Affinity chromatography: purification of bovine trypsin and thrombin

Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, K_i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low K_i values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.     

Recursive organizer (ROR): an analytic framework for sequence-based association analysis

The advent of next-generation sequencing technologies affords the ability to sequence thousands of subjects cost-effectively, and is revolutionizing the landscape of genetic research. With the evolving genotyping/sequencing technologies, it is not unrealistic to expect that we will soon obtain a pair of diploidic fully phased genome sequences from each subject in the near future. Here, in light of this potential, we propose an analytic framework called, recursive organizer (ROR), which recursively groups sequence variants based upon sequence similarities and their empirical disease associations, into fewer and potentially more interpretable super sequence variants (SSV). As an illustration, we applied ROR to assess an association between HLA-DRB1 and type 1 diabetes (T1D), discovering SSVs of HLA-DRB1 with sequence data from the Wellcome Trust Case Control Consortium. Specifically, ROR reduces 36 observed unique HLA-DRB1 sequences into 8 SSVs that empirically associate with T1D, a fourfold reduction of sequence complexity. Using HLA-DRB1 data from Type 1 Diabetes Genetics Consortium as cases and data from Fred Hutchinson Cancer Research Center as controls, we are able to validate associations of these SSVs with T1D. Further, SSVs consist of nine nucleotides, and each associates with its corresponding amino acids. Detailed examination of these selected amino acids reveals their potential functional roles in protein structures and possible implication to the mechanism of T1D.     

Biocatalysts for multicomponent Biginelli reaction: bovine serum albumin triggered waste-free synthesis of 3,4-dihydropyrimidin-2-(1H)-ones

Bovine serum albumin (BSA) promoted simple and efficient one-pot procedure was developed for the direct synthesis of 3,4-dihydropyrimidin-2(1H)-ones including potent mitotic kinesin Eg5 inhibitor monastrol under mild reaction conditions. The catalyst recyclability and gram scale synthesis have also been demonstrated to enhance the practical utility of process.     

Development and validation of a serological potency test for the release of Leptospira vaccines – Requirements in the European Union

Both European Pharmacopoeia Monograph 01/2008:0447 “Canine Leptospirosis vaccine (inactivated)” and the more recent Monograph 01/2008:1939 “Bovine Leptospirosis vaccine (inactivated)” explicitly allow for a sero-response test to assess batch potency. Test setup and requirements for in vivo and in vitro validation are described. Furthermore, the two main strategies to assess batch potency and their specific demands are addressed.     

Alzheimer’s Disease: Analyzing the Missing Heritability

Alzheimer’s disease (AD) is a complex disorder influenced by environmental and genetic factors. Recent work has identified 11 AD markers in 10 loci. We used Genome-wide Complex Trait Analysis to analyze >2 million SNPs for 10,922 individuals from the Alzheimer’s Disease Genetics Consortium to assess the phenotypic variance explained first by known late-onset AD loci, and then by all SNPs in the Alzheimer’s Disease Genetics Consortium dataset. In all, 33% of total phenotypic variance is explained by all common SNPs. APOE alone explained 6% and other known markers 2%, meaning more than 25% of phenotypic variance remains unexplained by known markers, but is tagged by common SNPs included on genotyping arrays or imputed with HapMap genotypes. Novel AD markers that explain large amounts of phenotypic variance are likely to be rare and unidentifiable using genome-wide association studies. Based on our findings and the current direction of human genetics research, we suggest specific study designs for future studies to identify the remaining heritability of Alzheimer’s disease.     

Animal Viruses Probe Dataset (AVPDS) for Microarray-Based Diagnosis and Identification of Viruses

AVPDS (Animal Viruses Probe dataset) is a dataset of virus-specific and conserve oligonucleotides for identification and diagnosis of viruses infecting animals. The current dataset contain 20,619 virus specific probes for 833 viruses and their subtypes and 3,988 conserved probes for 146 viral genera. Dataset of virus specific probe has been divided into two fields namely virus name and probe sequence. Similarly conserved probes for virus genera table have genus, and subgroup within genus name and probe sequence. The subgroup within genus is artificially divided subgroups with no taxonomic significance and contains probes which identifies viruses in that specific subgroup of the genus. Using this dataset we have successfully diagnosed the first case of Newcastle disease virus in sheep and reported a mixed infection of Bovine viral diarrhea and Bovine herpesvirus in cattle. These dataset also contains probes which cross reacts across species experimentally though computationally they meet specifications. These probes have been marked. We hope that this dataset will be useful in microarray-based detection of viruses. The dataset can be accessed through the link https://dl.dropboxusercontent.com/u/94060831/avpds/HOME.html.     

Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in-vitro culture in cryopreservation studies

Development of in vitro culture protocol for early stage ovarian follicles of zebrafish is important since cryopreserved early stage ovarian follicles would need to be matured in vitro following cryopreservation before they can be fertilised. Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in vitro culture of early stage zebrafish ovarian follicles in ovarian tissue fragments is reported here for the first time although some work has been reported for in vitro culture of isolated early stage zebrafish ovarian follicles. The main aim of the present study was to develop molecular markers in an optimised in vitro culture protocol for stage I and stage II zebrafish ovarian follicles in ovarian tissue fragments. The effect of concentration of the hormones human chorionic gonadotropin and follicle stimulating hormones, and additives such as Foetal Bovine Serum and Bovine Serum Albumin were studied. The results showed that early stage zebrafish ovarian fragments containing stage I and stage II follicles which are cultured in vitro for 24 h in 20% FBS and 100mIU/ml FSH in 90% L-15 medium at 28 °C can grow to the size of stage II and stage III ovarian follicles respectively. More importantly the follicle growth from stage I to stage II and from stage II to stage III were confirmed using molecular markers such as cyp19a1a (also known as P450aromA) and vtg1 genes respectively. However, no follicle growth was observed following cryopreservation and in vitro culture.     

Describing the intestinal microbiota of Holstein Fasciola-positive and -negative cattle from a hyperendemic area of fascioliasis in central Colombia

The ability to identify compositional changes in the intestinal microbiota of parasitized hosts is important for understanding the physiological processes that may affect animal productivity. Within the field of host–parasite interactions, many studies have suggested that helminths can influence the microbial composition of their hosts via their immunomodulatory effects. Bovine fascioliasis is a helminthiasis widely studied by immunologists, but with little information available regarding gut microbial communities. Thus, we aimed to describe the composition of the intestinal microbiota of Holstein Fasciola-positive and -negative cattle using parasitological methods and ELISA (enzyme-linked immunosorbent assay). Bovine fecal samples (n = 65) were obtained from livestock slaughter plants in the Cundi-Boyacense Colombian highlands (a hyperendemic region for bovine fascioliasis) and studied by amplicon-based next-generation 16S-rRNA and 18S-rRNA gene sequencing. From these samples, 35 were Fasciola hepatica-negative and, 30 were F. hepatica-positive in our detection analysis. Our results showed a reduction in the relative abundance of Bacteroidetes and Ascomycota in the Fasciola-positive samples, along with decreased relative abundances of the commensal taxa previously associated with fermentation and digestion processes. However, metabolomic approaches and functional analyzes of the intestinal microbiota are necessary to support these hypothesis. These findings are a small first step in the development of research aimed at understanding how microbial populations in bovines are modulated in liver helminth infections.     

In vitro prothrombin synthesis from a purified precursor protein III. Preparation of an acid-soluble substrate for vitamin K-dependent carboxylase by limited proteolysis of bovine descarboxyprothrombin

Bovine descarboxyprothrombin and descarboxyfragment-1 can be used as substrates for rat and bovine vitamin K-dependent carboxylase. In both enzyme systems, however, these substrates have a high K_m (0.3–0.4 mM). A better substrate (K_m = 0.001–0.003 mM) was prepared from bovine descarboxyprothrombin by limited proteolysis with subtilisin Carlsberg. This substrate is called Fragment-Su and is composed of the amino acids 13–29 of descarboxyprothrombin.     

GSEA-SNP identifies genes associated with Johne’s disease in cattle

SNP-based gene-set enrichment analysis from single nucleotide polymorphisms, or GSEA-SNP, is a tool to identify candidate genes based on enrichment analysis of sets of genes rather than single SNP associations. The objective of this study was to identify modest-effect genes associated with Mycobacterium avium subsp. paratuberculosis (Map) tissue infection or fecal shedding using GSEA-SNP applied to KEGG pathways or Gene Ontology (GO) gene sets. The Illumina Bovine SNP50 BeadChip was used to genotype 209 Holstein cows for the GSEA-SNP analyses. For each of 13,744 annotated genes genome-wide located within 50 kb of a Bovine SNP50 SNP, the single SNP with the highest Cochran-Armitage Max statistic was used as a proxy statistic for that gene’s strength of affiliation with Map. Gene-set enrichment was tested using a weighted Kolmogorov-Smirnov-like running sum statistic with data permutation to adjust for multiple testing. For tissue infection and fecal shedding, no gene sets in KEGG pathways or in GO sets for molecular function or cellular component were enriched for signal. The GO biological process gene set for positive regulation of cell motion (GO:0051272, q = 0.039, 5/11 genes contributing to the core enrichment) was enriched for Map tissue infection, while no GO biological process gene sets were enriched for fecal shedding. GSEA-SNP complements traditional SNP association approaches to identify genes of modest effects as well as genes with larger effects as demonstrated by the identification of one locus that we previously found to be associated with Map tissue infection using a SNP-by-SNP genome-wide association study.     

Generation of a Persistently Infected MDBK Cell Line with Natural Bovine Spongiform Encephalopathy (BSE)

Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrP^BSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrP^BSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.     

The reactivity of the disulphide bonds of bovine pancreatic ribonuclease with glutathione

1. Bovine pancreatic ribonuclease is not reduced by GSH at near-physiological concentrations and pH. 2. Disruption of the structure of ribonuclease by proteolytic enzymes leads to products that can be reduced by GSH. 3. At higher temperatures the disulphide bonds of ribonuclease are completely reduced by GSH in a coupled system. The T_tr is 51° and this has been found to be lower than the T_tr for the abnormal tyrosine residues under the same conditions.     

Bovidae (Mammalia) du Pliocène final d’Ahl al Oughlam, Casablanca, Maroc

Only seven bovid species are present at Ahl al Oughlam: aTragelaphus (rare), a Bovine close toPelorovis ? praeafricanus but mostly known by teeth, a new species of kob (perhaps of an endemic lineage), aParmularius slightly more primitive than at Olduvai,Beatragus (mentioned for the first time in North Africa),Gazella thomasi, and a new species of gazelle noticeable by its nasal region. The Ahl al Oughlam bovids are in good agreement with an age of 2.5 Ma. They point towards an open and probably rather unfavourable, perhaps cold, environment.     

Microarray Chip Based Identification of a Mixed Infection of Bovine Herpesvirus 1 and Bovine Viral Diarrhea 2 From Indian Cattle

Bovine herpesvirus 1 (BHV1) and bovine viral diarrhea virus 2 (BVD2) are endemic in India although no mixed infection with these viruses has been reported from India. We report first mixed infection of these viruses in cattle during routine screening with a microarray chip. 62 of the 69 probes of BHV1 and 42 of the 57 BVD2 probes in the chip gave positive signals for the virus. The virus infections were subsequently confirmed by RT-PCR. We also discuss the implications of these findings.     

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.     

Swelling and contraction of phaseolus hypocotyl mitochondria

Isolated Phaseolus mitochondria will swell spontaneously in buffered KCl and contract with an oxidizable substrate or ATP + Mg²⁺. The conditions under which the mitochondria are swollen affect subsequent contraction, substrate oxidation and ion accumulation, but not their oxidative phosphorylation ability. Bovine serum albumin reduces the rate of swelling and promotes substrate oxidation, contraction and ion accumulation. Swelling of these mitochondria is associated with the release of malic dehydrogenase and a loss of membrane integrity. The beneficial effects of bovine serum albumin in preserving the energy linked functions of Phaseolus mitochondria is discussed.     

Viral diagnosis in Indian livestock using customized microarray chips

Viral diagnosis in Indian livestock using customized microarray chips is gaining momentum in recent years. Hence, it is possible to design customized microarray chip for viruses infecting livestock in India. Customized microarray chips identified Bovine herpes virus-1 (BHV-1), Canine Adeno Virus-1 (CAV-1), and Canine Parvo Virus-2 (CPV-2) in clinical samples. Microarray identified specific probes were further confirmed using RT-PCR in all clinical and known samples. Therefore, the application of microarray chips during viral disease outbreaks in Indian livestock is possible where conventional methods are unsuitable. It should be noted that customized application requires a detailed cost efficiency calculation.