Haploinsufficiency of EXT1 and Heparan Sulphate Deficiency Associated with Hereditary Multiple Exostoses in a Pakistani Family.

Background and Objectives: Hereditary multiple exostoses (HME) is a disease characterized by cartilage-capped bony protuberances at the site of growth plates of long bones. Functional mutations in the exostosin genes (EXT1 and EXT2) are reported to affect the hedgehog signalling pathways leading to multiple enchondromatosis. However, the exact role of each EXT protein in the regulation of heparan sulphate (HS) chain elongation is still an enigma. In this study, a Pakistani family with HME is investigated to find out the genetic basis of the disease. Materials and Methods: Genotyping of eight members of the family by amplifying microsatellite markers, tightly linked to the EXT1 and EXT2 genes. Results: The study revealed linkage of the HME family to the EXT1 locus 8q24.1. Sanger sequencing identified a heterozygous deletion (c.247Cdel) in exon 1 of EXT1, segregating with the disease phenotype in the family. In silico analysis predicted a shift in the frame causing an early stop codon (p.R83GfsX52). The predicted dwarf protein constituting 134 amino acids was functionally aberrant with a complete loss of the catalytic domain at the C-terminus. Interestingly, an alternative open reading frame 3 (ORF3) caused by the frame shift is predicted to encode a protein sequence, identical to the wild type and containing the catalytic domain, but lacking the first 100 amino acids of the wild-type EXT1 protein. Conclusion: Consequently, haploinsufficiency could be the cause of HME in the investigated family as the mutated copy of EXT1 is ineffective for EXT-1/2 complex formation. The predicted ORF3 protein could be of great significance in understanding several aspects of HME pathogenesis.

Identification and association of recurrent ALOXE3 mutation with non-bullous congenital ichthyosiform erythroderma in two ethnically distinct Pakistani families.

Non-bullous congenital ichthyosiform erythroderma (NCIE) is characterized by skin scaling with erythema. In this study, two Pakistani families with NCIE are genetically characterized through Whole Exome and Sanger sequencing to identify molecular basis of the disease. We identified a nonsense homozygous c.2026C>T mutation of ALOXE3, causing premature termination of the eLOX3 protein (p.Q676X). In silico studies predicted impaired enzymatic activity of the premature truncated eLOX3, leading to abnormal synthesis of specific hepoxilin derivatives, essential for epidermal barrier formation. It is the first ever study reporting homozygotes of p.Q676X mutation in ethnically distinct two Pakistani families; otherwise, heterozygotes of the said mutation have been reported in South Asian population only. Hence, mutation seems to be region-specific and may be useful for molecular diagnosis of NCIE. Moreover, our findings should help in genetic counseling and career screening.

Identification and in silico characterization of a novel p.P208PfsX1 mutation in V-ATPase a3 subunit associated with autosomal recessive osteopetrosis in a Pakistani family.

BACKGROUND: Osteopetrosis is a rare inherited bone disorder mainly described as an increased bone density caused by defective osteoclastic bone resorption. To date, genetic variants of eleven genes have been reported so far to be associated with different types of osteopetrosis. However, malignant infantile osteopetrosis, a lethal form of the disease, is mostly (50%) caused by mutation(s) in TCIRG1 gene. In this study, we investigated a consanguineous Pakistani family clinically and genetically to elucidate underlying molecular basis of the infantile osteopetrosis. METHODS: DNA samples from five family members were subjected to SNP-array based whole genome homozygosity mapping. Data was analyzed and potentially pathogenic mutation was identified by Sanger sequencing of two affected as well as three phenotypically healthy individuals in the family. The significance of identified pathogenic variation and its impact on protein structure and function was studied using various bioinformatics tools. RESULTS: DNA samples from five family members were subjected to genome-wide SNP array genotyping and homozygosity mapping which identified ~4 Mb region on chr11 harboring the TCIRG1 gene. Sanger sequencing unveiled a novel homozygous deletion c. 624delC in exon 6 of the TCIRG1 gene encodes a3 subunit of V-ATPase complex. The identified deletion resulted in a frame shift producing a truncated protein of 208 aa. In silico analysis of premature termination of the a3 subunit of V-ATPase complex revealed deleterious effects on the protein structure, predicting impaired or complete loss of V-ATPase function causing infantile osteopetrosis. CONCLUSIONS: Since a3 subunit of V-ATPase complex plays a crucial role in bone resorption process, structurally abnormal a3 subunit might have adversely affected bone resorption process, leading to infantile osteopetrosis in Pakistani family. Therefore, the present study not only expands the genotypic spectrum of osteopetrosis but also improve understandings of the role of V-ATPase a3 subunit in bone resorption process. Moreover, our findings should help in genetic counseling and provide further insight into the disease pathogenesis and potential targeted therapy.

Identification and in silico characterization of p.G380R substitution in FGFR3, associated with achondroplasia in a non-consanguineous Pakistani family.

BACKGROUND: The dimerization efficiency of FGFR3 transmembrane domain plays a critical role in the formation of a normal skeleton through the negative regulation of bone development. Recently, gain-of-function mutations in the transmembrane domain of FGFR3 has been described associated with an aberrant negative regulation, leading to the development of achondroplasia-group disorders, including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). Here, we describe a non-consanguineous Pakistani family with achondroplasia to explain hereditary basis of the disease. METHODS: PCR-based linkage analysis using microsatellite markers was employed to localize the disease gene. Gene specific intronic primers were used to amplify the genomic DNA from all affected as well as phenotypically healthy individuals. Amplified PCR products were then subjected to Sanger sequencing and RFLP analysis to identify a potentially pathogenic mutation. The impact of identified mutation on FGFR3 protein's structure and stability was highlighted through different bioinformatics tools. RESULTS: Genetic screening of the family revealed a previously reported heterozygous c.1138 G > A (p.G380R) mutation in the coding exon 8 of FGFR3 gene. Identified genetic variation was confirmed in all affected individuals while healthy individuals and controls were found genotypically normal. The results were further validated by RFLP analysis as c.1138 G > A substitution generates a unique recognition site for SfcI endonuclease. Following SfcI digestion, the electrophoretic pattern of three bands/DNA fragments for each patient is indicative of heterozygous status of the disease allele. In silico studies of the mutant FGFR3 protein predicted to adversely affect the stability of FGFR3 protein. CONCLUSIONS: Mutation in the transmembrane domain may adversely affect the dimerization efficiency and overall stability of the FGFR3, leading to a constitutively active protein. As a result, an uncontrolled intracellular signaling or negative bone growth regulation leads to achondroplasia. Our findings support the fact that p.G380R is a common mutation among diverse population of the world and like other countries, can be used as a molecular diagnosis marker for achondroplasia in Pakistan.

Autologous mesenchymal stromal cell transplantation for spinal cord injury: A Phase I pilot study.

BACKGROUND AIMS: Mesenchymal stromal cell (MSC) transplantation has immerged as promising therapeutic approach to treat spinal cord injury (SCI). In this pilot study, we investigated the safety of intrathecal injection of autologous bone marrow-derived MSCs in nine patients with SCI. METHODS: Patients with complete SCI at the thoracic level were divided into two groups: chronic (>6 months, group 1) and sub-acute SCI (<6 months, group 2), according to time elapsed since injury. MSCs were isolated by density gradient separation of autologous bone marrow harvested from the iliac crest. Cells were cultured in a Good Manufacturing Practice-compliant facility to produce clinical scale dose. After quality control testing, MSCs were injected back to patients by intrathecal injection. Safety was defined as absence of adverse event and side effects after 1 month after receiving the injection. RESULTS: Six patients had chronic SCI with a median duration of 33 months since date of injury (range: 10-55 months), and three patients were in sub-acute phase of disease. Each patient received two or three injections with a median of 1.2 × 10(6) MSCs/kg body weight. No treatment-related adverse event was observed during median follow-up of 720 days (range: 630-826 days) in group 1 and 366 days (range: 269-367 days) in group 2, respectively. DISCUSSION: This pilot study demonstrated that autologous MSCs can be safely administered through intrathecal injection in spinal cord injury patients. Further investigation through randomized, placebo-controlled trials is needed.

Molecular diagnosis and phylogenetic analysis of human papillomavirus type-16 from suspected patients in Pakistan.

BACKGROUND: Human Papillomavirus (HPV) is well known pathogen that can cause benign and malignant tumors in humans, yet there is very little information regarding HPV types prevalent in Pakistan. METHODS: A total of 92 cervical secretions were collected from suspected married female patients and used for DNA isolation using a novel isolation method. The samples were tested through Polymerase Chain Reaction (PCR) using already reported primers MY09/MY11, GP5/GP6, GP5+/GP6+, CP65/CP70, CP66/CP69 and SPF1/SPF2 and with those developed in this study including HRT1 and HRT2 primer sets for typing HPV types and HACTB primer set for human beta actin gene as internal positive control. Sequencing and phylogenetic analyses were performed for two isolates to determine circulating HPV types. RESULTS: PCR with HRT1 and HRT2 indicated 2 (2.17 %) patients were positive for HPV type- 16 while 1 (1.08 %) with HPV type 18. Sequencing and phylogenetic analysis of isolates confirmed HPV type-16 in genus alpha 9 which have 99 % homology with already reported HPV from Japan and Costa Rica. CONCLUSION: This is the first report of HPV type-16 genus alpha 9 in Pakistan and the reported assay and sequence data will serve as valuable tools in further epidemiological studies for HPV surveillance to improve public health, especially of females in Pakistan.

Antioxidant and phytochemical analysis of Ranunculus arvensis L. extracts.

BACKGROUND: Ranunculus arvensis L. (R. arvensis) has long been used to treat a variety of medical conditions such as arthritis, asthma, hay fever, rheumatism, psoriasis, gut diseases and rheumatic pain. Here, we screened R. arvensis for antioxidant activity, phytochemical and high performance liquid chromatography (HPLC) analyses. METHODS: The chloroform, chloroform:methanol, methanol, methanol:acetone, acetone, methanol:water and water extracts of R. arvensis were examined for DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging assay, hydrogen peroxide scavenging assay, phosphomolybdenum assay, reducing power assay, flavonoid content, phenolic content and high performance liquid chromatography analysis. RESULTS: Significant antioxidant activity was displayed by methanol extract (IC 50 34.71 ± 0.02) in DPPH free radical scavenging assay. Total flavonoids and phenolics ranged 0.96-6.0 mg/g of extract calculated as rutin equivalent and 0.48-1.43 mg/g of extract calculated as gallic acid equivalent respectively. Significant value of rutin and caffeic acid was observed via high performance liquid chromatography. CONCLUSIONS: These results showed that extracts of R. arvensis exhibited significant antioxidant activities. Moreover, R. arvensis is a rich source of rutin, flavonoids and phenolics.

Antimicrobial, antitumor and brine shrimp lethality assay of Ranunculus arvensis L. extracts.

To investigate the antitumor activity, brine shrimp lethality assay, antibacterial and antifungal activity of Methanol Extract (ME), Water Extract (WE), Acetone Extract (AE), Chloroform Extract (CE), Methanol-Water Extract (MWE), Methanol-Acetone Extract (MAE), Methanol-Chloroform Extract (MCE) of Ranunculus arvensis (L.). Antitumor activity was evaluated with Agrobacterium tumefaciens (At10) induced potato disc assay. Cytotoxicity was evaluated with brine shrimp lethality assay. Antibacterial activity was evaluated with six bacterial strains including Escherichia coli, Enterobacter aerogenes, Bordetella bronchiseptica, Klebsiella pneumoniae, Micrococcus luteus and Streptococcus anginosus and antifungal screening was done against five fungal strains including Aspergillus niger, A. flavus, A. fumigates, Fusarium solani and Mucor species by using disc diffusion method. Best antitumor activity was obtained with ME and WE, having highest IC50 values 20.27 ± 1.62 and 93.01 ± 1.33µg/disc. Brine shrimp lethality assay showed LC50 values of AE, MAE and ME were obtained as 384.66 ± 9.42µg/ml, 724.11 ± 8.01µg/ml and 978.7 ±8.01 µg/ml respectively. WE of R. arvensis revealed weak antimicrobial result against the tested microorganisms. On the other hand, the antifungal activity of the plant extracts was found to be insignificant. These findings demonstrate that extracts of R. arvensis possesses significant antitumor activity. Further extensive study is necessary to assess the therapeutic potential of the plant.

Characterization of toxic metals in tobacco, tobacco smoke, and cigarette ash from selected imported and local brands in Pakistan.

In this study, concentrations of Cd, Ni, Pb, and Cr were determined in tobacco, tobacco smoke-condensate, and cigarette ash for selected brands used in Pakistan. Smoking apparatus was designed for metal extraction from cigarette smoke. Samples were digested through microwave digester and then analyzed by flame atomic absorption spectrophotometer (FAAS). Higher concentration of Ni was detected in imported brands than the counterparts in the local brands. Pb levels were however higher in local brands while significant concentration of Cd was observed in both brands. For Cr, the level in tobacco of local brands was higher than their emitted smoke, whereas imported brands showed higher level in smoke than in tobacco. The cigarette ash retained 65 to 75% of the metal and about 25 to 30% went into the body. While this study revealed the serious requirement to standardize the manufacturing of tobacco products, more importantly is the urgent need for stronger enforcements to put in place to alert the general population about the hazardous effects of cigarettes and the health risks associated with these toxic metals.

Identification of recurrent c.742G>T nonsense mutation in ECM1 in Pakistani families suffering from lipoid proteinosis.

Lipoid proteinosis (LP) is one of the rare, recessive autosomal disorders clinically characterized by widespread deposition of hyaline-like material in the skin, mucosa and viscera. Classical features include beaded eyelid papules, laryngeal infiltration and hoarseness of voice caused by pathogenic mutations in the ECM1 gene located on 1q21.2. In present study ethnically different, three consanguineous Pakistani families with typical cutaneous features of LP were analysed to investigate the underlying molecular basis. PCR based linkage analysis using microsatellite markers localized the families to locus 1q21.2, harboring ECM1 gene. To identify the mutation in the candidate gene (ECM1), Sanger sequencing was carried out. All the families were found to carry c.742 G>T nonsense mutation in exon 7 of the ECM1 gene that resulted in a truncated ECM1 protein containing 247 amino acids instead of 540 (p.E248X). To further investigate the impact and importance of mutation in LP pathogenesis we applied different bioinformatics tools. In silico studies has predicted lack of functional domains and 65 % shorter ECM1 mutant protein. It is the first report of recurrence mutation from Pakistan as c.742G>T nonsense mutation was found in three ethnically different Pakistani families with LP. Study strengthens the conclusion that c.742G>T mutation is the pathological cause of LP. Furthermore, data also support the fact that exon 7 is one of the most common hot spots of pathological mutations in ECM1. The absence of functional domains and truncated sequence most likely contribute to the lack of ECM1 function and thereby influence several aspects of dermal homeostasis that leads to LP pathogenesis.

Cattle egrets as a biosentinels of persistent organic pollutants exposure.

We investigated selected chlorinated pollutants (ß-HCH, γ-HCH, DDDs, DDEs, o,p'-DDT, p,p'-DDT, heptachlor, aldrin, dieldrin, and endrin) in the Lahore and the Sialkot districts of Pakistan, using eggs of cattle egret (Bubulcus ibis) collected during May and June 2007. The pollutant with highest level and frequency was ΣDDT, followed by ß-HCH, γ-HCH, heptachlor, aldrin, dieldrin, and endrin in descending order. The concentration(s) were significantly higher in Sialkot heronry for all the pollutants (except p,p'-DDT) than in Lahore. The values for DDTs, ß-HCH, γ-HCH, and heptachlor were significantly higher (p < 0.05) in the egg(s) than in sediment(s) and in the chicks' diet, due to biomagnification. Among DDTs analogues, p,p'-DDD was the major contaminant with >60 % of total DDT burden, reflecting the widespread aged as well as recent use of DDT as well as anaerobic degradation (DDD/DDE > 1 in many cases) in the nearby paddy soils. In few samples, p,p'-DDT/(DDD + DDE) > 0.5 suggested the recent emission patterns from surrounding contaminated areas of demolished DDT units and obsolete pesticide stores. The higher levels of HCHs (i.e., ß-HCH) in the samples collected from Sialkot indicate exposure from long-term agricultural use. Overall, concentrations of all studied POPs were less than the threshold levels known to affect reproduction. Nevertheless, total DDTs and/or HCHs burdens in some eggs contained concentrations of greater than what would educe adverse effects on birds. This is among few studies on OCPs exposure to avian species, which provide the evidence of Pakistan's contribution toward the Global POPs emission.

Evaluation of diagnostic potential of Rv3803c and Rv2626c recombinant antigens in TB endemic country Pakistan.

To overcome and eliminate tuberculosis (TB), definitive, reliable, and rapid diagnosis is mandatory. Presently, the diagnostic potential of acute and latent stage TB specific antigens i.e., Rv3803c and Rv2626c was determined. Immunogenic recombinant genes of Rv3803c and Rv2626c antigens were cloned in bacterial expression vector pET23b and expressed product was purified. The homogeneity and structural integrity was confirmed by Western blot analysis. Diagnostic potential of Rv3803c and Rv2626c antigens was analyzed using the sera of 140 active TB patients (AFB smear positive) by indirect ELISA. Ten patients of leprosy and 94 healthy individuals were taken as disease and normal control respectively. The data was analyzed using R statistical package. The sensitivity and specificity of Rv3803c in active TB patients was of 69.3% and 76.4% respectively with an area under ROC curve of 0.77, whereas sensitivity and specificity of Rv2626c 77.1% and 85.1%, respectively. The area under ROC curve of Rv2626c was 0.89 which was significantly higher than Rv3803c (p < 0.0001). Recombinant antigens Rv3803c and Rv2626c have potential to be used as diagnostic markers for TB and need to evaluate with other antigens for differential diagnosis of TB.

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum.

BACKGROUND: Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation. RESULTS: The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system. CONCLUSIONS: Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain.

Epidemiology and genetic diversity of rotavirus strains in children with acute gastroenteritis in Lahore, Pakistan.

Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008-2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus (p>0.05). A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P [4] = 3(6.81%); G1P [6] = 9(20.45%); G1P [8] = 1(2.27%); G2P [4] = 21(47.72%); G2P [8] = 1(2.27%); G9P [4] = 1(2.27%); G9P [6] = 1(2.27%) and G9P [8] = 7(15.90%). Phylogenetic analysis revealed that the VP7 and VP4 sequences clustered closely with the previously detected strains in the country as well as Belgian rotaviruses. Antigenic characterization was performed by analyzing major epitopes in the immunodominant VP7 and VP4 gene segments. Although the neutralization conferring motifs were found variable between the Pakistani strains and the two recommended vaccines strains (Rotarix™ and RotaTeq™), we validate the use of rotavirus vaccine in Pakistan based on the proven and recognized vaccine efficacy across the globe. Our findings constitute the first report on rotavirus' genotype diversity, their phylogenetic relatedness and epidemiology during the pre-vaccination era in Lahore, Pakistan and support the immediate introduction of rotavirus vaccine in the routine immunization program of the country.

Study of the effect of antiviral therapy on homocysteinemia in hepatitis C virus- infected patients.

BACKGROUND: Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease (CLD). About 80% of those exposed to the virus develop a chronic infection. Hyperhomocysteinemia, which is an independent risk factor for atherosclerotic vascular disease and thromboembolism, may develop in HCV-infected patients although altered alanine amino transferase (ALT) enzyme levels are generally associated with damage to liver cells. The gold standard therapy for chronic hepatitis C patients is pegylated interferon combined with an anti-viral drug (ribavirin). The current study aimed to investigate the effect of antiviral therapy on plasma homocysteine (Hcy) levels in HCV patients in addition to other parameters. METHODS: 532 HCV-infected patients and 70 healthy controls were recruited for the study. All patients were subjected to laboratory investigations including HCV-RNA levels, complete blood cell counts, serum levels of homocysteine, ALT, alkaline phosphatase (ALP), lipid profile and liver ultrasonographic examination. The outcome of treatment with pegylated interferon α plus ribavirin treatment and sustained virologic response (SVR) was determined 6-9 months post-therapy. RESULTS: Hyperhomocysteinemia was found in 91.35% of HCV-infected patients. The difference in plasma Hcy concentrations reached statistical significance between the patient and control groups. ALT, cholesterol and triglycerides (TGs) levels were found higher than normal in the patients group. After receiving a combined therapy for 24 weeks, 43.66% patients showed an SVR (responders); 30.98% patients were non-responders while 25.35% patients initially responded to therapy but again retrieved positive status of HCV infection six months post-therapy (relapse-cirrhotic patients). The mean levels of plasma Hcy, ALT and ALP were significantly reduced in responders within 10 weeks of therapy when compared with non-responders and relapse-cirrhotic patients. CONCLUSION: Elevated homocysteine levels in serum due to HCV infection can be reduced to normal range with the standard interferon α plus ribavirin treatment. This study highlights the significance of the measurement of serum homocysteine levels in the diagnosis and monitoring of HCV infection treatment in addition to other laboratory parameters.

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Kohat District, Pakistan.

Malaria is one of the serious diseases threatening human health in Pakistan and contributes to a large proportion of the total malaria deaths in South Asia. However, little is known about the nature and extent of genetic diversity of the malarial parasites circulating in Pakistan. This study was designed to assess the infection status of Plasmodium and the genetic diversity of Plasmodium vivax and Plasmodium falciparum by analyzing msp-3α, msp-3ß and msp-1, msp-2 genes respectively using allele specific nested PCR and RFLP assays. For this purpose, 130 field isolates were collected from the individuals who exhibited clinical symptoms associated with malaria in the Kohat region of Khyber Pakhtoonkhwa (KPK), Pakistan. Among 130 blood samples collected, P. vivax was detected in 105/130 (80.8%) and P. falciparum in 21/130 (16.2%). Mixed infections with both parasites were detected in 4/130 (3%) of the isolates. A large number of distinguishable alleles were found for msp genetic markers: 10 alleles for msp-3α and seven for msp-3ß with one mixed infection in case of msp-3ß. The genotyping of P. falciparum showed that K1+MAD20 mixed genotype was dominant in msp-1 and FC27 in msp-2. The results collectively suggest that P. vivax and P. falciparum populations in this region are highly polymorphic and mixed infections are prevalent.

Genetic characterization of rotavirus subtypes in Pakistan-first report of G12 genotype from Pakistan under WHO-Eastern Mediterranean region.

Rotaviruses are among the major causes of gastroenteritis and diarrhea among children in developed as well as the developing countries. The rapidly evolving strain prevalence and circulation have resulted in the emergence of novel strains over the period worldwide. The introduction of G12 prototype in 1987 from Philippines and subsequently re-emergence among most of the Asian countries along with USA and Europe has provoked new research horizons to address the global distribution of rotavirus serotypes. These newly emerging subtypes and their sustenance among the population have posed tremendous challenge to the development of an effectual vaccine with heterotypic protective efficacy. In Pakistan, no data is available regarding the prevalent rotavirus serotypes; therefore, this is the first study to report the prevalence of G12 strain in Pakistan in hospitalized children with diarrhea addressing a dire need of further large-scale epidemiological surveys to resolve the underlying rotavirus isolates in both the hospitalized and the community neonatal and child population before formulating the vaccine introduction policies in the country's routine immunization program.

Antimicrobial susceptibility and ESBL prevalence in Pseudomonas aeruginosa isolated from burn patients in the North West of Pakistan.

Pseudomonas aeruginosa is one of the most prevalent pathogen in burn infections. Infections with P. aeruginosa are associated with higher mortality rate and antibiotic costs in hospitalized patients. These bacteria also produce enzymes called Expanded Spectrum Beta-Lactamases (ESBL) which render penicillins and cephalosporins inactive. The aim of this study was to assess the antimicrobial susceptibility pattern and prevalence of ESBL in P. aeruginosa in Peshawar, North West of Pakistan. During 2005-2006, one hundred and six P. aeruginosa isolates were collected from burn patients at a tertiary care hospital. Antibiotic susceptibility testing and ESBL detection were carried out according to Clinical Laboratory and Standards Institute (CLSI) criteria. Eighteen antibiotics were tested in this study. A total of 38 (35.85%) isolates were found to be ESBL producers. Thirty one (29.24%) isolates were resistant to 3 or more antibiotics (multidrug resistance). Meropenem and imipenem showed high potency with 99% and 96% isolates being susceptible respectively. Susceptibility to amikacin was 70%; gentamicin 25%; ciprofloxacin 49%; enoxacin 47%; gatifloxacin 42%; doxycycline 21% and to co-trimoxazole only 16%. This study reveals that P. aeruginosa isolated from burns in this region are multidrug resistant and produce ESBL in large proportions.

Serum sialic acid changes in non-insulin-dependant diabetes mellitus (NIDDM) patients following bitter melon (Momordica charantia) and rosiglitazone (Avandia) treatment.

Diabetes mellitus is associated with an increase in sialic acid concentration along with other complications. Sialic acid changes in NIDDM patients were investigated following bitter melon (55 ml/24h) and rosiglitazone (4 mg/24h) treatment. A total of 25 patients of both sexes were used in each experimental group. Patients following bitter melon treatment showed no significant difference of serum sialic acid (57.95+/-4.90 vs. 57.6+/-5.56 mg/dl, p=0.17) and serum glucose concentration (93.7+/-9.63 vs. 88.35+/-6.31 mg/dl, p=0.78) as compared to control subjects. However, the concentration of total cholesterol was significantly high in these patients as compared to control subjects (192+/-14.23 vs. 170.6+/-15.1mg/dl, p<0.03) but within normal range (160-200mg/dl), suggesting the significant hypoglycemic and lipid-lowering properties of bitter melon. The patients following rosiglitazone treatment showed a significant increase of serum sialic acid concentration (60.2+/-5.80 vs. 57.6+/-5.56 mg/dl, p=0.01) along with glucose (112+/-6.2 vs. 88.35+/-6.31 mg/dl, p<0.04) and total cholesterol concentration (216.45+/-20.2 vs. 170.6+/-15.1mg/dl, p<0.01) as compared to control subjects. In addition six of the patients had retinopathy, two of whom were suffering also from myocardial infarction and they still had a higher serum sialic acid (61.05+/-1.20mg/dl), glucose (187+/-2.11 mg/dl), total cholesterol (239.10+/-5.04 mg/dl) and triglyceride (183+/-4.14 mg/dl) concentration, indicating a poor response of these patients to rosiglitazone. Comparison of serum sialic acid concentration of patients, following bitter melon and rosiglitazone treatment revealed no significant difference but the study showed that bitter melon could be more effective in the management of diabetes and its related complications as compared to rosiglitazone.

Evaluation of trace metals in tobacco of local and imported cigarette brands used in Pakistan by spectrophotometer through microwave digestion.

Uncontrolled exposure of active and passive smokers to trace metals causes increase in health risks. The primary objective of this study was to determine whether local and imported cigarette brands used in Pakistan, have elevated levels of metals or not. Six metals manganese (Mn), cobalt (Co), copper (Cu), cadmium (Cd), lead (Pb) and zinc (Zn) were determined in tobacco of twenty cigarette brands (local and imported) used in Pakistan by flame atomic absorption Spectrophotometry. To overcome contamination chances and for complete digestion of analytes a microwave digester was used. The analytical results showed highest concentration of Mn (84.78 microg/g dry weight), Cd (0.525 microg/g dry weight) and Zn (14.34 microg/g dry weight) metals in imported brands in relation to counterparts from the local brands. Certain elevated levels were observed for Co (3.344 microg/g dry weight), Pb (14.16 microg/g dry weight) and Cu (7.889 microg/g dry weight) metals in local brands. The inter-metal relationships in the tobacco of local and imported cigarette brands showed some integrated variation in the selected metal levels. In view of health risk associated with the above metals, there should be a strict quality control over monitoring of heavy metals during growing, processing and smoking of tobacco. Therefore, it is prudent to minimize exposure to toxic substances whenever possible because smoking and exposure to cigarette smoke is a confounder to be taken into account when carrying out epidemiological studies on human exposure to metals.