DEVELOPMENT AND VALIDATION OF RP- HPLC METHOD FOR METFORMIN HYDROCHLORIDE AND NATEGLINIDE IN BULK AND COMBINED DOSAGE FORM

Objective: To develop an accurate, precise and linear RP-HPLC method for simultaneous quantitative estimation of Metformin hydrochloride and Nateglinide in tablets and validate as per ICH guidelines.Methods: The method used a reverse phase column, Inertsil C18-ODS 3V (250×4.6 mm, 5 µm), a mobile phase comprising of phosphate buffer (pH 4.0): Acetonitrile: methanol (30:60:10) flow rate of 1.0 ml/min and a detection wavelength of 221 nm using a UV detector.Results: The developed method resulted in elution of Metformin hydrochloride at 2.45 min and Nateglinide at 4.21 min. The calibration curves were linear (r²=0.999) in the concentration range of 60-140 µg/ml and 14.4-33.2 µg/ml for Metformin hydrochloride and Nateglinide respectively. The percentage recoveries were found to be 99.59-101.36 for Metformin hydrochloride and 98.43-101.38 for Nateglinide. The LOD was found to be 2.18 µg/ml and 1.55 µg/ml for Metformin hydrochloride and Nateglinide respectively. LOQ was found to be 8.52µg/ml and 4.69µg/ml for Metformin hydrochloride and Nateglinide respectively.Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of Metformin hydrochloride and Nateglinide in bulk and pharmaceutical formulation and validated as per ICH guidelines. Hence, the method holds good for the routine analysis of Metformin hydrochloride and Nateglinide in various pharmaceutical industries as well as in academics.Keywords: Metformin hydrochloride, Nateglinide, RP-HPLC, Method development, Validatio

Citizen science, treatment and microbial compliance monitoring in rainwater harvesting in Namibia

Expected release date-April 202

Trophic relationships of hake (Merluccius capensis Castelnau, 1851 and M. paradoxus Franca 1960) from the Northern Benguela current ecosystem (Namibia) : inferences from stable isotopes and fatty acids

Two species of hake (Merluccius capensis and Merluccius paradoxus) account for most of Namibia’s fisheries catch, and they are important secondary consumers in the Benguela Current ecosystem. Inferences on their trophic relationships have been based mainly on stomach content analyses. However, such data are limited temporally because they represent only snapshots of recent feeding, and are quantitatively biased because of variation in the digestion rates of different prey. The principal aim of the thesis was to understand the trophic relationships of two hake species relative to each other, their known prey and top predators (demersal sharks) in the northern Benguela Current ecosystem (Namibia), using time-integrating trophic biomarkers. By using stable isotope (carbon and nitrogen) and fatty acid signatures of their muscle tissues, my overall objectives were to produce new knowledge about 1) hake ontogenic trophic relationships, 2) the contributions of different prey to hake diets, 3) hake dietary differences, and 4) some aspects of hake’s trophic relationships with demersal sharks. Tissues of hake (n=358), their potential prey (n=455), and demersal sharks (n=42) were collected between 2008 and 2012 during demersal bottom trawl surveys off Namibia, for stable isotope and fatty acid analyses. And more..

ESTIMATED DAILY INTAKE AND EXPOSURE OF SODIUM BENZOATE AND POTASSIUM SORBATE THROUGH FOOD PRODUCTS IN SCHOOL CHILDREN OF TIRUPATI, INDIA

Objective: According to Food Safety and Standards Authority of India (FSSAI) the preservatives, sodium benzoate and potassium sorbate belong to permitted class II preservatives. The aim of this study is to determine the concentration levels of these preservatives in food products that are consumed by school children and to assess the chronic dietary exposure by conducting the Total Diet Study (TDS).Methods: The quantitative determination was carried out by UV spectrophotometer. The absorbance for sodium benzoate and potassium sorbate were measured at 228 and 250 nm respectively. The 24-hour diet recall method was used to estimate the amount of food ate in last 24 hours. For estimation of preservative exposure dietary modelling techniques were utilized which combine the amount of preservative concentration present in that food with the amount of food consumed. Then the dietary exposure was assessed by considering the Acceptable Daily Intake (ADI).Results: The results include chemical concentration levels of the foods analyzed as well as estimated dietary exposures and contributions to the exposure from different foods. The obtained mean concentration of sodium benzoate was found to be425 ppm for sauces, 161 ppm for pickles and 80 ppm for soft drinks. Potassium sorbate was found to be 130 ppm for fruit juices,302 ppm for jellies and 380 ppm for jams. The highest mean dietary exposure for both the preservatives was observed in children of 2-7 years age group, the percentage exposure of sodium benzoate was 33% of the ADI and potassium sorbate was 17 % of the ADI.Conclusion: This study can enlighten the public on the consumption of preservative containing food products within the limit and encourages to eating fresh preservative free foods.Â

Is it feasible to integrate alcohol-related risk reduction counseling into VCT services? Findings from Kenya

In Kenya, an estimated 30 percent of men report to be alcohol users which may be fueling the HIV pandemic, due to its association with high-risk sexual behavior and reduced inhibitions. The Population Council’s Horizons program collaborated on a study to improve screening of clients for alcohol use, provision of feedback of screening results, and referrals to care and support during voluntary testing and counseling (VCT) for HIV. Overall, the results indicate that it is feasible to integrate alcohol risk reduction counseling into VCT, and that it is generally accepted by providers and clients alike. This study therefore supports the formal integration of alcohol risk reduction counseling into VCT services in Kenya

The feasibility of integrating alcohol risk-reduction counseling into existing VCT services in Kenya

This pretest-posttest separate-sample study with intervention and comparison groups documented the abilities and willingness of trained voluntary counseling and testing (VCT) service providers to integrate alcohol screening and risk reduction counseling into their routine service delivery. Pre-test (n=1073) and post-test data (n=1058) were collected from different clients exiting from 25 VCT centers. A 12-month intervention that required all VCT providers from the intervention groups to screen all VCT clients for their alcohol use and offer them brief risk reduction alcoholrelated counseling was implemented. At post-test, the intervention group clients (n=456) had better study outcomes than the comparison group clients (n=602). Intervention clients were more likely to report that their VCT service provider had: asked them about their alcohol use (83% vs. 41%:

Conservation of Garcinia imberti Bourd. through seeds

Garcinia imberti seeds were collected during 2015-2017 from Shangili, Cheenikkala and Bonaccord evergreen forests of Agasthyamala Biosphere Reserve, the only abode of this endangered endemic species. Germinability of seeds were analysed through decoating, Gibberellic acid (GA3) and light inductive pre-treatments on fresh (62.8 % moisture content; MC) and desiccated (fast; 23.3% MC and slow; 30.5 % MC) seeds. The seed germination with impermeable coat (0.7-1.2 mm) was restricted which on decoating got enhanced. Application of GA3 along with exposure to light breaked dormancy within 4-6 days compared to non-treated seeds that took 238-254 days to germinate. Stored seeds behaviour revealed that seed moisture content and rate of germination were negatively correlated. Seed storage was found to be more efficient only up to 80 days at controlled seed banking conditions (20 ± 20C, 20 % relative humidity; RH). Both fast and slow desiccated seeds stored for 60 days in seed bank conditions exhibited 50.4 and 43.4 % of germination compared 39.4% germination of non-desiccated seeds. Hence fast desiccated and decoated G. imberti seeds pre-treated with GA3 on subsequent exposure to light alleviated dormancy. For seed banking, fast desiccated seeds with MC in between 40-20% are found to be promising

Production of mycobacterial cell wall glycopeptidolipids requires a member of the MbtH-like protein family

Background Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production. Results Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility. Conclusions Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen

Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA

The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an alpha/beta architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.This work was supported by the European Commission Contract HEALTH-F3-2011-260872, the Spanish Ministry of Economy and Competitiveness Contract BIO2013-49022-C2-2-R, and the Basque Government (to M.E.G.); Slovak Research and Development Agency Contract No. DO7RP-0015-11 (to K.M.) and the NIH/NIAID grant AI064798 (to M.J.). D.A.-J. acknowledges the support from Fundacion Biofisica Bizkaia. We gratefully acknowledge Sonia Lopez-Fernandez (Unit of Biophysics, CSIC, UPV/EHU, Spain), Drs E. Ogando and T. Mercero (Scientific Computing Service UPV/EHU, Spain) for technical assistance. We thank the Swiss Light Source (SLS), and the Diamond Light Source (DLS) for granting access to synchrotron radiation facilities and their staff for the onsite assistance. We specially thank the BioStruct-X project to support access to structural biology facilities. We also acknowledge all members of the Structural Glycobiology Group (Spain) for valuable scientific discussions. The following reagent was obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, Strain H37Rv, Purified Phosphatidylinositol Mannosides 1 and 2 (PIM1,2), NR-14846