Ali Raza Awan

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.

Molecular classification of Pakistani collared dove through DNA barcoding

Pakistan is bestowed by a diversified array of wild bird species including collared doves of which the taxonomy has been least studied and reported. DNA barcoding is a geno-taxonomic tool that has been used for characterization of bird species using mitochondrial cytochrome c oxidase I gene (COI). This study aimed to identify taxonomic order of Pakistani collared dove using DNA barcoding. Purposely herein, we present a phylogenetic analysis of Pakistani collared dove based on 650 base pairs of COI gene sequences. Analysis of phylogenetic tree revealed that Pakistani collared dove shared a common clade with Eurasian collared dove (Streptopelia decaocto) and African collared dove (Streptopelia roseogrisea) which indicated a super-species group in Streptopelia genus. This is the first report of molecular classification of Pakistani collared dove using DNA barcoding.

Optimization of saccharification potential of recombinant xylanase from Bacillus licheniformis

ABSTRACT Saccharification potential of xylanase enzyme cloned from Bacillus licheniformis into E. coli BL21 (DE3) was evaluated against plant biomass for the production of bioethanol. The expression of cloned gene was studied and conditions were optimized for its large scale production. The parameters effecting enzyme production were examined in a fermenter. Recombinant xylanase has the ability to breakdown birchwood xylan to release xylose as well as the potential to treat plant biomass, such as wheat straw, rice straw, and sugarcane bagass. The saccharification ability of this enzyme was optimized by studying various parameters. The maximum saccharification percentage (84%) was achieved when 20 units of recombinant xylanase were used with 8% sugarcane bagass at 50°C and 120 rpm after 6 hours of incubation. The results indicated that the bioconversion of natural biomass by recombinant xylanase into simple sugars can be used for biofuel (bioethanol) production. This process can replace the use of fossil fuels, and the use of bioethanol can significantly reduce the emission of toxic gases. Future directions regarding pre-treatment of cellulosic and hemicellulosic biomass and other processes that can reduce the cost and enhance the yield of biofuels are briefly discussed.

Detection of methicillin-resistant Staphylococcus aureus of poultry and human sources

Staphylococcus aureus(S. aureus ) is a common gram positive anaerobic facultative pathogen of human and birds; and mostly resides on skin, wounds, mucous membranes, alimentary & urogenital tracts, soft tissue infections. Resistance in S. aureus against antibiotics has been increasingly reported though depending on the clonal lineage. Among the resistant strains methicillin resistant S. aureus (MRSA) is a major threat to the community as its genome is more proneto evolution. The emergence of MRSA is distressing in community that embodies a model for emergence of this uncontrollable super bugs. The molecular based detection and characterization of MRSA has been unstudied in the local population of human and poultry. In the present study50 isolates (n= 42 were recovered from poultry external naries, respiratory tract and feathers) and (n= 08 were recovered from naries and hands of poultry workers) were used after biochemical and bacteriological identifications. MecA gene (527 bp)was amplified for the detection of MRSA followed by and sequencing using specially designed primers. The obtained sequences were compared with reported data and phylogenetic analysis was done. Comparative analysis indicated 2 polymorphic sites in the local isolates. Phylogenetic analysis showed that local poultry isolates were mono-phyletically claded with S. aureus MRSA and shared the same clade with human MRSA isolates. This indicated that this organism has a zoonotic potential. This is the first report of detection and sequencing of MRSA isolates recovered from poultry and human samples.

Expression Profiling of Hspb1 and Tp53 Genes through RT-qPCR in Different Cancer Types of Canis familiaris

Background: Diagnostic molecular marker studies are in vogue to have insight of most prevalent animal diseases including cancer. Objectives: Gene expression profiling of pro and anti-apoptotic genes was conducted in dog Lymphoma, CTVT, SCC, granuloma, perianal adenocarcinoma and mammary tumors. Materials and Methods: Cancerous tissues of 21 affected animals were obtained. Total RNA was extracted followed by cDNA synthesis. Comparative Ct method via Taqman assay (RT-qPCR) was used to quantify corresponding mRNA molecules, Tp53 and Hspb1, as normalized by GAPDH as the reference gene . Results:Hspb1 showed ectopic expression in lymphoma, CTVT and mammary tumors; its down-regulation was observed in granuloma and oral SCC with fold difference (FD) of ±35. Similarly, Tp53 as the tumor suppressor gene with pro-apoptotic properties, showed up-regulation in all tumor types, notably 80% of mammary tumors and 60% of CTVT. The FD values were 33.31 and 2.27, respectively. Conclusion: Altered transcriptomic response of Hspb1 and Tp53 was observed in all cancer types of Canis familiaris. The resulting profile depicts the involvement of the genes in cancer pathways. Thus, the data might be helpful for diagnosis, prognosis, identification and classification of these widespread neoplasms in this species.

Expression of thermostable β-xylosidase in Escherichia coli for use in saccharification of plant biomass

ABSTRACT The present work is aimed to evaluate the saccharification potential of a thermostable β-xylosidase cloned from Bacillus licheniformis into Escherichia coli for production of bioethanol from plant biomass. Recombinant β-xylosidase enzyme possesses the ability of bioconversion of plant biomass like wheat straw, rice straw and sugarcane bagass. By using this approach, plant biomass that mainly constitute cellulose can be converted to reducing sugars that could then be easily converted to bioethanol by simple fermentation process. The production of bioethanol will help to overcome energy requirements due to depleting fossil fuels and will also help to protect environment by reducing greenhouse gas emission. In the end, future directions are briefly mentioned that can be utilized to reduce the cost and increase the yield of biofuels.

Hspb1 and Tp53 Mutation and Expression Analysis in Cat Mammary Tumors

Background Molecular marker based cancer diagnosis gaining more attention in the current genomics era. So, Hspb1 and Tp53 gene characterization and their mRNA expression might be helpful in diagnosis and prognosis of cat mammary adenocarcinoma. It will also add information in comparative cancer genetics and genomics. Objectives Eight tumors of Siamese cats were analyzed to ascertain germ-line and tissue-specific somatic DNA variations of Hspb1 and Tp53 genes along with the ectopic differential expression in tumorous and normal tissues were also analyzed. Materials and Methods Tumorous tissues and peripheral blood from mammary adenocarcinoma affected Siamese cats were collected from the Pet center-UVAS. DNA and RNA were extracted from these tissues to analyze the Hspb1 and Tp53 DNA variants and ectopic expression of their mRNA within cancerous and normal tissues. Results Exon 1 and 3 revealed as hotspots in Hspb1 gene. The 5´UTR region of the exon1 bear six mutation including 3 transitions, 2 transversion and one heterozygous synonymous transversion in two samples at locus c.34C>C/A. Exon 3 has 1 transversion at c.773A>A/T, 3´UTR of this exon harbor two point mutations at 1868A>T and 2193C>T loci. Intron 2 has two alterations at 1490C>C/T and GTCT4del at 1514. Overall up-regulation of Hspb1 gene was observed. While exons 3, 4 and 7 of Tp53 harbor a single variationat c.105A>A/G, c.465T>T/C and c.859G>T respectively. The locus c.1050G>G/A in exon 9 is a heterozygous (G/A) in 3 samples and homozygous (G) in 2 other tumours. Introns 3, 5, 7 and 9 harbor 3, 4, 2 and 7 altered loci respectively. Sixty percent of cancers showed up-regulated trend of Tp53 gene. Conclusions Tumor specific mutations and ectopic expression of Hspb1 and Tp53 genes might be helpful in the diagnosis of the mammary lesions and endorse their involvement in cat mammary neoplasm.

Polymorphic Status of PRKAA2 Gene in Pakistani Buffaloes

This study was designed to find single nucleotide polymorphism (SNPs) in coding and non-coding region of PRKAA2 gene in Nili-Ravi and Kundi buffaloes. The PRKAA2 gene (AMPKα2) of 100 animals from both buffalo breeds were sequenced for SNPs identification. A total number of 43 inter and intra-generic SNPs were found. Out of which 17 SNPs were detected among buffalo breeds (intra-generic). All SNPs were intronic and may have a role in gene regulation and splicing patterns. These SNPs might be associated with commercially important production traits. This is the first study to identify novel SNPs that are linked to energy metabolism and production traits of buffalo.