Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties.

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.

Genetic Susceptibility to Chronic Liver Disease in Individuals from Pakistan.

Chronic liver disease, with viral or non-viral etiology, is endemic in many countries and is a growing burden in Asia. Among the Asian countries, Pakistan has the highest prevalence of chronic liver disease. Despite this, the genetic susceptibility to chronic liver disease in this country has not been investigated. We performed a comprehensive analysis of the most robustly associated common genetic variants influencing chronic liver disease in a cohort of individuals from Pakistan. A total of 587 subjects with chronic liver disease and 68 healthy control individuals were genotyped for the HSD17B13 rs7261356, MBOAT7 rs641738, GCKR rs1260326, PNPLA3 rs738409, TM6SF2 rs58542926 and PPP1R3B rs4841132 variants. The variants distribution between case and control group and their association with chronic liver disease were tested by chi-square and binary logistic analysis, respectively. We report for the first time that HSD17B13 variant results in a 50% reduced risk for chronic liver disease; while MBOAT7; GCKR and PNPLA3 variants increase this risk by more than 35% in Pakistani individuals. Our genetic analysis extends the protective role of the HSD17B13 variant against chronic liver disease and disease risk conferred by the MBOAT7; GCKR and PNPLA3 variants in the Pakistani population.

Association study of Apolipoprotein A5 gene (APOA5 gene) variant with the metabolic syndrome in local Pakistani population.

OBJECTIVE: To explore the association of rs662799 variants of Apolipoprotein A5 gene with metabolic syndrome in Pakistani population. METHODS: The case-control study was conducted at Pakistan Institute of Medical Sciences, Islamabad, Pakistan from 2014 to2016, and comprised subjects enrolled from the out-patient clinics. Groups were formed on the basis of preliminary screening for risk factors like obesity, insulin resistance, hypertension, dyslipidemia and fasting blood glucose levels. Met S was diagnosed based on the international diabetes federation criteria. Blood samples were collected for biochemical testing and deoxyribonucleic acid extraction. Genotyping of rs662799 was performed a the Genome Research Centre of the University of Hong Kong using Sequenom Mass ARRAY, iPLEX Gold technology. Data was analysed using SPSS 16and Plink software. RESULTS: :There were 712 subjects in two groups of 356(50%) each. The overall mean age was 41.59}7.18 years. There was a significant association of risk allele C of rs662799 with metabolic syndrome (p=0.002). The risk showed strong association with dyslipidaemia (p=0.03) and obesity (p=0.01) which are risk phenotypes of metabolic syndrome in age- and gender-adjusted model. CONCLUSIONS: The association of risk allele C of genetic variant rs662799 of Apolipoprotein A5 gene with dyslipidaemia and obesity may lead to the development of metabolic syndrome in the Pakistan adult population.

Identification of Metabolic risk phenotypes predisposing to Non-Alcoholic Fatty Liver Disease in a Pakistani Cohort.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) has emerged in the last two decades with worldwide prevalence of 25.24%. Due to its increasing frequency in Pakistan, it was aimed to identify disease predisposing metabolic risks and their association with NAFLD. METHODS: Anthropometric and biochemical investigations were collected from 1366 subjects with minor metabolic disturbances. Comparative analyses were performed to compute frequencies of common metabolic risk phenotypes while their associations with NAFLD were explored using regression analyses. The prevalence of NAFLD was also estimated in total, age, and gender-based population cohorts. RESULTS: Among metabolic risk phenotypes obesity, hyperglycemia, hypertension, and dyslipidemia significantly associated (p<0.001) with NAFLD risk irrespective of age, gender, and BMI. Prevalence of NAFLD in total study cohort was 14.8%, 16.1% in males, 13.4% in females, 19.9% in ≥40 years and 8.7% in ≤40 years respectively. CONCLUSION: General Pakistani populations experiencing common metabolic disturbances are at high risk of NAFLD development, especially male gender and advanced age. Based on these parameters the stratified NAFLD population could be treated accordingly.

Germin-like protein 2 gene promoter from rice is responsive to fungal pathogens in transgenic potato plants.

Controlled transgene expression via a promoter is particularly triggered in response to pathogen infiltration. This is significant for eliciting disease-resistant features in crops through genetic engineering. The germins and germin-like proteins (GLPs) are known to be associated with plant and developmental stages. The 1107-bp Oryza sativa root GLP2 (OsRGLP2) gene promoter fused to a ß-glucuronidase (GUS) reporter gene was transformed into potato plants through an Agrobacterium-mediated transformation. The OsRGLP2 promoter was activated in response to Fusarium solani (Mart.) Sacc. and Alternaria solani Sorauer. Quantitative real-time PCR results revealed 4-5-fold increase in promoter activity every 24 h following infection. There was a 15-fold increase in OsRGLP2 promoter activity after 72 h of F. solani (Mart.) Sacc. treatment and a 12-fold increase observed with A. solani Sorauer. Our results confirmed that the OsRGLP2 promoter activity was enhanced under fungal stress. Furthermore, a hyperaccumulation of H2O2 in transgenic plants is a clear signal for the involvement of OsRGLP2 promoter region in the activation of specific genes in the potato genome involved in H2O2-mediated defense response. The OsRGLP2 promoter evidently harbors copies of GT-I and Dof transcription factors (AAAG) that act in response to elicitors generated in the wake of pathogen infection.

Banana bunchy top virus genetic diversity in Pakistan and association of diversity with recombination in its genomes.

Banana Bunchy top virus (BBTV) is a multipartite circular single strand DNA virus that belongs to genus Babuvirus and family Nanoviridae. It causes significant crop losses worldwide and also in Pakistan. BBTV is present in Pakistan since 1988 however, till now only few (about twenty only) sequence of genomic components have been reported from the country. To have insights into current genetic diversity in Pakistan fifty-seven genomic components including five complete genomes (comprises of DNA-R, -U3, -S, -M, -C and -N components) were sequenced in this study. The genetic diversity analysis of populations from Pakistan showed that DNA-R is highly conserved followed by DNA-N, whereas DNA-U3 is highly diverse with the most diverse Common Region Stem-loop (CR-SL) in BBTV genome, a functional region, which previously been reported to have undergone recombination in Pakistani population. A Maximum Likelihood (ML) phylogenetic analysis of entire genomes of isolates by using sequence of all the components concatenated together with the reported genomes around the world revealed deeper insights about the origin of the disease in Pakistan. A comparison of the genetic diversity of Pakistani and entire BBTV populations around the world indicates that there exists a correlation between genetic diversity and recombination. Population genetics analysis indicated that the degree of selection pressure differs depending on the area and genomic component. A detailed analysis of recombination across various components and functional regions suggested that recombination is closely associated with the functional parts of BBTV genome showing high genetic diversity. Both genetic diversity and recombination analyses suggest that the CR-SL is a recombination hotspot in all BBTV genomes and among the six components DNA-U3 is the only recombined component that has extensively undergone inter and intragenomic recombination. Diversity analysis of recombinant regions results on average one and half fold increase and, in some cases up to four-fold increase due to recombination. These results suggest that recombination is significantly contributing to the genetic diversity of BBTV populations around the world.

Functional characterization of the rice root Germin-like protein gene-1 (OsRGLP1) promoter in Nicotiana tabacum.

Germin (GER) and germin-like protein (GLP) genes play a very important role against various stresses. Promoter analysis provide significant insight into gene's function and regulation. Presently, upstream region (1228 bp) of the OsRGLP1 gene was functionally characterized via heterologous expression. It was fused with the Glucuronidase (GUS) reporter gene and the expression cassette was used to transform Nicotiana tabacum using Agrobacterium-mediated transformation. Transgenic plants were examined via quantitative real-time PCR (qPCR) to analyze its role in wounding, salinity, drought, abscisic acid (ABA) and circadian rhythm. OsRGLP1 was highly induced by ABA and drought by showing 28- and 25-fold changes in GUS mRNA level respectively as compared to wounding (fourfold change) and salinity (threefold change). However, no activity was observed in circadian rhythm. Histochemically, strong GUS activity was observed in leaf veins, midrib, epidermal hair, stomata guard cells, stem cortex, root hairs, xylem and phloem and at cellular level in cell wall, cytoplasm and its periphery. OsRGLP1 promoter can be used to develop agronomically important transgenic plants in future food program.

Transgenic Analysis Reveals 5' Abbreviated OsRGLP2 Promoter(s) as Responsive to Abiotic Stresses.

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5' deletion analysis of the promoter sequences was performed and the deletion fragments fused with the ß-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.

Trichophyton verrucosum infection in livestock in the Chitral district of Pakistan.

INTRODUCTION: Trichophyton verrucosum belongs to the dermatophyte fungi, closely related organisms that cause skin infections in animals and humans. T. verrucosum infection has been reported in livestock and people in different countries from all continents. Human cases have been reported in different areas of Pakistan, but there is little information about the animal source of the fungus. METHODOLOGY: Dermatological specimens collected in the Chitral district of Pakistan for a study on mange in livestock were retrospectively analyzed for the presence of T. verrucosum. In total, 5,873 animals (1,087 cows, 2,033 goats, and 2,753 sheep) were screened for evidence of dermatological lesions during two surveys performed in the summer and winter seasons. Skin scrapings collected from animals with lesions were analyzed by direct microscopic examination after digestion in sodium hydroxide and a real-time polymerase chain reaction (PCR) targeting pathogenic Trichophyton species. RESULTS: At microscopy, samples from 18 cows (1.6%), 3 sheep (0.1%), and 4 goats (0.2%) were positive for fungal elements consistent with T. verrucosum. PCR confirmed the microscopy results. The prevalence was lower than that reported in other countries in intensive breeding farms. Results agree with the literature regarding factors affecting T. verrucosum diffusion, i.e., infection was more prevalent in cattle, especially in younger animals during the winter season. CONCLUSIONS: This study reports, for the first time, the presence of T. verrucosum in livestock in Pakistan. A better knowledge of the animal role in the spread of this fungus may allow the adoption of more efficient control measures and prophylaxis.

Molecular diagnosis and phylogenetic analysis of human papillomavirus type-16 from suspected patients in Pakistan.

BACKGROUND: Human Papillomavirus (HPV) is well known pathogen that can cause benign and malignant tumors in humans, yet there is very little information regarding HPV types prevalent in Pakistan. METHODS: A total of 92 cervical secretions were collected from suspected married female patients and used for DNA isolation using a novel isolation method. The samples were tested through Polymerase Chain Reaction (PCR) using already reported primers MY09/MY11, GP5/GP6, GP5+/GP6+, CP65/CP70, CP66/CP69 and SPF1/SPF2 and with those developed in this study including HRT1 and HRT2 primer sets for typing HPV types and HACTB primer set for human beta actin gene as internal positive control. Sequencing and phylogenetic analyses were performed for two isolates to determine circulating HPV types. RESULTS: PCR with HRT1 and HRT2 indicated 2 (2.17 %) patients were positive for HPV type- 16 while 1 (1.08 %) with HPV type 18. Sequencing and phylogenetic analysis of isolates confirmed HPV type-16 in genus alpha 9 which have 99 % homology with already reported HPV from Japan and Costa Rica. CONCLUSION: This is the first report of HPV type-16 genus alpha 9 in Pakistan and the reported assay and sequence data will serve as valuable tools in further epidemiological studies for HPV surveillance to improve public health, especially of females in Pakistan.

Multiplex PCR assay for identification of commonly used disarmed Agrobacterium tumefaciens strains.

The success of Agrobacterium mediated plant transformation depends to a certain extent on appropriate selection of the A. tumefaciens strain for a particular plant species. Many stages in a plant transformation procedure are prone to bacterial contamination with similar antibiotic resistance that may compromise the identity of the A. tumefaciens strain used, in turn adversely affecting success of a transformation experiment. Different primer sets were designed to exploit genetic differences among different strains of A. tumefaciens which are commonly used for plant genetic transformation, to identity confirmation as well as to distinguish them from one another. The primer sets Ach5FtsZ-F/R specific for Ach5 and C58GlyA-F/R specific for C58 were designed on chromosomal DNA while primer sets pTiBo542-F/R and nptI-F/R specific for plasmid pTiBo542 are capable to identify and distinguish these strains from one another. These primer sets when used simultaneously in multiplex PCR, produce a pattern which uniquely identifies all these strains and distinguishes them except for GV3101 and C58C1, which can further be distinguished from each other by rifampicin screening. The multiplex PCR assay and primers being reported here serve as a valuable tool in determining the identity of A. tumefaciens strains at any stage of plant transformation procedure.

Evidence of recombination in the Banana bunchy top virus genome.

Viruses serve as good model for evolutionary studies, owing to their short generation times and small genomes. Banana bunchy top virus (BBTV) is a significant subject being multicomponent circular single stranded DNA virus. BBTV belongs to family Nanoviridae and contains DNA-R, -U3, -S, -M, -C, and -N as integral genomic components. Evolutionary studies have shown genetic re-assortment of components among its isolates and revealed a concerted type evolution in non-coding regions of its genome. The DNA U3 having been shown as the most diverse component in our previous studies, was subjected to sequencing from some Pakistani isolates for the first time. Sequence analysis revealed intergenomic recombination in DNA-U3 among the isolates of two sub-groups and a very rare intragenomic recombination in Pakistani BBTV population. This indicates that like other evolutionary processes including intergenomic recombination, intragenomic recombination among the genomic components of the same isolate may also have a significant contribution in the evolution of BBTV genome. Intragenomic recombination therefore appears to be a unique way to generate genetic diversity in the multicomponent ssDNA viruses.