Kinetic study of partially purified cellulase enzyme produced by <Emphasis Type="Italic">Trichoderma viride</Emphasis> FCBP-142 and its hyperactive mutants

Cellulases are the enzymes that cleave β-1,4 linkages of cellulose, and carbohydrate that is main part of plants’ cell walls. Presently, cellulase isolation and partial purification was executed through ammonium sulfate precipitation. The isolated protein of parental and derived mutants conferred molecular weights of 30, 45 and 55 kDa. The optimum temperature for maximal cellulase activity was 50°C with E _a for substrate hydrolysis of 77.73, 83.97 and 83.14 kJ mol⁻¹ and temperature quotient of 1.0020, 1.0022 and 1.0022 by Trichoderma viride FCBP-142, Tv-UV-5.6 and Tv-Ch-4.3, respectively. The enzyme was stable at 50°C for about 60 min but rapid denaturation occurred above 55°C. The enzyme showed optimum activity at pH 4.0 and involved two types of acidic and basic limbs with pKa₁ and pKa₂. The pKa1 of active site presented a significant shift from 2.55 to 2.9 and 3.1 by Tv-UV-5.6 and Tv-Ch-4.3, respectively in comparison to parental strain. Likewise, pKa₂ moved from 6.05 to 6.5 and 6.4. Enzyme kinetics displayed Michaelis-Menten constant K _m 0.6, 0.5 and 0.28 mg mL⁻¹ and V _max value of 8.33, 10 and 9.09 Units mL⁻¹ for parental, Tv-UV-5.6 and Tv-Ch-4.3, respectively.     

Pistacia integerrima alleviated Bisphenol A induced toxicity through Ubc13/p53 signalling

Molecular Biology Reports - Exposure to environmental toxicants such as Bisphenol A (BPA) has raised serious health issues globally particularly in developing countries. It is ubiquitously used in...     

Water-Pipe Smoking and Albuminuria: New Dog with Old Tricks

Water-pipe (WP) smoking is on rise worldwide for the past few years, particularly among younger individuals. Growing evidence indicates that WP smoking is as harmful as cigarette smoking. To date, most of the research has focused on acute health effects of WP smoking, and evidence remains limited when it comes to chronic health effects in relation to long-term WP smoking. Therefore, the aim of this study was to examine the association between WP smoking and albuminuria in apparently healthy individuals. This analysis was conducted on data of a population-based cross-sectional study—the Urban Rural Chronic Diseases Study (URCDS). The study sample was recruited from three sites in Pakistan. Trained nurses carried out individual interviews and obtained the information on demographics, lifestyle factors, and past and current medical history. Measurements of complete blood count, lipid profile, fasting glucose level, and 24-hour albuminuria were also made by using blood and urine samples. Albumin excretion was classified into three categories using standard cut-offs: normal excretion, high-normal excretion and microalbuminuria. Multiple logistic regression models were used to examine the relationship between WP smoking and albuminuria. The final analysis included data from 1,626 health individuals, of which 829 (51.0%) were males and 797 (49.0%) females. Of 1,626 individuals, 267 (16.4%) were current WP smokers and 1,359 (83.6%) were non-WP smokers. WP smoking was significantly associated with high-normal albuminuria (OR  =  2.33, 95% CI 1.68-3.22, p-value <0.001) and microalbuminuria (OR  =  1.75, 95% CI 1.18-2.58, p-value 0.005) after adjustment for age, sex, BMI, social class, hypertension, and diabetes mellitus. WP smoking was significantly associated with high-normal albuminuria and microalbuminuria when analysis was stratified on hypertension and diabetes mellitus categories. WP smoking has a strong association with albuminuria in apparently healthy individuals. More research is warranted to evaluate the temporality of this association between WP smoking and albuminuria.     

Under-reporting of road traffic mortality in developing countries: application of a capture-recapture statistical model to refine mortality estimates

Road traffic injuries are a major cause of preventable death in sub-Saharan Africa. Accurate epidemiologic data are scarce and under-reporting from primary data sources is common. Our objectives were to estimate the incidence of road traffic deaths in Malawi using capture-recapture statistical analysis and determine what future efforts will best improve upon this estimate. Our capture-recapture model combined primary data from both police and hospital-based registries over a one year period (July 2008 to June 2009). The mortality incidences from the primary data sources were 0.075 and 0.051 deaths/1000 person-years, respectively. Using capture-recapture analysis, the combined incidence of road traffic deaths ranged 0.192-0.209 deaths/1000 person-years. Additionally, police data were more likely to include victims who were male, drivers or pedestrians, and victims from incidents with greater than one vehicle involved. We concluded that capture-recapture analysis is a good tool to estimate the incidence of road traffic deaths, and that capture-recapture analysis overcomes limitations of incomplete data sources. The World Health Organization estimated incidence of road traffic deaths for Malawi utilizing a binomial regression model and survey data and found a similar estimate despite strikingly different methods, suggesting both approaches are valid. Further research should seek to improve capture-recapture data through utilization of more than two data sources and improving accuracy of matches by minimizing missing data, application of geographic information systems, and use of names and civil registration numbers if available.     

Socio-Economic Inequalities in Survival of Patients with Prostate Cancer: Role of Age and Gleason Grade at Diagnosis

In the United Kingdom, survival of prostate cancer patients has improved since the 1990s. A deprivation gap in survival (better survival for the least deprived compared with the most deprived) has been reported but it is not known if differential distribution of earlier age or lower grade disease at diagnosis might explain such patterns. We therefore investigated the impact of age and Gleason grade at diagnosis on the deprivation gap in survival of prostate cancer patients over time. Incident cases of prostate cancer (ICD-10 C61) from the West of Scotland were extracted from the Scottish Cancer Registry from 1991 to 2007. Socio-economic circumstances were measured using the Scottish Index for Multiple Deprivation 2004 (SIMD). Age and deprivation specific mortality rates were obtained from the General Registrar Office for Scotland (GRO(S)). The survival gradient across the five deprivation categories was estimated with linear regression, weighted by the variance of the relative survival estimate. We examined the data for 15,292 adults diagnosed with prostate cancer between 1991 and 2007. Despite substantial improvements in survival of prostate cancer patients, a deprivation gap persists throughout the three periods of diagnoses. The deprivation gap in five year relative survival widened from −4.76 in 1991–1996 to −10.08 in 2003–2007. On age and grade-specific analyses, a significant deprivation gap in five year survival existed between all age groups except among patients'' age ≥75 and both low and high grade disease. On multivariate analyses, deprivation was significantly associated with increased excess risk of death (RER 1.48, 95% CI 1.31–1.68, p-value<0.001) independent of age, Gleason grade and period of diagnosis. The deprivation gap in survival from prostate cancer cannot be wholly explained by socio-economic differentials in early detection of disease. Further research is needed to understand whether differences in comorbidities or treatment explain inequalities in prostate cancer outcomes.     

Sulfonate and sulfamate derivatives possessing benzofuran or benzothiophene nucleus as potent carbonic anhydrase II/IX/XII inhibitors

In the current work, we report the discovery of new sulfonate and sulfamate derivatives of benzofuran- and benzothiophene as potent inhibitors of human carbonic anhydrases (hCAs) II, IX and XII. A set of derivatives, 1a–t, having different substituents on the fused benzofuran and benzothiophene rings (R = alkyl, cyclohexyl, aryl, NH₂, NHMe, or NMe₂) was designed and synthesized. Most of the derivatives exhibited higher potency than acetazolamide as inhibitors of the purified hCAII, IX and XII isoforms. The most potent inhibitors for hCAII, hCAIX and hCAXII were 1g, 1b and 1d with an IC₅₀ ± SEM values of 0.14 ± 0.03, 0.13 ± 0.03 and 0.17 ± 0.06 µM, respectively. In addition, compounds 1d and 1n exerted preferential inhibitory effect against hCAXII isozyme with good potencies. Some selected compounds were docked within the active pocket of these isozymes and binding of the molecules revealed that sulfonate and sulfamate rings were located towards the active cavity and compounds coordinated to zinc ions.     

Evaluation of an Intranasal Virosomal Vaccine against Respiratory Syncytial Virus in Mice: Effect of TLR2 and NOD2 Ligands on Induction of Systemic and Mucosal Immune Responses

Introduction

RSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2) ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine.

Objective

To explore if intranasal (IN) immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity.

Methods

We produced RSV-virosomes carrying TLR2 (Pam₃CSK₄) and/or NOD2 (L18-MDP) ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice.

Results

Incorporation of Pam₃CSK₄ and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting) cells in vitro, as demonstrated by NF-κB induction. In vivo, incorporation of Pam₃CSK₄ in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam₃CSK₄-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam₃CSK₄/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced disease.

Conclusion

Mucosal immunization with RSV-virosomes, supplemented with incorporated TLR2- and/or NOD2-ligands, represents a promising approach to induce effective and safe RSV-specific immunity.     

Sensitivity to house dust mite allergens and prevalence of allergy-causing house dust mite species in Pothwar,Pakistan

This study is the first report on the epidemiological status of house dust mite (HDM) allergy in Pothwar, Pakistan. Allergy data of 2087 symptomatic patients were obtained, of whom 1706 (81.7%) patients were skin-prick-test positive for HDM allergens. This percentage was significantly higher than for pollen and food allergens. In the results of this study Dermatophagoides farinae (61%) and D. pteronyssinus (29%) were the predominant species in the study area. Besides these pyroglyphids, predatory Cheyletus sp. (10%) and an oribatid mite sp. (1%) were also observed. Random and patients’ houses showed 87.4 and 87.1% positive mite infestation, respectively. Mean (±?SEM) D. farinae counts per g of dust in random samples was 235.4 ± 7.93 compared to 274.7 ± 10.78 from patients’ homes. Mean D. pteronyssinus counts from random houses compared to patients’ houses were 115.0 ± 4.57 and 124.6 ± 5.76, respectively. Mite counts depicted seasonal variation, with peaks during monsoon season. ELISA results of dust samples demonstrated that of the dust samples with?>?10 µg/g of dust, the threshold value described as a risk factor for developing asthma, 57.6% had Der f1 and 20% Der p1 allergen load. Mean Der f1 burden was significantly higher than Der p1, with maximum levels during monsoon and autumn seasons. This research established a better awareness about the epidemiological status of HDM allergy and prevalence of allergy causing HDM species in Pakistan.     

As(V) Reduction,As(III) Oxidation,and Cr(VI) Reduction by Multi-metal-resistant Bacillus subtilis,Bacillus safensis,and Bacillus cereus Species Isolated from Wastewater Treatment Plant

Industrial progress has resulted in threatening concentrations of toxic metals in various areas of the world. Bioremediation is an economical alternative to chemical methods. Bacteria resistant to As(III), As(V), Cr, Co, Cu, Cd, Hg, Ni, Pb, Se, and Zn were isolated from the wastewater treatment plant of Kasur, Pakistan. Highest resistance against all metals was exhibited by MX-1, MX-3, MX-4, and MX-5. The isolates possessed dual ability to oxidize as well as to reduce As. Highest As(V) reduction (454 µM) was exhibited by MX-1, while most As(III) oxidation was shown by MX-3 (170 µM). The isolates were also capable of reducing Cr(VI), and maximum Cr(VI) reduction (500 µM) was exhibited by MX-3. Transformation of DH5α with MX-1 plasmid showed that resistance genes for As(III), As(V), Cr, Cd, Se, Hg, and Ni were plasmid borne, while in case of MX-3, resistance genes for As(III), As(V), Co, Cu, Se, Pb, Zn, and Ni were present on plasmid. MX-1 and MX-3 were also positive for auxin production (37 and 32.96 µg ml^?1, respectively). MX-3 was also found to produce hydrogen cyanide (HCN) and solubilized phosphate. These isolates promoted plant (Vigna radiata) growth both in the presence and absence of the metals. MX-1, MX-3, and MX-5 were identified as Bacillus subtilis, Bacillus safensis, and Bacillus cereus through 16S rRNA gene sequencing, respectively. Such bacteria having multiple traits of resisting multiple metals, dual ability to oxidize/reduce As, and reduce Cr(VI) along with the ability to support plant growth are good tools for remediation of metal-contaminated sites and its cultivation.     

Phytochemical and Biological Activities of Pseudocalymma elegans: A False Garlic

Evaluation of phytochemical constituents and antioxidant and antimicrobial activities of hexane (PELH), dichloromethane (PELDCM), ethyl acetate (PELEA), and MeOH (PELM) extracts of young leaves of Pseudocalymma elegans have been carried out. Moreover, extracts have also been explored for the presence of sulphur containing compounds, 1,2‐dithiolane ( 33 ), diallyl disulfide ( 35 ), 3‐vinyl‐1,2‐dithiacyclohex‐5‐ene ( 37 ), and diallyl trisulfide ( 38 ) responsible for the garlic like smell of P. elegans. All the extracts were found to be antioxidant and showed potent inhibition with IC₅₀ values of 0.168 ± 0.001, 0.128 ± 0.002, 0.221 ± 0.011, and 0.054 ± 0.001, respectively, as compared to standard drugs ascorbic acid (AA) and butylated hydroxytoluene (BHT). The ethyl acetate extract (PELE) showed excellent activities against few Gram‐positive and Gram‐negative bacteria and some fungi as compared with standard drug ceftriaxone (3rd generation cephalosporin) and nystatin, respectively. Chemical constituents of hexane, dichloromethane, and ethyl acetate extracts were identified by gas chromatography‐mass spectrometry and mass spectral library search. Over all 55 chemical constituents were first time identified from the leaves which included branched and n‐hydrocarbons, fatty acids, fatty acid methyl esters, fatty alcohols, terpenes, alkaloid, vitamins, glycosides, aromatic compounds, and sulfur containing compounds. Two known chemical constituents, ursolic acid ( 1 ) and β‐amyrin ( 2 ), were also purified for the first time from the MeOH extract. To elucidate the structures of these compounds, UV, IR, EI‐MS, ¹H‐ and ¹³C‐NMR spectroscopy were used.