Center for Applied Molecular Biology

Lahore, Pakistan

Center for Applied Molecular Biology

Lahore, Pakistan
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Azeemi S.T.,The University of Lahore | Shaukat S.F.,The University of Lahore | Azeemi K.S.,Khanwada Silsila e Azeemia | Khan I.,Center for Applied Molecular Biology | And 2 more authors.
African journal of traditional, complementary, and alternative medicines : AJTCAM | Year: 2017

RESULTS: The analysis of the microscopic and SEM images of irradiated E. coli samples with six different visible range radiations is representative of The fact that E. coli responded differently to every applied radiation in the visible region and the most profound inhibitory effects were that of 538nm Visible Range Radiation (Green) which proved to be bactericidal and 590nm Visible Range Radiation (yellow) which was bacteriostatic. The enhanced growth of E. coli with varying degrees was clearly observed in 610nm (orange), 644nm (red), 464nm (Purple) and 453nm (blue).CONCLUSION: It can be concluded that 538nm (Green) and 590nm (Yellow) can effectively be used for treating E. coli borne diseases.BACKGROUND: Escherichia coli is the agent responsible for a range of clinical diseases. With emerging antimicrobial resistance, other treatment options including solar/photo-therapy are becoming increasingly common. Visible Range Radiation Therapy/Colour Therapy is an emerging technique in the field of energy/vibrational medicine that uses visible spectrum of Electromagnetic Radiations to cure different diseases. In this study, our goal was to understand the effect of Visible Range Electromagnetic Radiations on E. coli (in vitro) and therefore find out the most appropriate visible range radiation for the treatment of diseases caused by E. coli.MATERIALS AND METHODS: A total of 6 non-repetitive E. coli isolates were obtained from urine samples obtained from hospitalized patients with UTI. Single colony of E. coli was inoculated in 3 ml of Lysogeny Broth (LB) and 40 μl of this E. coli suspension was poured into each of the plastic tubes which were then irradiated with six different wavelengths in the visible region (Table. 1) after 18 hours with one acting as a control. The Optical Densities of these irradiated samples were then measured. Furthermore, scanning electron microscopy (TEFCAN ZEGA3) was carried out.

Ali A.,University of Malakand | Rehman H.U.,District Head Quarter Hospital | Nisar M.,University of Malakand | Rafique S.,Center for Applied Molecular Biology | And 7 more authors.
International Journal of Infectious Diseases | Year: 2013

Background: Dengue is the most important vector-borne disease in many different parts of the world and is expanding into other areas of the globe without hindrance. The morbidity and mortality due to dengue complications are increasing globally at an alarming rate. Although transmission of the dengue virus has been documented in well-characterized areas of Pakistan, its incidence in Khyber Pakhtunkhawa has not been characterized. To address this issue we aimed to determine the seroprevalence of dengue (IgM and IgG) antibodies and the disease symptoms in the population of Khyber Pakhtunkhawa, and to investigate the incidence of dengue fever in different seasons and in urban as well as in rural areas. Methods: From August to October 2011, data of suspected dengue patients were collected from different primary, secondary, and tertiary collection centers situated in Khyber Pakhtunkhawa in order to determine the actual seroprevalence of dengue antibodies (IgM and IgG) in Khyber Pakhtunkhawa. Results: A total 612 subjects with a suspected infection were enrolled in our study. Of the 612 suspected cases, 319 were found positive for dengue IgG, IgM, or both IgG and IgM. The overall weighted prevalence of dengue-specific antibodies (IgM and/or IgG) was 52.12%. Overall, of the 52.12%, 31.86% (95% confidence interval (CI) 28.17-35.55) were positive for dengue IgM and 20.26% (95% CI 17.03-23.39) were positive for dengue IgG. Only 23 (3.75%) samples showed both IgG and IgM antibodies. A higher prevalence of IgM (39.35%, 95% CI 32.84-45.86) and IgG (22.42%, 95% CI 16.86-27.98) antibodies was found in the age group 21-30 years as compared to the children age group (≤10 years) and the oldest age group (≥51 years). The mean age of the febrile cohort was 53.16. ±. 44.22 years, ranging from 4 to 85 years. Age group was not statistically associated with IgM (p= 0.64) or IgG (p= 0.49) positivity. A higher seroprevalence of IgM (37.24%, 95%CI 32.84-45.86) was observed in males as compared to females (IgM 17.88%, 95% CI 11.11-24.65) while higher seroprevalnce of IgG (22.76%, 95% CI 15.35-30.17) was seen in females as compared to males (IgG 17.58%, 95% CI 14.21-20.95). Gender was not significantly associated with IgM (. p=. 0.06) or IgG (p= 0.53) positivity. Dengue IgM (35.38%, 95% CI 38.61-62.91) and IgG (50.76%, 95% CI 38.61-62.91) were higher in patients who had a history of travel to a dengue endemic area as compared to those who did not (IgM 33%, 95% CI 29.06-36.94, and IgG 15%, 95% CI 12.01-17.99). History of travel to an endemic area was significantly associated with IgM (p= 0.023) and IgG (p= 0.041) positivity. A higher incidence of IgM (41.13%, 95% CI 35.55-46.71) and IgG (27.42%, 95% CI 22.36-32.48) was observed in urban areas than in rural areas (IgM 23%, 95% CI 18.34- 27.66, and IgG 13.41%, 95% CI 9.63-17.19). IgM (p= 0.0005) and IgG (p= 0.0007) positivity was significantly associated with area of residence. Symptoms including fever (p= 0.007), headache (p= 0.001), Skin rash (0.005), joint pain (0.004) and Fatigue were significantly linked to dengue fever. IgM and IgG antibodies were more frequently seen in the post-monsoon season (68.33%) than in the monsoon period (31.68%). The death ratio in the overall weighted prevalence was 2.19%. Conclusion: The results of the present cohort study of febrile subjects show that young people and males are more susceptible to dengue fever. Dengue infection was most prominent in the post-monsoon season, in urban areas, and in patients with a history of travel to an endemic locality. Furthermore seven deaths were found in our cohort study. © 2013 International Society for Infectious Diseases.

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 | Year: 2015

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.

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 | Year: 2016

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.

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 | Year: 2011

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.

Shafique M.,University of Punjab | Shahzad M.S.,The University of Lahore | Shan M.A.,Center for Applied Molecular Biology | Ali A.,The University of Lahore | And 2 more authors.
International Journal of Legal Medicine | Year: 2015

Ten MiniSTR loci were analyzed on 250 unrelated individuals of Punjabi population from Punjab province of Pakistan. The product amplification range is from 65 to 280 bp. Allele frequency for a total of 98 observed alleles is from 0.002 to 0.41. Forensic and paternity statistical parameters were investigated including combined power of discrimination (PD), combined power of exclusion (PE), and cumulative probability of matching (PM) equaled to 0.99999999999824, 0.999824, and 1.75931 × 10−12, respectively. These MiniSTRs on the basis of high degree polymorphism and fragment size reduction will be highly informative in most of the forensic cases where DNA of the samples is degraded, mass disasters, and dead body identification. Significant differences were observed in all loci when comparing with same STR loci in previously published different world populations. © 2014, Springer-Verlag Berlin Heidelberg.

Kakar N.,BUITEMS | Kakar N.,University of Ulm | Goebel I.,University of Ulm | Daud S.,Center for Applied Molecular Biology | And 7 more authors.
European Journal of Medical Genetics | Year: 2012

Autosomal recessive intellectual disability is believed to be particularly prevalent in highly consanguineous populations and genetic isolates and may account for a quarter of all non-syndromic cases. Mutations in more than 50 genes have been reported to be involved in autosomal recessive intellectual disability, including TRAPPC9 (MIM 611966), mutations of which have been identified in six families from different geographical origins. We performed a clinical and molecular genetic study of a consanguineous Pakistani family segregating intellectual disability and microcephaly. SNP-array-based homozygosity mapping revealed suggestive linkage to four genomic regions including one on chromosome 8 that contained TRAPPC9. We detected a homozygous TRAPPC9 splice donor site mutation (c.1024+1G>T) that cosegregated with intellectual disability in the family and led to skipping of exon 3 and exons 3 and 4 in blood-derived patient RNA. We have thus identified a novel splice site mutation leading to exon skipping and premature termination of TRAPPC9 translation. These data further suggest that TRAPPC9 mutations -unlike mutations in the vast majority of the known intellectual disability-associated genes- constitute a more frequent cause of autosomal-recessive cognitive deficits, especially when microcephaly is also present. © 2012 Elsevier Masson SAS.

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 | Year: 2014

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.

PubMed | Center for Applied Molecular Biology
Type: Journal Article | Journal: Pakistan journal of pharmaceutical sciences | Year: 2013

Di-branched (Y-shaped) polyethylene glycols (PEGs) are considered more effective than linear molecules to enhance the efficacy of the conjugated drug. In the present study interferon -2a was conjugated with three different 40 KDa di-branched PEGs. The results of this study show that length and/or the structure of linker between PEG and the protein is also involved in the synthesis, in vitro biological activity and stability of the conjugate. Three conjugates i.e., mPEG2L-IFN, mPEG2P-IFN and mPEG(2)M-IFN yielded 25%, 24% and 17%, with bioactivities 2.8 x 10(6) IU/mg, 3.95 x 10(6) IU/mg and 6.7 x 10(6) IU/mg, respectively. The order of bioactivity stability is mPEG2L-IFN > mPEG2P-IFN > mPEG2M-IFN > IFN (native). We report that although lengthy linkers are more reactive in terms of conjugation, they have opposite effect on the in vitro bioactivity of the conjugate. PEGylation as a whole increases the stability of the conjugate, and linkers also add in stability.

PubMed | University of Punjab and Center for Applied Molecular Biology
Type: Journal Article | Journal: Biotechnology and applied biochemistry | Year: 2015

Recombinant human consensus interferon (rh-cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high-level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high-level expression, shake-flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l-arginine were optimized. A one-step chromatography method was developed for high-yield purification of consensus IFN. rh-cIFN was characterized by SDS-PAGE, Western blot, and high-performance liquid chromatography. Purified protein has a molecular weight of 19.5kDa and specific activity was 2.0 10(8) as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100mg/L of biologically active rh-cIFN, which is highest ever reported according to available data.

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