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Gulati B.R.,National Research Center on Equines | Kumar R.,National Research Center on Equines | Kumar P.,University of Veterinary and Animal Sciences | Yadav P.S.,Central Institute for Research on Buffaloes
Cells Tissues Organs | Year: 2014

Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polymerase chain reaction (PCR) and immunocytochemistry, but did not express haematopoietic markers (CD34, CD45 and CD14). In flow cytometry, the expression of CD29, CD44, CD73 and CD90 was observed in 68.83 ± 1.27, 93.66 ± 1.80, 96.96 ± 0.44 and 93.7 ± 1.89% of AF-MSCs, respectively. Osteogenic, chondrogenic and adipogenic differentiation of MSCs was confirmed by von Kossa and Alizarin red S, Alcian blue and oil red O staining, respectively. Upon supplementation of MSC growth media with 50 ng/ml bone morphogenetic protein (BMP)-12, AF-MSCs differentiated to tenocytes within 14 days. The differentiated cells were more slender, elongated and spindle shaped with thinner and longer cytoplasmic processes and showed expression of tenomodulin and decorin by RT-PCR and immunocytochemistry. In flow cytometry, 96.7 ± 1.90 and 80.9 ± 6.4% of differentiated cells expressed tenomodulin and decorin in comparison to 1.6 and 3.1% in undifferentiated control cells, respectively. Our results suggest that AF is an easily accessible and effective source of MSCs. On BMP-12 supplementation, AF-MSCs can be differentiated to tenocytes, which could be exploited for regeneration of ruptured or damaged tendon in race horses.


Prakash S.,University of Warwick | Singh R.,National Research Center on Equines | Lodhi N.,Fox Chase Cancer Center
Cellular and Molecular Biology | Year: 2014

Covalent histone modifications, chromatin remodeling and incorporation of histone variants regulate the dynamics of chromatin structure. Among covalent histone modifications, histone methylation mediates by histone methylases that influence the gene expression in heterochromatin silencing, genomic imprinting and transcription. In contrast to methylases, histone demethylases remove the methyl groups from lysine or arginine residues of histones and have enormous impact on gene expression via modified chromatin structures. Two types of histone lysie demethylases have been identified, including lysine specific demethylases 1 (LSD1) and Jmj (Jumonji) domain containing family proteins. The human demethyliminase (PADI4) converts monomethyl arginine residue to citrulline by the arginine demethylimination. In this review we summarize recent advances to understand the mechanism of demethylases in regulation of plant gene expression. In addition we are highlighting the function of four human like LSD1 (LDL) and jmj domain containing genes of Arabidopsis that regulate the defense related, flowering controlling and brassinosteroid response genes. © 2015.


Arangasamy A.,Washington State University | Arangasamy A.,National Research Center on Equines | Kasimanickam V.R.,Washington State University | DeJarnette J.M.,Select Sires Inc. | Kasimanickam R.K.,Washington State University
Theriogenology | Year: 2011

The objective was to determine the association of mRNA expression of cystine rich secretary protein 2 (CRISP2), chaperonin containing T-complex protein 1, subunit 8 (CCT8), and phosphatidylethanolamine-binding protein 1 (PEBP1), in sperm of Holstein bulls with Sire Conception Rate (SCR) scores between -4 and +4. These proteins were involved in sperm capacitation and sperm-egg fusion. Samples of sperm obtained on a single day from Holstein bulls (N = 34) in a commercial AI centre were used to evaluate relative mRNA expression of CRISP2, CCT8, and PEBP1. The mRNA abundance of CRISP2 was positively correlated (r = 0.88; P < 0.002), CCT8 was negatively correlated (r = -0.87; P < 0.002), and PEBP1 was positively correlated (r = 0.83; P < 0.006) with SCR-scores. The means of CRISP2 mRNA abundance was greater among positive SCR-score bulls (2.5 to 8 fold), the means of CCT8 mRNA abundance was greater among the negative SCR-score bulls (9.5 to 3.5 fold), and the means of PEBP1 mRNA abundance was greater for the positive SCR-score bulls (5.4 to 7.7 fold). In multivariate regression models predicting SCR-scores, mRNA abundance of CCT8 was significantly associated with SCR-score in all models. In the presence of CRISP2 mRNA abundance in the model, the SCR score's predictability of PEBP1 was insignificant. However, in the absence of CRISP2 mRNA abundance in the model, the SCR-score's predictability of PEBP1 was significant. In multivariate regression models, CRISP2 and CCT8 mRNA expression in sperm accounted for 95% of the variance in Holstein bull's SCR-scores. In conclusion, Holstein bulls with greater CRISP2 and lower CCT8 mRNA expression in sperm had higher probabilities of siring calves. © 2011 Elsevier Inc.


Pal V.,Canadian Department of National Defence | Kumar S.,Canadian Department of National Defence | Malik P.,National Research Center on Equines | Rai G.P.,Canadian Department of National Defence
Clinical and Vaccine Immunology | Year: 2012

Glanders is a contagious disease caused by the Gram-negative bacillus Burkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins of B. mallei were used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Pallavi R.,Indian Institute of Science | Roy N.,Indian Institute of Science | Nageshan R.K.,Indian Institute of Science | Talukdar P.,Indian Institute of Science | And 8 more authors.
Journal of Biological Chemistry | Year: 2010

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Balamurugan V.,Project Directorate on Animal Disease Monitoring and Surveillance PD ADMAS | Hemadri D.,Project Directorate on Animal Disease Monitoring and Surveillance PD ADMAS | Gajendragad M.R.,Project Directorate on Animal Disease Monitoring and Surveillance PD ADMAS | Singh R.K.,National Research Center on Equines | Rahman H.,Project Directorate on Animal Disease Monitoring and Surveillance PD ADMAS
Indian Journal of Virology | Year: 2014

Peste des petits ruminants (PPR) is an acute, highly contagious, world organization for animal health (OIE) notifiable and economically important transboundary viral disease of sheep and goats associated with high morbidity and mortality and caused by PPR virus. PPR is considered as one of the main constraints in augmenting the productivity of small ruminants in developing countries and particularly severely affects poor farmer's economy. The disease is clinically manifested by pyrexia, oculo-nasal discharges, necrotizing and erosive stomatitis, gastroenteritis, diarrhoea and bronchopneumonia. The disease can be diagnosed from its clinical signs, pathological lesions, and specific detection of virus antigen/antibodies/genome in the clinical samples by various serological tests and molecular assays. PPR is the one of the priority animal diseases whose control is considered important for poverty alleviation in enzootic countries. Availability of effective and safe live attenuated cell culture PPR vaccines and diagnostics have boosted the recently launched centrally sponsored control programme in India and also in other countries. This review article primarily focus on the current scenario of PPR diagnosis and its control programme with advancement of research areas that have taken place in the recent years with future perspectives. © 2013 Indian Virological Society.


Khurana S.K.,National Research Center on Equines | Srivastava S.K.,National Research Center on Equines | Prabhudas K.,National Research Center on Equines
Indian Journal of Animal Sciences | Year: 2012

Brucellosis is a zoonotic disease of immense economic importance affecting many species of animals. Serosurveillance of animals from 45 randomly selected villages of 11 districts of Southern Haryana was carried out. Representative serum samples were collected randomly from 5% of the total population of cattle and buffaloes. Overall prevalence of brucellosis was 12.13% (142 cases) out of 1170 serum samples screened by avidin-biotin serum ELISA. It was 14.44% (26 cases) and 11.71% (116 cases) out of 180 cattle and 990 buffalo serum samples, respectively. However, 101(8.6%) and 66 (5.64%) serum samples were found positive by STAT and RBPT, respectively. Prevalence of Brucella antibodies was higher in female animals (16.02% in cattle and 12.32% in buffaloes) than in males (4.14% in cattle and 4.10% in buffaloes). Similarly, sexually mature animals (>3 years of age) were found to be affected more (16.94% in cattle and 13.68% in buffaloes) than the immature ones (<3 years of age (9.67% in cattle and 6.56% in buffaloes), Jind district with prevalence rate of 22.72% and 25.74% and Faridabad with prevalence rate of 13.88% and 14.62% were affected more against overall prevalence rate of 14.44% and 11.71% in cattle and buffaloes respectively, in Southern Haryana.


Kumar D.,ICAR Central Institute for Research on Buffaloes | Talluri T.R.,National Research Center on Equines | Anand T.,National Research Center on Equines | Kues W.A.,Friedrich Loeffler Institute
Histology and Histopathology | Year: 2015

Induced pluripotent stem (iPS) cells represent a recent innovation in the field of stem cells. Commonly, iPS cells are generated by viral transduction of core reprogramming genes, such as Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28. However, integrating viruses, like retro- and lentiviral vectors, may cause insertional mutagenesis and may increase the risk of tumor formation. Therefore, alternative methods which avoid these safety concerns are intensively investigated. Here, we review the current status of transposon-based methods to induce pluripotency. DNA transposons are non-viral elements, which can be effectively integrated into a genome by their corresponding transposase enzyme. The advantages of transposon-based gene transfer are their increased safety, their large cargo capacity, their relatively simple design, and the availability of hyper-active and mutated transposase enzymes. For example, integration-deficient, excisioncompetent transposase variants allow the complete removal of the reprogramming transposon after successful reprogramming to obtain transposon-free reprogrammed cells. Transposon-based reprogramming broaden the toolbox for iPS cell production and will advance the establishment of safe, non-viral methods. © 2015, Histology and Histopathology. All rights reserved.


Manuja A.,National Research Center on Equines | Manuja B.K.,National Research Center on Equines | Kaushik J.,National Research Center on Equines | Singha H.,National Research Center on Equines | Singh R.K.,National Research Center on Equines
Immunopharmacology and Immunotoxicology | Year: 2013

Innate immunity plays a critical role in host defense against infectious diseases by discriminating between self and infectious non-self. The recognition of infectious non-self involves germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). The PAMPs are the components of pathogenic microbes which include not only the cell wall constituents but also the unmethylated 2′-deoxy-ribo-cytosine-phosphate- guanosine (CpG) motifs. These CpG motifs present within bacterial and viral DNA are recognized by toll-like receptor 9 (TLR9), and signaling by this receptor triggers a proinflammatory cytokine response which, in turn, influences both innate and adaptive immune responses. The activation of TLR9 with synthetic CpG oligodeoxynucleotides (ODNs) induces powerful Th1-like immune responses. It has been shown to provide protection against infectious diseases, allergy and cancer in laboratory animal models and some domestic animal species. With better understanding of the basic biology and immune mechanisms, it would be possible to exploit the potential of CpG motifs for animal welfare. The research developments in the area of CpG and TLR9 and the potential applications in animal health have been reviewed in this article. © 2013 Informa Healthcare USA, Inc. All rights reserved.


Chauhan M.,National Research Center on Equines | Gupta A.K.,National Research Center on Equines | Dhillon S.,CCS Haryana Agricultural University
Molecular Biology Reports | Year: 2011

The genetic relationships of three Indian horse breeds-Marwari, Spiti, and Kathiawari were studied by genotyping 96 individuals with 20 polymorphic microsatellite markers. A total of 157 alleles were detected across 20 polymorphic loci. The Marwari population showed the highest allelic diversity (A = 5.7 and Ar = 5.14), followed by Spiti (A = 4.9 and Ar = 4.74) and Kathiawari (A = 4.1 and Ar = 3.82). The gene diversity was highest in the Spiti population (He = 0.67), followed by Marwari (He = 0.66) and Kathiawari (He = 0.59). Within population inbreeding estimates (f) in Marwari, Spiti and Kathiawari breeds were 0.18, 0.08, and 0.07, respectively, suggesting high level of inbreeding in these breeds. Analysis of bottleneck revealed evidence of recent bottleneck in Spiti and Kathiawari populations. Pair-wise Fst analysis, AMOVA and assignment tests demonstrated high genetic differentiation and low gene flow between populations. The information about genetic diversity and population structure will be useful for the future development of effective breeding management in order to preserve these Indian horse breeds. © Springer Science+Business Media B.V. 2010.

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