Diet-induced obesity precipitates kidney dysfunction and alters inflammatory mediators in mice treated with Shiga Toxin 2.

Shiga Toxin (Stx)-producing E. coli (STEC) continue to be a prominent cause of foodborne outbreaks of hemorrhagic colitis worldwide, and can result in life-threatening diseases, including hemolytic uremic syndrome (HUS), in susceptible individuals. Obesity-associated immune dysfunction has been shown to be a risk factor for infectious diseases, although few studies have addressed the role of obesity in foodborne diseases. We hypothesized that obesity may affect the development of HUS through an alteration of immune responses and kidney function. We combined diet-induced obese (DIO) and HUS mouse models to look for differences in disease outcome between DIO and wild-type (WT) male and female C57 B l/6 mice. Following multiple intraperitoneal injections with endotoxin-free saline or sublethal doses of purified Stx2, we examined DIO and WT mice for signs of HUS development. DIO mice receiving Stx2 injections lost more body weight, and had significantly higher (p < 0.001) BUN, serum creatinine, and neutrophil counts compared to WT mice or DIO mice receiving saline injections. Lymphocyte counts were significantly (p < 0.05) lower in Stx2-treated obese mice compared to WT mice or saline-treated DIO mice. In addition to increased Stx2-induced kidney dysfunction, DIO mouse kidneys also had significantly increased expression of IL-1α, IL-1ß, IL-6, TNF-α, MCP-1, and KC RNA compared to saline controls (p < 0.05). Serum cytokine levels of IL-6 and KC were also significantly higher in Stx2-treated mice compared to saline controls, but there were no significant differences between the WT and DIO mice. WT and DIO mice treated with Stx2 exhibited significantly higher degrees of kidney tubular dilation and necrosis as well as some signs of tissue repair/regeneration, but did not appear to progress to the full pathology typically associated with human HUS. Although the combined obesity/HUS mouse model did not manifest into HUS symptoms and pathogenesis, these data demonstrate that obesity alters kidney function, inflammatory cells and cytokine production in response to Stx2, and may play a role in HUS severity in a susceptible model of infection.

Purification of lipase from Aspergillus fumigatus using Octyl Sepharose column chromatography and its characterization.

Lipases are ubiquitous biological macromolecules which have many industrial and environmental applications. The purpose of this study was to purify lipase from Aspergillus fumigatus by using Octyl Sepharose column chromatography. The enzyme was purified by ammonium sulfate precipitation and hydrophobic interaction chromatography which resulted in sevenfold purification. The molecular weight of protein using Native-PAGE was found to be 70 kDa. The apparent molecular weight by SDS-PAGE was found to be 35 kDa which indicated that the enzyme was homodimer. The optimum temperature and pH for activity of the enzyme was found to be 40 °C and 9.0, respectively. The kinetic parameters Vmax and Km of the purified lipase were 10.42 µmol min-1 mg-1 and 9.89 mM, respectively. Detergents Tween-20 and Triton X-100 inhibited enzyme activity. However, there was minimal loss of enzyme activity with SDS and Tween-80. All metal ions inhibited the enzyme activity. Among solvents, maximum loss (65%) in the enzyme activity was in hexane.

Isolation, characterization and identification of pesticide degrading bacteria from contaminated soil for bioremediation.

In this study, malathion and chlorpyrifos degrading bacteria were isolated from agricultural soil samples taken from the Himachal region in India. A total of 52 organisms were isolated which were further screened for their efficiency for chlorpyrifos and malathion degradation. Screening was done by checking the growth on Nutrient Agar, Mineral Salt Medium and MacConkey agar plates containing chlorpyrifos and malathion; 37 isolates showed growth in these. Biomass assay and minimum inhibitory concentration (MIC) determination were carried out for the selection of most efficient bacterial isolates. Out of the seven isolates which showed good biomass assay and MIC, only three isolates (PDM-2, PDM-15 and PDM-20) were selected for further studies. These were characterized by various biochemical tests, Gram staining, indole test, methyl red test, Voges-Proskauer test, citrate utilization test and carbohydrate fermentation test. Out of three isolates, PDM-15 showed good resistance against the antibiotics such as erythromycin, chloramphenicol, ampicillin and penicillin and identified as Kocuria assamensis. Degradation of 71.3% of chlorpyrifos and 85% of malathion was observed by the gas chromatography. Therefore, the Kocuria assamensis can be used in the bioremediation of pesticide-contaminated soil.

Purification of high molecular weight thermotolerant esterase from Serratia sp. and its characterization.

In the present study, an extracellular esterase from Serratia sp. was purified 24.46 fold using an initial ammonium sulphate precipitation step (optimized concentration of 30-40%), followed by Diethylaminoethyl cellulose (DEAE-cellulose) chromatography and size exclusion Sephadex G-200 column chromatography steps. The molecular weight of the esterase using native polyacrylamide gel electrophoresis (PAGE) was determined to be 236 kDa and by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was found to be 60 kDa suggesting that the enzyme was a tetramer of 4 subunits. The purified esterase was able to catalyze the hydrolysis of p-nitrophenyl esters, especially p-nitrophenyl acetate. Maximum esterase activity was achieved in 0.15 M Tris-HCl buffer of pH 8.5 at 50 °C after 10 min. The enzyme was stable for at least 8 h at 4 and 35 °C but the half-life was determined to be 4.5 h at 50 °C and 3 h at 60 °C. The esterase activity was inhibited by detergents (1 mM) (Triton X-100, Tween 60, Tween 80, ethylenediamine tetraacetic acid and SDS) except Tween 20. The esterase activity was inhibited by organic solvents (1 mM) such as ethanol, methanol, acetone, acetonitrile and was stable in the presence of glycerol, isopropanol but the organic solvent dimethyl sulfoxide (DMSO) significantly (p < 0.05) enhanced esterase activity. The matrix-assisted laser desorption ionization-time of flight mass spectrometry showed that the enzyme exhibited similarity with the pimeloyl-[acyl carrier protein] methyl ester esterase of Serratia marcescens.

Influence of Culture Conditions on the Production of Extracellular Esterase from Bacillus licheniformis and Its Characterization.

Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. In the present study, thirty-seven bacterial isolates were isolated from soil contaminated with waste cooking oil, dairy waste etc. from Shimla and Solan district of H.P. Out of 37 isolates, the isolate RL-1, which gave maximum activity, was identified as Bacillus licheniformis MH061919. The optimization of various production parameters resulted in maximum activity at inoculum age of 24 h and inoculum size of 1.5% (v/v). Esterase gave considerable activity in production medium containing sodium chloride (0.5 % w/v), galactose (1%, w/v), coconut oil (2.0%, v/v) and beef extract (0.3%, w/v) at a temperature of 45℃ and pH 8.5.The enzyme production was enhanced by 3-fold after optimization of production parameters. Further, on optimizing reaction conditions, enzyme gave maximum activity at a temperature of 45℃ and pH 8.5. The para-nitrophenyl acetate (p-NPA) was found to be optimum substrate and metal ions and detergents have inhibitory effect on esterase activity.

Application of Lipase Purified from Aspergillus fumigatus in the Syntheses of Ethyl Acetate and Ethyl Lactate.

Microbial lipases are used for the synthesis of various short chain esters such as octyl acetate, methyl salicylate, ethyl acetate and ethyl lactate. In this study, a purified lipase of Aspergillus fumigatus was utilized for the synthesis of two esters i.e. ethyl acetate and ethyl lactate. The purified lipase from Aspergillus fumigatus performed esterification of ethanol and acetic acid (at a molar ratio of 1:1) when incubated at 40℃ under shaking (130 min-1) for 12 h resulting in the formation of ethyl acetate (89%). In case of ethyl lactate maximum esterification (87.32%) was achieved when ethanol and lactic acid (500:100 mM ) was used in heptane resulting in the synthesis of ethyl lactate at 40°C under shaking (120 rpm) after 12 h of reaction time. These esters of short chain carboxylic acid and alcohols belong to the highly important natural aroma compounds and are used as green solvents in food and pharmaceutical industry.

Imaging findings in Möbius-Poland syndrome.

Poland syndrome refers to a chest wall disorder in which there is a deficiency of the pectoral musculature. Möbius syndrome is a rare disorder in which there is absence or hypoplasia of the facial or abducens nerve, either unilaterally or bilaterally. Described here is a case in a newborn male in which both conditions manifest simultaneously as Poland-Möbius syndrome. The imaging findings here serve as a useful guide for the radiologist and ordering providers by reinforcing the need for dedicated cranial nerve imaging in patients who have deficiencies in anterior chest wall musculature.

Synthesis of Methyl Butyrate Catalyzed by Lipase from Aspergillus fumigatus.

Lipase is a potential biocatalyst and can be exploited for various applications such as food, pharmaceutical, oleochemistry, organic chemistry, biofuels and in detergent industries. In the present study, lipase from Aspergillus fumigatus was purified to homogeneity by SDS and Native PAGE and evaluated as biocatalyst for the synthesis of methyl butyrate which is a flavor ester. A purification fold of 6.96 was achieved by using Octyl Sepharose column chromatography. Methyl butyrate was synthesized by trans-esterification of vinyl butyrate with methanol, in a medium containing n-hexane as a solvent. The molar ratio of 2:2 (vinyl butyrate:methanol) was found to be optimum for the synthesis of methyl butyrate. The yield of methyl butyrate was maximum when reactants were incubated for 16 h at an incubation temperature of 40°C. The maximum yield (86%) of ester was obtained with 30 µg/ml of purified lipase.

Recurrent painful ophthalmoplegic neuropathy: MRI findings in 2 patients.

Recurrent painful ophthalmoplegic neuropathy is a form of cranial neuralgia and rare source of pediatric headache. We present 2 children who presented with headaches accompanied by visual symptoms including eye pain, blurry vision, and diplopia. MRI in both patients demonstrated enhancement of the cisternal segment of the oculomotor nerve in the affected side, correlating with the observed symptoms.

Teaching and learning of medical biochemistry according to clinical realities: A case study.

To foster medical students to become physicians who will be lifelong independent learners and critical thinkers with healthy skepticism and provide high-quality patient care guided by the best evidence, teaching of evidence-based medicine (EBM) has become an important component of medical education. Currently, the teaching and learning of biochemistry in medical schools incorporates its medical relevance and applications. However, to our knowledge there have been no reports on integrating EBM with teaching and learning medical biochemistry. Here, we present a case study to illustrate the significance of this approach. This case study was based on a biochemistry/nutrition question in a popular board review book about whether a homeless alcoholic man is at risk of developing a deficiency of vitamin E. The possible answers and explanation provided in the book raised a question about the correct answer, which provided us with an opportunity to adapt the philosophy and certain basic EBM principles to find evidence for the clinical applicability of a commonly taught biochemistry topic. The outcome of this case study not only taught us how to conduct an EBM exercise to answer a specific patient question, but also provided us with an opportunity for in-depth teaching and learning of the medical relevance of a specific biochemistry topic based on the best clinical evidence obtained from a systematic research of medical literature.

Isolation of lipase producing thermophilic bacteria: optimization of production and reaction conditions for lipase from Geobacillus sp.

Lipases catalyze the hydrolysis and the synthesis of esters formed from glycerol and long chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. In the present study, thirty-two bacterial strains, isolated from soil sample of a hot spring were screened for lipase production. The strain TS-4, which gave maximum activity, was identified as Geobacillus sp. at MTCC, IMTECH, Chandigarh. The isolated lipase producing bacteria were grown on minimal salt medium containing olive oil. Maximal quantities of lipase were produced when 30 h old inoculum was used at 10% (v/v) in production medium and incubated in shaking conditions (150 rpm) for 72 h. The optimal temperature and pH for the bacterial growth and lipase production were found to be 60°C and 9.5, respectively. Maximal enzyme production resulted when mustard oil was used as carbon source and yeast extract as sole nitrogen source at a concentration of 1% (v/v) and 0.15% (w/v), respectively. The different optimized reaction parameters were temperature 65°C, pH 8.5, incubation time 10 min and substrate p-nitrophenyl palmitate. The Km and Vmax values of enzyme were found to be 14 mM and 17.86 µmol ml-1min-1, respectively, with p-nitrophenyl palmitate as substrate. All metal ions studied (1 mM) increased the lipase activity.