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    ABSTRACT: Human interferon alpha-2b (hIFNα2b) is the most important member of interferon family. Escherichia coli, yeast, mammalian cell cultures and baculovirus infected insect cells have been used for expressing recombinant human interferon. Recently Pichia pastoris based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered, an important aspect to get the optimum expression of recombinant protein which may vary from one protein to another. In present study, we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b expressing cassettes were created via in vitro multimerization approach. P. pastoris host strain X-33 was transformed with these expression cassettes. Groups of P. pastoris clones transformed with different copies of IFNα2b expression cassette were screened for intrachromosomal integration. IFNα2b expression level of stable transformants was checked. Copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that increase in copy number generally have positive effect on expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b. This was also observed that increase in drug resistance of a clone not guarantee its high expression, as integration of marker gene do not always correlate with integration of gene of interest. This article is protected by copyright. All rights reserved.
    Yeast 01/2014; 31(1). DOI:10.1002/yea.2990
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    Public health 08/2013; 127(9). DOI:10.1016/j.puhe.2013.03.003
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    ABSTRACT: To identify pathogenic mutations responsible for retinal dystrophy in three consanguineous Pakistani families. A thorough ophthalmic examination including fundus examination and electroretinography was performed, and blood samples were collected from all participating members. Genomic DNA was extracted, and genome-wide linkage and/or exclusion analyses were completed with fluorescently labeled short tandem repeat microsatellite markers. Two-point Lod scores were calculated, and coding exons along with exon-intron boundaries of RPE65 gene were sequenced, bidirectionally. Ophthalmic examinations of the patients affected in all three families suggested retinal dystrophy with an early, most probably congenital, onset. Genome-wide linkage and/or exclusion analyses localized the critical interval in all three families to chromosome 1p31 harboring RPE65. Bidirectional sequencing of RPE65 identified a splice acceptor site variation in intron 2: c.95-1G>A, a single base substitution in exon 3: c.179T>C, and a single base deletion in exon 5: c.361delT in the three families, respectively. All three variations segregated with the disease phenotype in their respective families and were absent from ethnically matched control chromosomes. These results strongly suggest that causal mutations in RPE65 are responsible for retinal dystrophy in the affected individuals of these consanguineous Pakistani families.
    Molecular vision 07/2013; 19:1554-64.
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    ABSTRACT: OBJECTIVE AND DESIGN: This study evaluated the effect of an antioxidant, Vitamin E, on cultured chondrocytes against H2O2-induced damage in vitro. MATERIAL: Rat chondrocytes isolated from articular cartilage. TREATMENT: Chondrocytes were pretreated with either 50 or 100 μM Vitamin E or serum-free medium for 24 h followed by their exposure to 200 μM H2O2 for 3 h. Chondrocytes without exposure to H2O2 served as control group. METHODS: The effect of Vitamin E pretreatment was evaluated by examining proteoglycan contents, nitrite levels, viability, apoptosis, and senescence of cultured chondrocytes. RESULTS: Proteoglycan contents increased in groups treated with Vitamin E. Semi-quantitative real-time PCR data also correlated with these results and demonstrated that Vitamin E up-regulated expression of Agc1, Col2a1, and PCNA genes along with down-regulation in the expression of Col1a1 and Casp3 genes. The differentiation index improved after Vitamin E pretreatment. Nitrite levels were reduced with a corresponding increase in cell viability. Reduction in apoptosis and senescence was also observed after Vitamin E pretreatment. Moreover, a dose-dependent effect of Vitamin E was seen. In contrast to 50 μM Vitamin E, 100 μM was more potent in inducing protection of chondrocytes from H2O2-induced oxidative damage. CONCLUSION: Vitamin E reversed the oxidant-induced alterations in chondrocytes and may be a good option to pretreat chondrocytes before transplantation.
    Inflammation Research 05/2013; DOI:10.1007/s00011-013-0635-y
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    ABSTRACT: IntroductionThe clinical correlation between hepatitis C virus (HCV) NS5A gene mutations and response to antiviral treatment in HCV-3a-infected patients is not as well known as that in HCV-1a-infected and HCV-1b-infected patients.Objectives This study attempted to determine mutations in the NS5A gene in isolates of HCV-3a obtained from infected Pakistani patients treated with interferon- standard therapy and to determine its association with several host factors.MethodsA total of 71 consecutive chronically infected HCV patients [43 men and 28 women, mean age 38 years (range, 16-70 years)] without previous antiviral therapy, who fulfilled other criteria of the current study were enrolled for antiviral therapy.ResultsOf the 71 patients enrolled, 53 completed the course of antiviral therapy. Among them, 23 (43.3%) patients showed an end-of-treatment response, 20 (41.4%) showed a sustained virological response, and three (5.6%) patients were HCV-RNA negative at the end of treatment but developed a relapse thereafter. Only seven (13.2%) patients were virologic nonresponders (NR). Probable transmission risk factors were previous major/minor surgery (20%), transfusion of blood or blood products (2%), dental surgery (10%), or sporadic (unknown; 60%). The estimated duration of infection varied from 6 months to 20 years. The correlation between the number of mutations and several factors was also studied. The first factor that was associated with the number of mutations in the NS5A gene was age older than 40 years, where the difference was statistically significant between viral sequences of patients showing an end-of-treatment response and NR (P<0.001). Further, the difference in the average number of mutations was statistically significant between patients showing sustained virological response and NR (P<0.005). The second and third factors that were found to be associated with number of mutations in the NS5A gene were sex and viral load, respectively.Conclusion Along with host factors such as age older than 40 years, female sex, and low baseline viral load, the less number of mutations in the NS5A gene of HCV-3a is associated with a positive outcome of treatment response in HCV patients receiving interferon plus ribavirin therapy.
    European journal of gastroenterology & hepatology 04/2013; 25(10). DOI:10.1097/MEG.0b013e328360aeb4
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    ABSTRACT: Membrane fusion is the central molecular event during the entry of enveloped viruses into cells. The critical agents of this process are viral surface proteins, primed to facilitate cell bilayer fusion. The important role of Dendritic-cell-specific ICAM3-grabbing non-integrin (DC-SIGN) in Dengue virus transmission makes it an attractive target to interfere with Dengue virus Propagation. Receptor mediated endocytosis allows the entry of virions due to the presence of endosomal membranes and low pH-induced fusion of the virus. DC-SIGN is the best characterized molecule among the candidate protein receptors and is able to mediate infection with the four serotypes of dengue virus (DENV). Unrestrained pair wise docking was used for the interaction of dengue envelope protein with DC-SIGN and monoclonal antibody 2G12. Pre-processed the PDB coordinates of dengue envelope glycoprotein and other candidate proteins were prepared and energy minimized through AMBER99 force field distributed in MOE software. Protein-protein interaction server, ZDOCK was used to find molecular interaction among the candidate proteins. Based on these interactions it was found that antibody successfully blocks the glycosylation site ASN 67 and other conserved residues present at DC-SIGN-Den-E complex interface. In order to know for certain, the exact location of the antibody in the envelope protein, co-crystallize of the envelope protein with these compounds is needed so that their exact docking locations can be identified with respect to our results.
    PLoS ONE 03/2013; 8(3):e59211. DOI:10.1371/journal.pone.0059211
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    ABSTRACT: BACKGROUND AIMS: Stem cell therapies can provide an alternative approach for repair and regeneration of tissues and organs. Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies. Although bone marrow-derived MSCs have multi-lineage differentiation potential, bone marrow is not an optimal source because of the isolation process and low yield. The goal of this study was to investigate comparatively for the first time the in vitro regenerative potential of human MSCs from two other sources: umbilical cord tissue and adipose tissue. METHODS: Cells from each tissue were isolated with 100% efficiency and characterized by fluorescence activated cell sorting (FACS) analysis for CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105. Growth characteristics were investigated by population doublings, saturation density and plating efficiency. MSCs derived from both types of tissues were assessed for differentiation potential qualitatively and quantitatively. RESULTS: FACS analysis showed no differences in expression of CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105 between cord tissue MSCs (CT-MSCs) and adipose tissue MSCs (AT-MSCs). CT-MSCs showed more proliferative potential than AT-MSCs. When cultured in low numbers to determine colony-forming units (CFUs), CT-MSCs showed less CFUs than AT-MSCs. Cells from both sources efficiently differentiated into adipose, bone, cartilage and neuronal structures as determined with histochemistry, immunofluorescence and real-time reverse transcriptase polymerase chain reaction. CONCLUSIONS: MSCs can easily be obtained from umbilical cord and adipose tissues, and it appears that both tissues are suitable sources of stem cells for potential use in regenerative medicine.
    Cytotherapy 01/2013; 15(3). DOI:10.1016/j.jcyt.2012.11.010
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    ABSTRACT: Our aim was to improve the survival and reduce the apoptosis of chondrocytes derived from mesenchymal stem cells from Wharton's jelly of human umbilical cord (WJMSCs) by Lovastatin supplementation under hydrogen-peroxide-induced injury conditions to simulate the osteoarthritic micro-environment. Chondrocytes were differentiated in vitro from WJMSCs. The cultured WJMSCs expressed CD90 (84.07%), CD105 (80.84%), OCT4 (26.90%), CD45 (0.42%) and CD34 (0.48%) as determined by flow cytometry. Increased aggregation of proteoglycans observed by Safranin-O staining accompanied by increased expression of COL2A1, ACAN, SOX9 and BGN shown by immunocytochemistry and reverse transcription with the polymerase chain reaction (PCR) confirmed the chondrogenic differentiation of the WJMSCs. The in vitro differentiated chondrocytes were subjected to oxidative stress by exposure to 200 μM hydrogen peroxide, either in the presence or absence of Lovastatin (2 μM) for 5 h. Lovastatin treatment resulted in decreased apoptosis, senescence and LDH release and in increased viability and proliferation of WJMSC-derived chondrocytes. Real time PCR analysis showed markedly up-regulated expression of prosurvival, proliferation and chondrogenic genes (BCL2L1, BCL2, AKT, PCNA, COL2A1, ACAN, SOX9 and BGN) and significantly down-regulated expression of pro-apoptotic genes (BAX, FADD) in the Lovastatin-treated group in comparison with injured cells. The reduced expression of VEGF and p53 as determined by enzyme-linked immunosorbent assay and PCR suggests the suitability of the use of Lovastatin in adjunct to WJMSC-derived chondrocytes for the treatment of osteoarthritis. We conclude that Lovastatin protects WJMSC-derived chondrocytes from hydrogen-peroxide-induced in vitro injury.
    Cell and Tissue Research 12/2012; 351(3). DOI:10.1007/s00441-012-1540-3
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    ABSTRACT: Human hereditary deafness at the DFNB29 locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetramembrane spanning that localizes to the apical tight junctions of organ of Corti hair cells and in many other tissues. Typically, the DFNB29 phenotype is characterized by prelingual, bilateral, sensorineural hearing loss. The goal of this study was to define the identity and frequency of CLDN14 mutations and associated inner ear phenotypes in a cohort of 800 Pakistani families segregating deafness. Hearing loss in 15 multi-generational families was found to co-segregate with CLDN14-linked STR markers. The sequence of the six exons and regions flanking the introns of CLDN14 in these 15 families revealed five likely pathogenic alleles. Two are novel missense substitutions (p.Ser87Ile and p.Ala94Val), whereas p.Arg81His, p.Val85Asp and p.Met133ArgfsX23 have been reported previously. Haplotype analyses indicate that p.Val85Asp and p.Met133ArgfsX23 are founder mutations. The p.Val85Asp accounts for ∼67% of the mutant alleles of CLDN14 in our cohort. Combined with the previously reported data, CLDN14 mutations were identified in 18 of 800 Pakistani families (2.25; 95% CI, 1.4-3.5). Hearing loss in the affected individuals homozygous for CLDN14 mutations varied from moderate to profound. This phenotypic variability may be due to environmental factors (for example drug and noise exposure) and/or genetic modifiers.Journal of Human Genetics advance online publication, 13 December 2012; doi:10.1038/jhg.2012.143.
    Journal of Human Genetics 12/2012; 58(2). DOI:10.1038/jhg.2012.143
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    ABSTRACT: Hepatitis C is the major health problem over the globe affecting approximately 200 million people worldwide and about 10 million Pakistani populations. Developing countries are especially facing the problems of HCV infection. Hence the goal of the study was to find out the antigenic epitopes that could be effective vaccine targets of HCV genotype 1 of Asian origin against HLA alleles frequently distributed in Asian countries. A total of 85 complete genome sequences of HCV 1 of Asian origin were retrieved from the HCV sequence database. Using in silico tools, T cell epitopes were predicted from conserved regions of all the available HCV 1 subtypes against Asian HLA alleles. Using 10 MHC I supertypes 51 epitopes was predicted as promiscuous binders. MHC class I supertypes A2 and B7 were found to be good promiscuous binders for a large number of predicted epitopes. Other alleles of MHC I supertypes (B57, B27, BX, B44) either were not respondent as promiscuous binders or responded only to a limited number of epitopes. Against 8 predominantly found Asian alleles of DRB1 supertype, 42 epitopes was predicted as promiscuous binders. MHC class II alleles DRB1-0101, DRB1-0701 and DRB1-1501 were the highest binders to promiscuous predicted epitopes while DRB1-0301 was the least binder for the predicted promiscuous epitopes of HCV 1 genotype of Asian origin. Literature review survey of predicted epitopes via IEDB also confirmed that great numbers of predicted epitopes are true positive. Hence, sophisticated selection of viral proteins and MHCs provided conserved promiscuous epitopes that can be used as effective vaccine candidates for all Asian counties. ABBREVIATIONS: HCV - hepatitis C virus, MHC - major histocompatability complex, HLA - human leukocyte antigen, CTL - cytotoxic T lymphocytes.
    Bioinformation 10/2012; 8(20):957-62. DOI:10.6026/97320630008957
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