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Sadiq H.M.,Center for Applied Molecular Biology | Jahangir G.Z.,Center for Applied Molecular Biology | Nasir I.A.,University of Punjab | Iqtidar M.,LCWU | Iqbal M.,Center for Applied Molecular Biology
Biotechnology and Biotechnological Equipment

Different strains of phosphate-solubilizing bacteria (PSB) were isolated from the rhizosphere of different plants of Lahore District, Pakistan. The objective of the study was to explore the capabilities of PSB and evaluate their efficiency to enhance growth of sugarcane plants under greenhouse condition. The purified isolates were identified as Proteous vulgaris, Klebsiella pneumoniae, Enterobacter aerogenes, Burkholederi acepacia, Citrobacter freundii, Acinetobacter lwoffi, and Pseudomonas fuorescens, and the identification was based on the characteristic morphological and biochemical behavior. The efficiency of different PSB isolates for phosphate solubilization was evaluated from the zones they formed on agar plates of Pickovaskaya growth medium (PVK) by solubilizing the tricalcium phosphate of the medium. The efficiency of purified PSB was evaluated from pot experiments of two different genetically modified sugarcane varieties (SCMV resistant CAMB-I and CAMB-II) under greenhouse condition for their positive role in plant growth promotion. All the six PSB enhanced the growth rate significantly over that in the non-inoculated control. A significant increase in plant height, number of leaves, root length and dry matter contents was recorded and E. aerogenes and C. freundii were found significantly superior over the rest of the isolates for all the tested parameters. The efficiency gradient of different isolates for CAMB-I and CAMB-II varieties was recorded as C. freundii> K. pneumoniae> E. aerogenes> B. cepacia> A. lwoffi> P. vulgaris and E. aerogenes> C. freundii> A. lwoffi> B. cepacia> P. vulgaris, respectively. The results of this greenhouse evaluation are encouraging and need to be confirmed under field condition in combination with organic and chemical fertilizers. © Biotechnol. & Biotechnol. Source

Majeed R.A.,University of Punjab | Shahid A.A.,University of Punjab | Ashfaq M.,University of Punjab | Saleem M.Z.,Center for Applied Molecular Biology | Haider M.S.,University of Punjab
Plant Disease

A leaf spot of rice (Oryza sativa L.) was observed in a survey conducted in different districts of Punjab, Pakistan, from August to October 2012. Symptoms appeared as brown spots on the leaf blade and on the small emerging leaves. Primary symptoms were brown small, round-ovoid spots with a chlorotic halo, evenly distributed over the leaf. The size of spots varied from 10 mm to 1 cm. Over time, spots merged and became large. Infected leaves were collected and washed in running water thoroughly for 5 to 10 min. Small pieces (0.5 to 2 cm) of leaf tissue were then surface disinfected with 2% NaOCl for 2 min, rinsed three to four times in sterile distilled water (SDW), blotted dry on sterile filter paper, and placed on potato dextrose agar (PDA) plates. PDA plates were incubated at 28°C for 5 to 7 days. A single-spore fungal isolate was maintained on PDA for further investigation. Morphological and microscopic characteristics and phylogenetic analysis were used for the identification of the fungal isolate. On PDA, fungal colony attained a 14.5-cm diameter in 5 days, was zonate and felted with a greenish-gray or grayish-black conidial area and a 3-mm sterile advancing margin. The reverse side of the colony was zonate, black at the center, and lighter outward. Microscopically, the fungal hyphae were branched, septate, subhyaline to light-brown to dark-brown, and smooth walled. Conidiophores generally were dark-brown, septate, unbranched, occasionally bent, and having genticulation at the tip and sometimes down the length of conidiophore. Generally conidia were of light- to dark-brown; boat-shaped or hook shaped; 17 to 26.5 × 8.5 to 14.0 μm; rounded at the tip; frequently constricted at the base; smooth-walled, with three septa, the middle second cell larger than the first, third, and fourth; bent on the second cell and endured at the tip; arranged in a flower-like whorl or in thick panicles. Molecular identification was accomplished by amplifying the rDNA ITS region (da Cunha et al. 2013) using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers and then determining the sequence. A BLASTn search showed greatest homology (98% similarity) with the ITS sequence of Curvularia lunata (GenBank Accession No. KC662107). The sequence was deposited in GenBank (KP940576). Pathogenicity was confirmed by following Koch’s postulates. Greenhouse-grown rice plants cv. Super Basmati were inoculated at the two leaf stage with a conidial suspension (106 spores/ml) prepared in SDW with a hand sprayer and then covered with plastic bags for 24 h. Control plants were sprayed with SDW and covered with plastic bags. Plants were incubated at 28°C for 1 week in Green house. Disease severity on inoculated leaves was estimated 7 days after inoculation using a 0 to 5 severity scale (0 = healthy plants and 5 = severely diseased plants). Typical symptoms were observed on all the inoculated leaves after 1 week, which was similar to the symptoms observed during the survey, whereas no symptoms appeared on the control leaves. The pathogen reisolated from inoculated leaves was identical in all respects to the isolates used to inoculate the plants, which confirmed Koch’s postulates. Based on microscopic, morphological, and molecular characteristics, the fungal isolates were identified as C. lunata (Wakker) Boedijin (Ellis 1971). Pure cultures were submitted to Frist Fungal Culture Bank of Pakistan (FCBP). To our knowledge, this is the first report of leaf spots caused by C. lunata on rice in Punjab, Pakistan. © The American Phytopathological Society. Source

Ur Rehman K.,University of Punjab | Akhtar T.,University of Punjab | Sabar M.F.,Center for Applied Molecular Biology | Tariq M.A.,COMSATS Institute of Information Technology
Experimental and Therapeutic Medicine

Drug resistance is a phenomenon that has become a critical issue in medical practice. Such is the case in the response to clopidogrel treatment, which is variable inter-individually and inter-ethnically due to genetic polymorphisms in the cytochrome P40 (CYP) gene. Clopidogrel is an anti-platelet agent administered to cardiac patients in the form of a prodrug, which is further metabolized into an active form by CYP enzymes. There are many allelic variants of the CYP gene that are involved in clopidogrel resistance, of which CYP2C19*2 has been demonstrated to be one of the most significant loss-of-function alleles. In the present study, 100 cardiac patients with percutaneous coronary intervention (PCI) or acute coronary syndrome (ACS) who were undergoing treatment with clopidogrel were selected and the patients were analyzed for CYP2C19*2 allelic variants using an allele-specific primer extension polymerase chain reaction method. The variant amplicons were visualized on gel and validated by Sanger sequencing. The observed allelic frequency distribution of CYP2C19*2 variants was 18% heterozygous for CYP2C19*2 A/C/G variants, 35% heterozygous for A/G variants, 13% heterozygous for C/G variants, 6% heterozygous for A/C variants, 7% homozygous for A variant, 5% homozygous for C variant and 16% homozygous for G wild-type. Furthermore, tri-allelic single nucleotide polymorphisms (SNPs) were identified in the CYP2C19*2 allele in cardiac patients for the first time, to the best of our knowledge; these were CYP2C19*2 A/C/G SNPs (18%). The overall frequency observed for new allelic variant C of CYP2C19*2 was 42%. These results suggested that there are significant inter-ethnic variations in the allelic frequencies of CYP2C19*2, which may be responsible for the variable clopidogrel response in cardiac patients. © 2015, Spandidos Publications. All rights reserved. Source

Butt F.A.,University of Punjab | Amin I.,University of Punjab | Idrees M.,University of Punjab | Iqbal M.,Center for Applied Molecular Biology
European Journal of Gastroenterology and Hepatology

INTRODUCTION: Hepatitis delta virus (HDV) and hepatitis B virus (HBV) have been identified as major causes of morbidity and mortality in Pakistan because HDV causes infection only in the presence of HBV. Coinfection with both hepatitis viruses can lead to a more severe acute form of disease and to an increased risk of fulminant hepatitis. HDV infection differs in its distribution and severity depending on the geographical distribution, and several genotypes of HDV have been identified so far. OBJECTIVES: The aim of the present study was to establish the HDV and HBV genotypes in chronically infected Pakistani patients and to determine whether there is any correlation between HDV and HBV genotypes. PATIENTS AND METHODS: We studied samples from a total of 46 chronically infected HBV and HDV patients for HBV and HDV genotype analysis out of a total of 75 chronic HBV carriers enrolled. HBV and HDV genotypes were determined using type-specific PCR, followed by sequencing of PCR amplified products. RESULTS: The results of HBV genotyping showed that 33 of 46 (71.7%) patients had genotype D, five (10.9%) had A+D mixed genotypes, whereas eight (17.3) samples were untypable. We could detect only one HDV genotype (HDV-1) prevalent in the Pakistani population. The HDV-1 genotype isolate was associated with HBV genotype D alone or in combination with A (HBV-A+D). CONCLUSION: The present study concludes that HDV/HBV coinfection is very high in the Pakistani population and was previously underestimated. The most prevalent circulating genotypes of HBV and HDV are HDV-1 and HBV-D, respectively, in the studied area. There is no specific interaction between HBV and HDV genotypes as suggested by HDV-1/HBV-D or HDV-1/HBV-A+D coinfection. Coinfection of HDV-1 and HBV-D simply reflects the most frequent genotypes circulating in this specific geographical region of the world. © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Source

Khan S.,Kohat University Kohat Khyber Pakhtunkhwa | Attaullah S.,University of Peshawar | Ayaz S.,Kohat University Kohat Khyber Pakhtunkhwa | Niaz Khan S.,Kohat University Kohat Khyber Pakhtunkhwa | And 4 more authors.
Virology Journal

Background: Studies of the molecular epidemiology and risk factors for hepatitis C virus (HCV) in health care workers (HCWs) of Peshawar, Khyber Pakhtunkhwa region are scarce. Lack of awareness about the transmission of HCV and regular blood screening is contributing a great deal towards the spread of hepatitis C. This study is an attempt to investigate the prevalence of HCV and its possible association with both occupational and non-occupational risk factors among the HCWs of Peshawar. Results: Blood samples of 824 HCWs, aged between 20-59 years were analysed for anti-HCV antibodies, HCV RNA and HCV genotypes by Immunochromatographic tests and PCR. All relevant information was obtained from the HCWs with the help of a questionnaire. The study revealed that 4.13% of the HCWs were positive for HCV antibodies, while HCV RNA was detected in 2.79% of the individuals. The most predominant HCV genotype was 3a and 2a. Conclusion: A program for education about occupational risk factors and regular blood screening must be implemented in all healthcare setups of Khyber Pakhtunkhwa province in order to help reduce the burden of HCV infection. © 2011 Khan et al; licensee BioMed Central Ltd. Source

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