Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus.

INTRODUCTION: Dengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines. MATERIAL AND METHODS: Blood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors. RESULTS: The gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells. CONCLUSION: Recombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.

Synergistic potential of Zingiber officinale and Curcuma longa to ameliorate diabetic-dyslipidemia.

To find the cure of world's one of the leading morbid and mortal disorders; diabetes mellitus and its most prevalent complication, 'diabetic-dyslipidemia', is one of the leading health challenges of 21st century. The use of phytomedicine is a glimmer of hope in this scenario. Studies of current decade have shown that methanolic extracts of Zingiber officinale and Curcuma longa have highly effective therapeutic potentials against the aforesaid disorders, however, which of the extracts has more potential is still unclear. Furthermore, synergistic effect of the extracts has never been studied. Forty-eight Albino adult rats of either sex were randomly divided into eight groups. A-D groups were containing healthy rats while E-H groups were of induced diabetic-dyslipidemic rats. For forty-two days, rats of each group were given either distilled water or Zingiber officinale methanolic extract (ZOME) or Curcuma longa methanolic extract (CLME) or ZOME+CLME therapies at dose rate of 300mg/100 mL dist. H2O/kg body wt/day. FPG and lipid profiles were estimated before and after the trial, and were statistically analyzed by one-way ANOVA along with Post-hoc Tukey's multiple comparison tests. Although, ZOME and CLME significantly (P<0.05) lowered fasting plasma glucose (FPG) levels and controlled lipid profiles in diabetic-dyslipidemic rats; yet, synergistic therapy of both extracts (ZOME+CLME) most significantly (P<0.05) controlled all parameters of diabetic-dyslipidemia (78.00±1.06mg/dL FPG, 62.00±0.58mg/dL TG, 66.50±0.76mg/dL cholesterol, 32.00±0.36mg/dL HDL, 22.43±0.64 mg/dL LDL, and 12.40±0.12mg/dL VLDL). Our findings may be useful to formulate new medicines having multiple potentials to control diabetes mellitus, dyslipidemia, and diabetic-dyslipidemia.

Osteopontin gene polymorphism association with milk traits and its expression analysis in milk of riverine buffalo.

Osteopontin gene is regarded as a plausible candidate in mammary gland differentiation and development, expressed by variety of cells, tissues, and biological fluids including milk. The current study was performed in two phases. In the first phase, Osteopontin gene polymorphisms were identified and associated with milk composition such as ash, milk fat, SNF, lactose, and protein. In the second phase, milk samples from five healthy mastitis-free Nili Ravi buffaloes were analyzed for expression of Osteopontin gene at transition (day 15), mid (day 90), and end (day 250) stage of their second lactation. Briefly, blood samples were collected from Nili Ravi buffalo to isolate the genomic DNA, specific primers were designed for PCR amplification. The amplified PCR products were sequenced bi-directionally. Six polymorphisms were identified in the coding region and four in the intronic region of the gene. The results showed that SNP g.38329758 T > C causing substitution of valine to alanine (V127A) was associated with high milk protein. For mRNA expression analysis, somatic cells were separated from milk samples for RNA isolation. Analysis of differential gene expression data has permitted us to illustrate the expression pattern of osteopontin gene in lactating buffalo. The Osteopontin gene was found to be transcribed among all three lactation stages, but expression was observed with the highest value (fold change) in peak lactation and remained elevated till the end of lactation. Identified gene marker may be helpful for the prediction of superior animal for selection. The presented study also gave an insight into the genetic screening and lactation biology of riverine buffalo, offering direction for future research in lactating buffalo.

Polymerase Chain Reaction-Based Detection Method Is Suitable for Dengue Infection During Epidemics.

Over the years, dengue fever has become a significant infectious disease in different parts of the world. Medical and public health services have been unable to deal with infection as there is no vaccine available for the prevention of this infection. With dengue, effective treatments are not available due to which severe symptoms may develop. To deal with this challenge, a sensitive and specific technique is required for its early diagnosis along with the knowledge of dengue serotype to increase the specificity of diagnosis and treatment. This study was designed to check the usefulness of immunological and nucleic acid-based molecular determination of dengue virus. The study recommends polymerase chain reaction as a suitable method for the rapid detection of dengue virus as it was found more sensitive than other utilized techniques, including antibodies detection. Nucleic acid analysis may also help to define the common serotypes/genotypes of dengue virus circulating in any region.

Identification of two novel ALS2 mutations in infantile-onset ascending hereditary spastic paraplegia.

Biallelic mutations of ALS2 cause a clinical spectrum of overlapping autosomal recessive neurodegenerative disorders: infantile-onset ascending hereditary spastic paralysis (IAHSP), juvenile primary lateral sclerosis (JPLS), and juvenile amyotrophic lateral sclerosis (ALS2). We report on eleven individuals affected with IAHSP from two consanguineous Pakistani families. A combination of linkage analysis with homozygosity mapping and targeted sequencing identified two novel ALS2 mutations, a c.194T > C (p.Phe65Ser) missense substitution located in the first RCC-like domain of ALS2/alsin and a c.2998delA (p.Ile1000*) nonsense mutation. This study of extended families including a total of eleven affected individuals suggests that a given ALS2 mutation may lead to a phenotype with remarkable intrafamilial clinical homogeneity.

Identification of functional consequence of a novel selection signature in CYP11b1 gene for milk fat content in Bubalus bubalis.

Genomic selection for traits of economic importance is an emerging approach carrying tremendous potentials. Many of polygenic traits as milk fat, protein and yield have been characterize at genomic level and important selection signatures have been identified. Cytochrome P450 enzymes are potential loci for affecting many of dairy capabilities. Present study was conducted for genomic dissection of CYP11b1 gene in riverine buffaloes and seven genetic variations were identified. Out of these, one novel polymorphism (p.A313T) was found well associated with milk fat %age. AB genotyped buffaloes were found to have higher milk fat %age (8.9%) for this loci. p.A313T was further validated at larger data set by restriction digestion using CviAII enzyme. Functional consequences of this locus were also predicted by studying three dimensional structure of CYP11b1 protein. For this purpose, 3D protein model was predicted by homology modeling, secondary structural attributes were determined, signal peptide was predicted and a transmembrane helix was also identified. One of polymorphism (p.Y205L) was found in the vicinity of functionally significant F-G loop region, which is the part of protein gets attached to the inner mitochondrial membrane. But this variation could not be associated and needs further investigation. p.A30V, a popular selection marker in cattle, was found in buffaloes as well but could not be associated and might need further confirmation on larger data set. Results of this study illustrate the impending potential of this gene in determining dairy capabilities of buffaloes and might have a role in selection of superior dairy buffaloes.

Report: Bioconversion of agriculture waste to lysine with UV mutated strain of brevibacterium flavum and its biological evaluation in broiler chicks.

Lysine executes imperative structural and functional roles in body and its supplementation in diet beneficial to prevent the escalating threat of protein deficiency. The physical mutagenesis offers new fascinating avenues of research for overproduction of lysine through surplus carbohydrate containing agriculture waste especially in developing countries. The current study was aimed to investigate the potential of UV mutated strain of Brevibacterium flavum at 254 nm for lysine production. The physical and nutritional parameters were optimized and maximum lysine production was observed with molasses (4% substrate water ratio). Moreover, supplementation of culture medium with metal cations (i.e. 0.4% CaSO4, 0.3% NaCl, 0.3% KH2PO4, 0.4% MgSO4, and 0.2% (NH4) 2SO4w/v) together with 0.75% v/v corn steep liquor significantly enhanced the lysine production up to 26.71 ± 0.31 g/L. Though, concentrations of urea, ammonium nitrate and yeast sludge did not exhibit any profound effect on lysine production. Biological evaluation of lysine enriched biomass in terms of weight gain and feed conversion ratio reflected non-significant difference for experimental and control (+ve) groups. Conclusively, lysine produced in the form of biomass was compatible to market lysine in its effectiveness and have potential to utilize at commercial scale.

DNA fingerprinting of Pakistani buffalo breeds (Nili-Ravi, Kundi) using microsatellite and cytochrome b gene markers.

Cytochrome b gene markers have been proved as an efficient and powerful tool for breed characterization and species identification of buffaloes. This study represents the substantial analysis of mitochondrial DNA variation in Pakistani buffalo breeds and provides information about their genetic diversity. In this study partial amplification of cytochrome b gene of 1,061 bp was done and sequencing results showed ten haplotypes. Comparing all fifty samples from two buffalo breeds of Pakistan, fifteen polymorphic sites were observed out of which, twelve codons 42, 71, 118, 120, 199, 235, 269, 297, 318, 327, 350, 355 of mitochondrial cytochrome b gene are monomorphic which translate same amino acids as in the reference protein sequence due to silent mutation while different in DNA sequence. Similarly three codons 163, 246, 337 of mitochondrial cytochrome b are polymorphic and different from the reference sequence with respect to DNA as well as protein sequence. For the further confirmation a panel of nine microsatellite markers was used with high polymorphism information content (PIC). The frequency distribution of these alleles varies from three to eight allele at locus CSSM66 and ILST029 respectively. The results obtained from this study may contribute to the establishment of routine genotyping service of buffalo breeds for buffalo farmers for animal forensic application in case of any dispute. Additionally this study may help for breed characterization and phylogeny of aforementioned breeds of buffalo.

Modified picrate method for determination of cyanide in blood.

Cyanide (CN-) is widely distributed in the ecosystem and has been associated with toxic effects in humans and animals. Most outbreaks of CNÖ¿ poisoning in animals result from ingestion of plants containing cyanogenic glycosides. Various analytical techniques for estimating cyanide in blood are available. A simple picrate method was developed to determine blood CN- in goats. This assay is a modification of commonly available methods using picrate paper and those using Conway diffusion cells. Cyanide in blood was measured during and after IV administration of KCN at 0.6 mg/min for 1 h. Blood CN- levels in rabbits were determined after oral administration of KCN for 10, 20, 30 and 40 days. The CN- concentration in blood of goats was time-dependent and continued rising during infusion followed by gradual decline after infusion stopped. A calibration curve set by dissolving various concentrations of KCN in distilled water showed a linear relationship between CN- concentration and absorbance (R=0.995) ranging from 0.3-120 mg CN-/L. Blood CN- levels in rabbits showed time-dependent increase with maximum concentration (1.34 mg/L) at 40 days. This is a simple and inexpensive tool for the determination of blood CN- in the laboratory and under field conditions as well.