Formulation of novel chitosan guargum based hydrogels for sustained drug release of paracetamol.

The report presents the formulation of hydrogel based on biopolymers chitosan and guar gum after cross-linking for sustained release of a commonly used orally prescribed analgesic Paracetamol. The oral ingestion of Paracetamol is associated with complications of the gastric tract and liver metabolism that can be effectually avoided by using transdermal drug delivery systems. The formulated transdermal patch was characterized for physicochemical properties including swelling, bonding pattern (using FTIR Fourier Transform Infra-Red and Scanning Electron Microscopy SEM) and antimicrobial activity. Biocompatibility and cytotoxicity was examined in vitro using cell culture in HeLa cell lines. After characterizing the novel formulated hydrogel were employed for the preparation of drug encapsulated in alginate beads as a transdermal patch. After formulation of the transdermal patch, the drug release was studied using an avian skin model. The results followed zero order kinetics and Non-Fickian law for diffusion. Paracetamol due to its small molecular mass (151.163g/mol) released in a sustained manner. The released drug successfully retained its biological effects including anti-inflammatory and anti-protease activity, indicating no interaction between the drug and the formulated hydrogel. It was shown that the formulated hydrogels could be safely used as a dermal patch for the sustained drug release of Paracetamol.

Genotype CC of rs12979860 is providing protection against infection rather than assisting in treatment response for HCV genotype 3a infection.

Recent discovery of single-nucleotide polymorphisms located in the upstream region of interleukin-28B (IL28B) has shown association with interferon (IFN) treatment response especially in hepatitis C virus (HCV) genotype 1-infected patients. Pakistan, being the country with second highest prevalence of HCV with predominantly 3a genotype infection, bears a significant disease burden. The present study was conducted to evaluate the effect of rs12979860 genotypes on treatment response in HCV-3a-infected patients. This study shows that the CC genotype is providing protection against infection to HCV. But once infected, the CC genotype patients show viral persistence following IFN therapy. The TT genotype is assisting the 3a patients in viral clearance after IFN treatment. To our knowledge, this is the first study showing rs12979860 genotype association with IFN response in Pakistani HCV-3a-infected patients.

Spectrum of K ras mutations in Pakistani colorectal cancer patients.

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

Spectrum of K ras mutations in Pakistani colorectal cancer patients

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

SP-D polymorphisms and the risk of COPD.

INTRODUCTION: There are limited data linking serum levels of surfactant protein D, its genetic polymorphisms to the risk of Chronic Obstructive Pulmonary Disease (COPD). OBJECTIVES: We sought to investigate these relationships using a case control study design. METHODS: Post bronchodilator values of FEV1/FVC < 0.7 were used to diagnose COPD patients (n=115). Controls were healthy subjects with normal spirometry (n=106) Single nucleotide polymorphisms (rs721917, rs2243639, rs3088308) were genotyped using polymerase chain reaction (PCR) and restriction analysis. Serum SP-D levels were measured using a specific immunoassay. RESULTS: Allele 'A' at rs3088308 (p < 0.00, B= -0.41) and 'C' allele at rs721917 (p=0.03; B= -0.30) were associated with reduced serum SP-D levels. Genotype 'T/T' at rs721917 was significantly associated with risk of COPD (p=0.01). Patients with repeat exacerbations had significantly higher serum SP-D even after adjusting for genetic factors. CONCLUSIONS: We report for the first time that rs3088308 is an important factor influencing systemic SP-D levels and confirm the previous association of rs721917 to the risk of COPD and serum SP-D levels.

Cry genes profiling and the toxicity of isolates of Bacillus thuringiensis from soil samples against American bollworm, Helicoverpa armigera.

AIMS: The aim of this study was to search for Bacillus thuringiensis (Bt) harbouring cry1A gene which could effectively control cotton pest, American bollworm, Helicoverpa armigera. METHODS AND RESULTS: cry gene profiling of 50 Bt isolates showed the presence of cry1, cry2, cry3, cry4, cry7, cry8 and cry9 genes. None of the isolates harboured cry1 gene alone. It was always found in combination with cry3. There was no isolate positive for cry10 gene. Considering isolates with single cry genes, the frequency of cry4 was predominant (22%) followed cry2 (6%), cry3 (4%) and cry8 (2%). Isolates having two cry genes in combination had 14% incidence for cry2 + cry4, 12% for cry3 + cry4 and 10% for cry1 + cry3. The most dominant three gene linkage was cry1 + cry3 + cry4. Further profiling of cry1 gene showed that cry1K gene was abundantly present in all combinations such as cry1A, cry1D, cry1F and cry1I. However, cry1C existed independent of other subtypes. Finally, the Bt isolates with cry1A were analyzed for 16S rRNA gene, which showed two distinct groups of isolates on the basis of sequence homology. Bioassays of spore-crystal mixtures of SBS-Bt4, 8, 17, 21 and 26 harbouring cry1 against neonate larvae of H. armigera showed LC(50) 1288, 1202, 467·7, 524·8 and 108·5 µg ml(-1) . The SBS-Bt26 showed fourfold higher toxicity than the cry 1Ac harbouring positive control, HD-73. CONCLUSIONS: None of the isolates harboured single cry 1 gene. They were always in combination of two or three genes. A Bt isolate (Bt26) had fourfold higher toxicity against H. armigera larvae compared with the positive control HD 73 and hence can be commercially exploited to control insect pest. SIGNIFICANCE AND IMPACT OF THE STUDY: The inter relationship between the cry genes content and the toxicity may allow better understanding of Bt ecology.

Heavy metal uptake by Euplotes mutabilis and its possible use in bioremediation of industrial wastewater.

A ciliate protozoan, Euplotes mutabilis, isolated from heavy metal laden industrial wastewater, has been shown to tolerate multiple heavy metals thus suggesting its significance in bioremediation of industrial effluents. This ciliate tolerated Zn(2+) up to 33 microg/mL, Cd(2+) up to 22 microg/mL and Ni(2+) up to 18 microg/mL. The ciliate could uptake 85% Zn(2+), 84% of Cd(2+) and 87% of Ni(2+) after 96 h of inoculation of growth medium containing 10 microg/mL of Zn(2+) and 5 microg/mL of Cd(2+) and Ni(2+), with actively growing ciliates. After 6 days of incubation the ciliate removed 87% Cd(2+), 92% Ni(2+), and 93% Zn(2+) from the wastewater. The heavy metal uptake capability of Euplotes mutabilis may be employed for metal detoxification operations.

Aetiology of shigellosis in northern Pakistan.

People of northern Pakistan face health hazards because of poor sanitation practices. Bacterial gastrointestinal infections are very common, and sometimes outbreaks occur. The present study was aimed at evaluating and analyzing infestation of Shigella spp. in patients with suspected gastroenteritis and ascertaining the status of antibiotic therapy. Five hundred and eighty-five faecal samples of patients with suspected gastroenteritis, referred to the District Headquarter Hospital Gilgit, were investigated for common enteropathogenic bacteria from July 1997 to September 1999. Seventy-seven (13.2%) of the faecal specimens were infected with different strains of Shigella spp., 61% of which were Shigella dysenteriae, 15.6% were S. flexneri, and 23.4% were Shigella sp. All Shigella strains were sensitive to ceftriaxone, cefotaxime, ciprofloxacin, and enoxacin. Sixty-one percent of the strains were resistant to both ampicillin and chloramphenicol, and 3.9% to ampicillin and nalidixic acid, while 10.4% were resistant to ampicillin alone and 14.3% to chloramphenicol only. Only 10.4% of the strains were sensitive to all the antibiotics tested. Sixty strains of Shigella spp. were processed for isolation of plasmids, and 58 (97%) of these antibiotic-resistant bacteria harboured at least one plasmid. The number of plasmids varied from 1 to 9. Escherichia coli C600 were transformed with the isolated plasmids. Transformants, containing 23-kb plasmid, resisted growth in media containing antibiotics, thereby indicating that antibiotic resistance is plasmid-borne. Based on the findings of the study, it is concluded that the infestation of Shigella spp. is high in northern Pakistan, the aetiological agents are highly resistant to chloramphenicol and ampicillin, and the antibiotic resistance is mediated by the 23-kb plasmid.

Copper-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater.

Six copper-resistant bacterial strains were isolated from wastewater of tanneries of Kasur and Rohi Nala. Two strains tolerated copper at 380 mg/L, four up to 400 mg/L. Three strains were identified as members of the genus Salmonella; one strain was identified as Streptococcus pyrogenes, one as Vagococcus fluvialis and the last was identified as Escherichia coli. The pH and temperature optimum for two of them were 7.0 and 30 degrees C, respectively; four strains had corresponding optima at 7.5 and 37 degrees C, respectively. All bacterial isola-tes showed resistance against Ag+ (280-350 mg/L), Co2+ (200-420), CrVI (280-400), Cd2+ (250-350), Hg2+ (110-200), Mn2+ (300-380), Pb2+ (300-400), Sn2+ (480-520) and Zn2+ (300-450). Large-sized plasmids (> 20 kb), were detected in all of the strains. After the isolates were cured of plasmids with ethidium bromide, the efficiency of curing was estimated in the range of 60-90%. Reference strain of E. coli was transformed with the plasmids of the bacterial isolates which grew in Luria-Bertani medium containing 100 mg/L Cu2+. The capability to adsorb and afterwards accumulate Cu2+ inside their cells was assayed by atomic absorption spectrophotometer; all bacterial cells had the ability to adsorb 50-80% of the Cu2+ and accumulate 30-45% Cu2+ inside them after 1 d of incubation.

Hexavalent chromium reduction by a dichromate-resistant gram-positive bacterium isolated from effluents of tanneries.

A gram-positive, chromium (Cr)-resistant bacterial strain (ATCC 700729) was isolated from effluent of tanneries. It was grown in media containing potassium dichromate concentration up to 80 mg ml(-1) of the medium. The dichromate reducing capability of the bacterium was checked by estimating the amount of Cr VI in the medium before and after introduction of bacterial culture. The influence of factors like pH of the medium, concentration of Cr, and the amount of the inoculum was studied to determine the ability of the bacterium to reduce Cr VI in the medium under various conditions. In a medium containing dichromate 20 mg ml(-1) more than 87% reduction of dichromate ions was achieved within 72 h. The feasibility of the use of this bacterial strain for detoxification of dichromate in the industrial wastewater has been assessed. The isolated strain can be exploited for specific environmental clean-up operations.

Effect of dichromate on population and growth of various protozoa isolated from industrial effluents.

Three protozoa belonging to genera Euglena, Vorticella and Stylonychia collected from industrial wastes were cultured in a medium containing inorganic salts, basically meant for the growth of algae. Protozoa showed rapid growth in the medium. Hexavalent chromium (K2Cr2O7) at a concentration of 5 micrograms/L in the medium adversely affected the growth of protozoa. At the end of eight days of Cr administration, the population of Euglena, Vorticella and Stylonychia increased 8-, 4.5- and 10-fold, respectively, as against 30-, 6.75- and 50-fold increase in the control cultures. No apparent death phase and no change in activity or morphology of protozoa was observed at this Cr concentration. The protozoa were also exposed to different metal ions, viz. Pb (2.42 mmol/L), Cr (0.48 mmol/L), Cd (0.36 mmol/L), administered in the culture medium for a period of 2 years. The metal tolerance for S. mytilus and V. microstoma was Pb > Cr > Cd. E. proxima could not tolerate any of the long-term metal treatments. Because of the ability of these protozoa to tolerate high concentrations of heavy metals, their potential role in remediation of heavy metals from industrial wastewater is considered.

Microorganisms resistant to heavy metals and toxic chemicals as indicators of environmental pollution and their use in bioremediation.

Microorganisms present in water samples from various industrial effluents were analysed for their resistance to lead, chromium, and cadmium. The ability of these microorganisms to grow on or metabolize toxic hydrocarbons and pesticides was also checked. Microorganisms in samples from the steel and tanning industries were generally resistant to metal ions but were not capable of metabolizing toxic hydrocarbons. Conversely, microorganisms found in samples of pesticide and from the chemical industry were capable of metabolizing hydrocarbons and pesticides but were not much resistant to metal ions. Microorganisms from effluents of the paint industry and urban wastes were resistant to lead. A correlation between the population of microorganisms and the type of pollution was observed. Indigenous microorganism could be regarded as indicators of pollution and be used in various operations to resist, process, metabolize, and detoxify toxic industrial wastes.