are

installed in MSc labs of Biotech and Med Biotech courses. The students are well abreast with the concept of working on pathogenic bacteria at various biosafety levels. All the basic instruments i.e. PCR, Gel

documentation system, ELISA reader, cold centrifuge, microfuge, etc. were purchased in different labs/Depts of Life Sciences such as Microbiology, Botany, Genetics and Biochemistry Depts Equipments purchased (as per sanction order): Pl see Asset certificate

3. Research Details (not more than ten pages including some good figures; kindly note that this information should not be mere reproduction of research articles or combining yearly report): Pl see Annexure 1

4. Research Outcome details - List of Publications 12 (Attached) 1. Singh N, Sreenivas V, Gupta KB, Chaudhary A, Mittal A, Varma-Basil M, Prasad R, Gakhar SK, Khuller

GK, Mehta PK (2015). Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR. Diagn Microbiol Infect Dis.; 83:359-64. (Impact factor 2.457)

2. Raj A, Singh N, Gupta KB, Chaudhary D, Yadav A, Chaudhary A, Agarwal K, Varma-Basil M, Prasad R, Khuller GK, Mehta PK (2016). Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis. Yonsei Med J ; 57:88-96. (Impact factor 1.554)

3. Singh N, Sreenivas V, Sheoran A, Sharma S, Gupta KB, Khuller GK, Mehta PK (2016). Serodiagnostic potential of immuno-PCR using a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor in tuberculosis patients. J Microbiol Methods. ; 120:56-64. (Impact factor 2.338)

4. Sharma D, Lather M, Mallick PK, Adak T, Dang AS, Valecha N, Singh OP (2015). Polymorphism in drug resistance genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium falciparum in some states of India. Parasit Vectors.; 8:471. (Impact factor 3.43)

5. Sachdeva V, Hooda V (2016). Effect of changing the nanoscale environment on activity and stabilityof nitrate reductase. Enzyme and Microbial Technology ; 89: 52–62 (Impact factor 2.28).

6. Sachdeva V, Hooda V (2015) Immobilization of nitrate reductase onto epoxy affixed silvernanoparticles for determination of soil nitrates. International Journal of Biological Macromolecules: 79; 240–247. (Impact factor 3.09)

7. Mehta PK, Singh N, Dharra R, Dahiya B, Sharma S, Sheoran A, Gupta KB, Chaudhary D, Mehta N, Varma-Basil M. Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR. J Microbiol Methods. 2016;127: 24-27 (5-Year Impact Factor: 2.247).

8. Mehta PK (2016) Immuno-PCR: Its Role in Serodiagnosis of Tuberculosis. Mycobact Dis 6: 219-220 (Impact Factor: 0.993).

9. Sharma S, Raj A, Singh N, Dahiya B, Sheoran A, Gupta KB, Mehta PK (2017) Development of real-time immuno-PCR for the quantitative detection of mycobacterial PstS1 in tuberculosis patients. J Microbiological Methods 132: 134-138 (5-Year Impact Factor: 2.247).

10. Khatri S et al (2016). Chemical composition, antioxidant, antibacterial and cytotoxicity analysis of Blumea lacera (Burm. F.) Dc. International journal of Pharmacy and Pharmaceutical Sciences, 1:313-319. ISSN 0975-1491.

11. Mehta PK, Dahiya B, Sharma S, Singh N, Dharra R, Thakur Z, Mehta N, Gupta KB, Gupta MC, Chaudhary D. (2017). Immuno-PCR, a new technique for the serodiagnosis of tuberculosis. J Microbiol Methods.139:218-229. (5-Year Impact Factor: 2.247).

12. Miglani, M and Gakhar, S. (2017). Vitellogenin gene expression during the development of anautogenous malaria vector, Anopheles culicifacies A. Int J Current Res Review (Impact factor 4.016)

- List of Patents (filed or granted) NA

- List of Technology Developed/Transferred/Commercialized: A robust and accurate immuno-PCR test has been developed for an early diagnosis of both pulmonary and extrapulmonary TB patients, which showed superiority over ELISA though it needs further validation in a large number of samples and commercialized.

5. Grants received from other agencies after establishing the required infrastructure :

i) Detection of mycobacterial RD antigens by nanoparticle based Immuno-PCR for an early detection of pulmonary and extrapulmonary tuberculosis (39.8 lakhs DBT funded, 2015-18) to Dr P Mehta (PI).

ii) Cloning, expression, purification and characterization of chromatin assembly factor I from human malaria parasite Plasmodium falciparum (9.36 lakhs, 2011-14, UGC funded) to Dr Ritu Gill (PI)

iii) Molecular Cloning and Characterization of type III HSP40- a chromatin related factor 1 from Human Malarial Parasite Plasmodium falciparum (25 lakhs, 2014-17, DST-SERB funded) to Dr Ritu Gill (PI)

iv) Development and Evaluation of One Step Single Tube Multiplex RT-PCR for Rapid Detection & Typing of Dengue Viruses [10 lakhs, 2013-15, Haryana State Council for Science & technology (HSCST)] to Dr Samender Kaushik (PI).

v) Development of an ideal pre-treatment and saccharification process for rice straw using microbial cellulases (10 lakhs, 2016-19) to Dr Bijender Singh (PI)

6. Number of Departments within an institution or outside institution benefitted from the infrastructure established i) Centre for Biotechnology ii) Centre for Medical Biotechnology iii) Microbiology Dept

iv) Genetics Dept v) Botany Dept vi) Biochemistry Dept of Faculty of Life Sciences, MDU, Rohtak. 7.Extension of the project with justification (kindly note project will not be extended in the absence of sound reason e.g. reasons like extension for supporting the manpower only will not be considered) NA 8. Enlosures - Summary and Conclusions:

This study provides a comprehensive in silico analysis of P. falciparum chromatin remodelling protein, thus highlighting the parasite specific features as compared to human host. This provides a scaffold for further experimental dissection of the chromatin remodulator. In addition, P falciparum PfCAF-1 and PfZRF1 were cloned, expressed and purified; which can be further exploited for the functional and structural characterization. Detection of mycobacterial Ag85B (Rv1886c) by immuno-PCR (I-PCR) showed the highest sensitivity, followed by cocktail (Ag85B, cord factor and ESAT-6), ESAT-6 and cord factor and that was superior as compared to ELISA. Furthermore, real-time I-PCR was designed based on the detection of PstS1 (Rv0934) in sputum and pleural fluids of TB patients. A wide dynamic range varying from10 ng/mL to 1 pg/mL of PstS1 could be detected by real-time I-PCR, which may facilitate monitoring the dynamics of diseases in patients. We made an attempt to further raise the sensitivity by the detection of a combination of PstS1 and MPB64 (Rv1980c).