Insilico modeling and analysis of small molecules binding to the PHLPP1 protein by molecular dynamics simulation.

The PHLPP (Pleckstrin homology domain leucine-rich repeat protein phosphatases) is a newly discovered group of genes which includes PHLPP1 and PHLPP2 and plays an integral part in several cellular processes like apoptosis, cell signaling cell survival, and cell proliferation etc. Both the activation and deactivation of these genes can have vital role in several ailments like heart diseases, circadian rhythm and most importantly the cancer, hence encouraging the growth of novel therapeutic elements. To give new directions into the development of PHLPP1- targeting drugs, the interaction mechanism between PHLPP1 and five important ligands 4IP, B39, 635, ATP and GTA were investigated through docking and Molecular Dynamics Simulation. It is also noteworthy to be mentioned here that there is no previous crystal structure of PHLPP1 available. The in-silico results can provide potential base for advancements in development of new therapeutic elements targeting different diseases, mainly cancer. In this study, we employed homology modeling technique to develop a high-quality structure model of PHLPP1. The PHLPP1 model was then used in docking interaction analysis and Molecular Dynamics Simulation, to study binding pockets and interactions of PHLPP1 ligands and finding actively contributing residues in binding pocket. In final step, Free Energy Estimation was performed to observe ligand binding's quantitative characteristics.

Pathotyping and genetic characterization of avian avulavirus-1 from domestic and wild waterfowl, geese and black swans in Pakistan, 2014 to 2017.

Twenty-nine avian avulavirus-1 viruses (AAvV-1s) from healthy domestic and wild ducks, geese and black swans collected in Pakistan between 2014-2017 have been pathotyped and genetically characterized. A phylogenetic analysis revealed that 21 of the isolates belonged to sub-genotype VIIi, whereas eight isolates were highly similar to vaccine-like viruses of genotype II. In addition to confirming the continued presence of sub-genotype VIIi AAvV-1s in Pakistan, this study identifies the probable spill-over of vaccine-like viruses from vaccinated poultry to wild and domestic waterfowl and, as such, has important implications for the control and management of Newcastle disease in Pakistan.

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.

Review - MicroRNAs: A new paradigm towards mechanistic insight of diseases.

In 1993 miRNAs were discovered during a research on Caenorhabditis elegans conducted by Victor Ambros and Gary Ruvkun. The gene lin-4 that played important role in development in C. elgans was observed not encoding any protein but a very small RNA molecule of just 22 nucleotides. Main objective of this review is to highlight the significance of miRNAs in regulating the expression of many genes, which are either directly or indirectly involved in many diseases. One of the major causes of illness and death in developed countries of the world is cardiovascular disease. Some of the miRNAs have certain role to play in heart that are not specified for heart. So miRNAs have been found to be in other tissues like fibroblasts, endothelial cells and smooth muscle cells that are part of physiological study of cardiovascular system. Adult heart has limited capacity of regeneration therefore lost cardiomyocytes due to myocardial ischemia or infarction can result in low performance of heart. miRNAs have been shown to play a role in apoptotic regulation of cardiomyocytes in vivo. Many studies have shown that miR146a and 155 are up regulated in peripheral blood mononuclear cells, synovial fibroblasts, synovial fluid and Th-17 cells from rheumatoid arthritis patients as compared to healthy persons. Several types of miRNAs are playing important roles in type 1 diabetes mellitus including miR-375 and miR-375 with intolerance to glucose and decreased beta cells account due to impaired proliferation. Up regulation of miR-125a in WAT of type 2 Diabetes mellitus have been observed. miRNAs have proved to be the important regulators of cytokines and growth factor expression. Thus, suggested as a good biomarker and target of therapy. miRNA profiling techniques have revealed the role of miRNAs in Multiple sclerosis.

Worldwide patterns of ancestry, divergence, and admixture in domesticated cattle.

The domestication and development of cattle has considerably impacted human societies, but the histories of cattle breeds and populations have been poorly understood especially for African, Asian, and American breeds. Using genotypes from 43,043 autosomal single nucleotide polymorphism markers scored in 1,543 animals, we evaluate the population structure of 134 domesticated bovid breeds. Regardless of the analytical method or sample subset, the three major groups of Asian indicine, Eurasian taurine, and African taurine were consistently observed. Patterns of geographic dispersal resulting from co-migration with humans and exportation are recognizable in phylogenetic networks. All analytical methods reveal patterns of hybridization which occurred after divergence. Using 19 breeds, we map the cline of indicine introgression into Africa. We infer that African taurine possess a large portion of wild African auroch ancestry, causing their divergence from Eurasian taurine. We detect exportation patterns in Asia and identify a cline of Eurasian taurine/indicine hybridization in Asia. We also identify the influence of species other than Bos taurus taurus and B. t. indicus in the formation of Asian breeds. We detect the pronounced influence of Shorthorn cattle in the formation of European breeds. Iberian and Italian cattle possess introgression from African taurine. American Criollo cattle originate from Iberia, and not directly from Africa with African ancestry inherited via Iberian ancestors. Indicine introgression into American cattle occurred in the Americas, and not Europe. We argue that cattle migration, movement and trading followed by admixture have been important forces in shaping modern bovine genomic variation.

Genetic analysis of prolactin gene in Pakistani cattle.

Prolactin (PRL) is a polypeptide hormone, secreted mainly by the anterior pituitary gland. It is involved in many endocrine activities. The key functions of PRL are related to reproduction and lactation in mammals. To ascertain the presence of polymorphisms in the bovine PRL gene (bPRL), the bPRL gene was sequenced. Five mutations were identified in exonic region and eleven in associated intronic regions in 100 cattle from four Pakistani cattle breeds. Haplotype of predicted amino acid changes represent a common alteration at codon 222 from R-Arginine into K-Lysine in all four breeds. Significant statistical variations were observed in the distribution of single nucleotide polymorphism (SNP) in various cattle populations. However, on basis of present study, an association of these SNPs with milk performance traits in four Pakistani cow breeds cannot be truly replicated but at least can be effective DNA markers for some of the breeds studied. Linkage analysis between these SNPs on larger populations can be useful for the association with milk production traits. Furthermore, present study may be used for marker-assisted selection and management in cattle breeding program in local cattle breeds.

Global genetic diversity, introgression, and evolutionary adaptation of indicine cattle revealed by whole genome sequencing.

Indicine cattle, also referred to as zebu (Bos taurus indicus), play a central role in pastoral communities across a wide range of agro-ecosystems, from extremely hot semiarid regions to hot humid tropical regions. However, their adaptive genetic changes following their dispersal into East Asia from the Indian subcontinent have remained poorly documented. Here, we characterize their global genetic diversity using high-quality whole-genome sequencing data from 354 indicine cattle of 57 breeds/populations, including major indicine phylogeographic groups worldwide. We reveal their probable migration into East Asia was along a coastal route rather than inland routes and we detected introgression from other bovine species. Genomic regions carrying morphology-, immune-, and heat-tolerance-related genes underwent divergent selection according to Asian agro-ecologies. We identify distinct sets of loci that contain promising candidate variants for adaptation to hot semi-arid and hot humid tropical ecosystems. Our results indicate that the rapid and successful adaptation of East Asian indicine cattle to hot humid environments was promoted by localized introgression from banteng and/or gaur. Our findings provide insights into the history and environmental adaptation of indicine cattle.

Molecular phylogeny of Pakistani riverine buffalo based on genetic variability of mitochondrial cytochrome b gene.

Mitochondrial cytochrome b gene is considered to be one of the best markers for breed characterization as well as studying the ancestry in the vertebrates due to its exclusive maternal inheritance. DNA fingerprinting by single nucleotide polymorphism is most reliable and widely used molecular technique in modern forensics and is being considered in this study. Partial sequencing of 1,061 bp of aforementioned gene from 14580 to 15643 was conducted in two famous Pakistani buffalo breeds named Nili-Ravi and Kundi. In which we explore seven haplotypes within earlier and none in the latter breed. Nili-Ravi is polymorphic at four codons of this gene, and the protein translation is also different from the reference sample while monomorphic at three codons with no amino acid replacement. Haplotypes frequency distribution of these four haplotypes named NR3, NR4, NR5, NR7 revealed that the prevalence of each haplotype is 0.04 % in the Pakistani buffalo population of this Nili-Ravi breed while complete homoplasmy was observed in the Kundi breed population. Nili-Ravi breed of buffalo is genetically more variable than the Kundi breed as far as the gene in subject is concerned. It means later breed has spent more time to propagate its wild type haplotype which make this breed more ancestral as compare to Nili-Ravi. Secondly both breeds share their common ancestors with regional water buffalo rather than the swamp one.

Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome.

Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at -30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (-30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.

DNA typing of Pakistani cattle breeds Tharparkar and Red Sindhi by microsatellite markers.

Microsatellite markers are used for any individual identity and breed characterization in animals that is an efficient and successful way of investigation. They are used for multiple purposes as genetic detectors including, rapid mutation rate, high level of polymorphism, and range of variety of microsatellite markers available. A panel of 19 microsatellite markers was developed for breed characterization in Tharparkar and Red Sindhi breeds of cattle in Pakistan. Forty four blood samples of cattle (each breed) were collected from Department of Livestock Management, Sindh Agriculture University, Tandojam, Tando Qaiser, Tharparkar Cattle Farm Nabi sar Road, Umer Kot, Sindh, and Govt. Red Sindhi Cattle Breeding Farm, Tando Muhammad Khan Pakistan. Breed characterization was 100% successful. Average PIC, He and Power of Exclusion values were found to be 0.91, 0.62 and 13.28, respectively. Pattern of allelic frequencies of most of the microsatellite markers were clearly distinct between two breeds. As a result of present study a reliable, efficient and very informative panel of microsatellite markers was successfully developed which was capable to interpret individual identity, forensic cases and breed characterization in cattle. This facility is ready to be provided to local cattle breeder at commercial level for DNA testing of cattle. This study will also be highly helpful for breed conservation of cattle. In addition this study can also become a basis to open up new disciplines of animal forensics in Pakistan.

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.

Effect of varying dietary energy levels during the last trimester of pregnancy on subsequent first lactation performance in Sahiwal heifers.

The aim of the study was to determine optimum dietary energy level during the last trimester of pregnancy for Sahiwal heifers in subtropical Pakistan. Sixteen Sahiwal heifers, 5-6 months pregnant, were assigned to four dietary treatments with four heifers on each treatment. Isonitrogenous (CP = 14.1%) diets having varying energy, namely, ME 88%, ME 100% (Control), ME 112% and ME 124% of NRC recommended level for pregnant heifers, were fed until calving. All were fed a similar diet after calving. Precalving weight gain was highest (P < 0.05) in heifers fed ME 112 and 124% (486 ± 13 and 497 ± 5 g/day, respectively) followed by ME 100% (444 ± 7 g/day), and the lowest weight gain was recorded for ME 88% (397 ± 8 g/day). A similar trend was observed for feed efficiency. Body condition score at calving in groups ME 124% and ME 112% was higher than ME 88% and ME 100%. Nutrient digestibility, birth weight of calves and milk composition except fat content were not influenced by energy levels. The highest daily milk yield was observed in heifers fed ME 100% followed by ME 112, 124, and 88%. We conclude that the NRC recommendation is applicable to the subtropical region.

The study of gene GJB2/DFNB1 causing deafness in humans by linkage analysis from district Peshawar.

Families with at least 2 or more individuals having hereditary hearing loss were enrolled from different areas of Khyber Pakhtoonkhwa, mainly from district Peshawar. Detailed history was taken from each family to minimize the presence of other abnormalities and environmental causes for deafness. Families were questioned about skin pigmentation, hair pigmentation, and problems relating to balance, vision, night blindness, thyroid, kidneys, heart, and infectious diseases like meningitis, antibiotic usage, injury, and typhoid. The pedigree structures were based upon interviews with multiple family members, and pedigrees of the enrolled families were drawn using Cyrillic program (version 2.1). All families showed recessive mode of inheritance. I studied 8 families of these 10. For linkage analyses, studies for DFNB1 locus, 3 STR markers (D13S175, D13S292, and D13S787) were genotyped using polyacrylamide gel electrophoresis (PAGE) and haplotypes were constructed to determined, linkage with DFNB1 locus. From a total of 8 families, a single family-10 showed linkage to DFNB1 locus.

Promyelocytic leukemia zinc finger protein (PLZF) enhances glucocorticoid-induced apoptosis in leukemic cell line NALM6.

Glucocorticoids (GC) actuate apoptosis as well as cell cycle arrest in lymphocytes, and included as core element in the lymphoid malignancy treatment. Despite clinical significance of GC and considerable efforts to understand it, the molecular basis of GC regulated cell death and the resistance phenomenon remains, however, poorly understood. Using Affymetrix-based whole genome expression profiling our group has previously identified a number of prominent glucocorticoid-response genes (Blood 107: 2061, 2006). Promyelocytic leukemia zinc finger (PLZF) was one of the best candidate genes. This study was proposed to investigate the possible role of PLZF in GC regulated cell death in leukemic model cell line NALM6. To this end, we generated NALM6 cell line (bulk) transduced with a retroviral expression vectors, pHR-SFFV-PLZF-IRES-Puro (U426) and pHR-SFFV-Venus-IRES-Puro (U417), as control, for constitutive gene-expression. HEK293T cells were transfected transiently to generate viral particles. These cell lines were characterized by Western blotting and used to assay the effect of constitutive PLZF expression. In conclusion, we report that bona fide transcription repressor PLZF, which turned out as prominent GC-regulated gene both in vivo and in vitro situations was found to enhance the GC-induced cell death (basal) in leukemic model cell line NALM6 after 48 and 72h time points.

Double-Stranded Break Repair in Mammalian Cells and Precise Genome Editing.

In mammalian cells, double-strand breaks (DSBs) are repaired predominantly by error-prone non-homologous end joining (NHEJ), but less prevalently by error-free template-dependent homologous recombination (HR). DSB repair pathway selection is the bedrock for genome editing. NHEJ results in random mutations when repairing DSB, while HR induces high-fidelity sequence-specific variations, but with an undesirable low efficiency. In this review, we first discuss the latest insights into the action mode of NHEJ and HR in a panoramic view. We then propose the future direction of genome editing by virtue of these advancements. We suggest that by switching NHEJ to HR, full fidelity genome editing and robust gene knock-in could be enabled. We also envision that RNA molecules could be repurposed by RNA-templated DSB repair to mediate precise genetic editing.

Myostatin increases the expression of matrix metalloproteinase genes to promote preadipocytes differentiation in pigs.

ABSTACTMyostatin (MSTN) resulted in reduced backfat thickness in MSTN-knockout (MSTN-KO) pigs, whereas the underlying mechanism remains elusive. In this study, RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) in porcine fat tissues. We identified 285 DEGs, including 4 adipocyte differentiation-related genes (ADRGs). Matrix Metalloproteinase-2/7 (MMP-2/7), fibronectin (FN), and laminin (LN) were differentially expressed in MSTN-KO pigs compared with wild-type (WT) pigs. To investigate the molecular mechanism, we treated the preadipocytes with siRNA and recombinant MSTN protein. The results indicated that MSTN increased the expression of MMP-2/7/9 and promoted the preadipocyte differentiation. To further validate the effect of MSTN on MMP-2/7/9 expression, we treated MSTN-KO PK15 cells with recombinant MSTN protein and detected the expression of MMP-2/7/9. The data showed that MSTN increases the expression of MMP-2/7/9 in PK15. This study revealed that MSTN promoted preadipocyte differentiation and provided the basis for the mechanism of fatty deposition in pigs.

Compound Homozygous Rare Mutations in PLCE1 and HPS1 Genes Associated with Autosomal Recessive Retinitis Pigmentosa in Pakistani Families.

Background: Retinitis pigmentosa (RP) belongs to pigmentary retinopathies, a generic name for all retinal dystrophies with a major phenotypical and genotypical variation, characterized by progressive reduction of photo-receptor functionality of the rod and cone. Global prevalence of RP is ~ 1/4000 and it can be inherited as autosomal dominant (adRP), autosomal recessive (arRP) or X- linked (xlRP). We designed this study to identify causative mutations in Pakistani families affected with arRP. Methods: In 2019, we recruited two unrelated Pakistani consanguineous families affected with progressive vision loss and night blindness from Punjab region. Clinical diagnosis confirmed the; bone spicule pigmentation of the retina, and an altered electroretinogram (EGR) response. Proband and healthy individual from each family were subjected for whole-exome sequencing (WES). Various computational tools were used to analyze the Next Generation Sequencing (NGS) data and to predict the pathogenicity of the identified mutations. Results: WES data analysis highlighted two missense homozygous variants at position c.T1405A (p.S469T) in PLCE1 and c.T11C (p.V4A) in HPS1 genes in proband of both families. Healthy individuals of two families were tested negative for p.S469T and p.V4A mutations. The variant analysis study including molecular dynamic simulations predicted mutations as disease causing. Conclusion: Compound effect of mutations in rarely linked PLCE1 and HPS1 genes could also cause RP. This study highlights the potential application of WES for a rapid and precise molecular diagnosis for heterogeneous genetic diseases such as RP.

Identification of a novel LCA5 mutation in a Pakistani family with Leber congenital amaurosis and cataracts.

PURPOSE: To determine the cause of Leber congenital amaurosis (LCA) and developmental cataracts in a consanguineous Pakistani family. METHODS: The diagnosis was established in all affected individuals of a Pakistani LCA family by medical history, funduscopy, and standard ERG. We performed genome-wide linkage analysis for mapping the disease locus in this family. RESULTS: Congenitally severely reduced visual acuity and nystagmus were reported for all patients who, in the later phase of the disease, also developed cataracts. LCA in the family cosegregated with homozygosity for a single nucleotide polymorphism (SNP) haplotype on chromosome 6p14.1. The respective candidate region contained Leber congenital amaurosis 5 (LCA5), a gene previously reported to underlie LCA. We subsequently identified a novel truncating mutation in exon 4 of LCA5, c.642delC, in homozygous state in all affected persons of the family. CONCLUSIONS: We report a novel LCA5 mutation causing LCA in a Pakistani family. Developmental cataracts were present in two of the four patients, raising the possibility that LCA5 mutations may predispose to this additional ocular pathology.

Novel single nucleotide polymorphism identification in interleukin-6 gene of Pakistani sheep.

The present study aimed to identify single-nucleotide polymorphism (SNP) in coding and non-coding regions of interleukin-6 (IL-6) gene of Pakistani sheep. The IL-6 gene of 205 animals from nine sheep breeds were sequenced for screening of SNP. Characterizing the IL-6 gene revealed thirteen SNP sites within the intronic region of IL-6 gene. The novel SNPs found in the present study can serve as genetic marker for association studies with susceptibility/resistance to parasite infection in sheep. This is first report of SNP polymorphism of IL-6 gene of Pakistani sheep.

Molecular investigation of mental retardation locus gene PRSS12 by linkage analysis.

The present study was carried out to determine the prevalence of families having mental retardation in Pakistani population. We enrolled seven mentally retarded (MR) families with two or more affected individuals. Family history was taken to minimize the chances of other abnormalities. Pedigrees were drawn using the Cyrillic software (version 2.1). The structure of pedigrees shows that all the marriages are consanguineous and the families have recessive mode of inheritance. All the families were studied by linkage analysis to mental retardation locus (MRT1)/gene PRSS12. Three STR markers (D4S191, D4S2392, and D4S3024) in vicinity of mental retardation (MR) locus (MRT1)/gene PRSS12 were amplified on all the sample of each family by PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). The Haplotype were constructed to determine the pattern of inheritance and also to determine that a family was linked or unlinked to gene PRSS12. One out of the seven families was potentially linked to gene PRSS12, while the other six families remain unlinked.