Purification and Characterization of Soluble (Cytosolic) and Bound (Cell Wall) Isoforms of Invertases in Barley (Hordeum vulgare) Elongating Stem Tissue

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (β-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organomercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, K_m of 12 millimolar, and a V_max of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, K_m of millimolar, and a V_max of 9 micromole per minute per milligram of protein.     

Partitioning studies of α-lactalbumin in environmental friendly poly (ethylene glycol)—citrate salt aqueous two phase systems

Aqueous two-phase systems (ATPS) formed by polymer and salt have been utilized to enrich the desired biomolecule into one of the phase with higher yield and purity. The eco-friendly, biodegradable poly ethylene glycol (PEG) and different citrate salts were chosen as ATPS phase components to investigate the partitioning behavior of α-lactalbumin (α-La). System factors and process parameters such as type and concentration of salt, molecular weight and concentration of PEG, pH, temperature and the effect of additives were studied and the results are discussed in detail. PEG 1000–tri-potassium citrate system yields high partition coefficient of 20 with a better yield of 98 % in the top phase. The addition of NaCl as an additive and acidic pH lowers the yield of α-La in the top phase. Influence of phase volume ratio (V _r) on partitioning was studied and found that the partition coefficient remains almost constant along the tie line. High yield was achieved at a V _r of 3.5 at the tie line length of 50.63 (%, w/w).     

Metallic silver nanoparticle: a therapeutic agent in combination with antifungal drug against human fungal pathogen

Silver nanoparticles (Ag-NPs) are known to have inhibitory and fungicidal effects. Resistance against fungal infection has emerged as a major health problem in recent years, which needs great and immediate concern. Here, we report the extracellular biological synthesis of silver nanoparticles through a simple green route approach using a marine mangrove (Rhizophora mucronata) and silver nitrate. Aqueous extract of marine mangrove helped in reduction and was used as capping agent in biological synthesis. Nanoparticles were characterized using microscopy and spectroscopy techniques such as HRTEM, UV–Vis absorption spectroscopy and FTIR spectroscopy. X-ray diffraction analysis showed that the nanoparticles had face centered cubic structure with crystalline nature. FTIR spectroscopy showed the presence of different functional groups, such as hydroxyl and carbonyl, involved in the synthesis of nanoparticles. The antifungal activity of fluconazole and itraconazole was enhanced against the tested pathogenic fungi in the presence of Ag-NP and confirmed from increase in fold area of inhibition. This environmentally friendly method of biological synthesis can be easily integrated for various medical applications.     

Small Intestine Inflammation in Roquin-Mutant and Roquin-Deficient Mice

Roquin, an E3 ubiquitin ligase that localizes to cytosolic RNA granules, is involved in regulating mRNA stability and translation. Mice that have a M199R mutation in the Roquin protein (referred to as sanroque or Roquin^san/san mice) develop autoimmune pathologies, although the extent to which these occur in the intestinal mucosa has not been determined. Here, we demonstrate that Roquin^san/san mice reproducibly develop intestinal inflammation in the small intestine but not the colon. Similarly, mice generated in our laboratory in which the Roquin gene was disrupted by insertion of a gene trap cassette (Roquin^gt/gt mice) had small intestinal inflammation that mimicked that of Roquin^san/san mice. MLN cells in Roquin^san/san mice consisted of activated proliferating T cells, and had increased numbers of CD44^hi CD62L^lo KLRG1⁺ short-lived effector cells. Proportionally more small intestinal intraepithelial lymphocytes in Roquin^san/san mice expressed the ICOS T cell activation marker. Of particular interest, small intestinal lamina propria lymphocytes in Roquin^san/san mice consisted of a high proportion of Gr-1⁺ T cells that included IL-17A⁺ cells and CD8⁺ IFN-γ⁺ cells. Extensive cytokine dysregulation resulting in both over-expression and under-expression of chemotactic cytokines occurred in the ileum of Roquin^san/san mice, the region most prone to the development of inflammation. These findings demonstrate that chronic inflammation ensues in the intestine following Roquin alteration either as a consequence of protein mutation or gene disruption, and they have implications for understanding how small intestinal inflammation is perpetuated in Crohn''s disease (CD). Due to the paucity of animal models of CD-like pathophysiology in the small intestine, and because the primary gene/protein defects of the Roquin animal systems used here are well-defined, it will be possible to further elucidate the underlying genetic and molecular mechanisms that drive the disease process.     

Incidence and Prevalence of Tuberculosis among Household Contacts of Pulmonary Tuberculosis Patients in a Peri-Urban Population of South Delhi,India

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of mortality and morbidity across all age groups throughout the world, especially in developing countries.

Methodology/Principal Findings

In this study, we have included 432 open index cases with their 1608 household contacts in a prospective cohort study conducted from May 2007 to March 2009. The follow-up period was 2 years. All Index cases were diagnosed on the basis of suggestive signs and symptoms and sputum being AFB positive. Among the 432 index patients, 250 (57.9%) were males and 182 (42.1%) females; with mean age of 34±14.4 yr and 26±11.1 yr, respectively. Out of 1608 household contacts, 866 (53.9%) were males and 742 (46.1%) females; with mean age of 26.5±15.8 and 26.5±16.0 yr, respectively. Of the total 432 households, 304 (70.4%) had ≤4 members and 128 (29.6%) had ≥5 members. The median size of the family was four. Of the 1608 contacts, 1206 were able to provide sputum samples, of whom 83 (6.9%) were found MTB culture positive. Household contacts belonging to adult age group were predominantly (74, 89.2%) infected as compared to the children (9, 10.8%). On screening the contact relationship status with index patients, 52 (62.7%) were first-degree relatives, 18 (34.6%) second-degree relatives and 12 (14.5%) spouses who got infected from their respective index patients. Co-prevalent and incident tuberculosis was found in 52 (4.3%) and 31 (2.6%) contacts, respectively. In incident cases, the diagnosis could be made between 4 to 24 months of follow-up, after their baseline evaluation.

Conclusion

Active household contact investigation is a powerful tool to detect and treat tuberculosis at early stages and the only method to control TB in high-TB-burden countries.     

Modes of neuronal migration in the developing cerebral cortex

The conventional scheme of cortical formation shows that postmitotic neurons migrate away from the germinal ventricular zone to their positions in the developing cortex, guided by the processes of radial glial cells. However, recent studies indicate that different neuronal types adopt distinct modes of migration in the developing cortex. Here, we review evidence for two modes of radial movement: somal translocation, which is adopted by the early-generated neurons; and glia-guided locomotion, which is used predominantly by pyramidal cells. Cortical interneurons, which originate in the ventral telencephalon, use a third mode of migration. They migrate tangentially into the cortex, then seek the ventricular zone before moving radially to take up their positions in the cortical anlage.     

Enhanced transformation of polycyclic aromatic hydrocarbons using a combined Fenton's reagent,microbial treatment and surfactants

The potential for using Fenton's reagent (H₂O₂ + Fe²⁺) as an advanced oxidation pretreatment process to enhance microbial transformation of two model polycyclic aromatic hydrocarbons, anthracene and benzo[a]pyrene, in an aqueous system was evaluated. Fenton's reagent at a concentration of 0.5% H₂O₂ and 10 mM Fe²⁺ (molar ratio, 15:1) was most effective in transforming anthracene at pH 4. Application of non-ionic surfactants during Fenton's pre-treatment was found to be more effective in the transformation of both anthracene and benzo[a]pyrene. The extent of removal of substrates by a combined Fenton's–biotreatment was 2–4 times higher than with Fenton's treatment or biotreatment alone. In a chemical–biological treatment train, 48 h of Fenton's pre-treatment in the presence of a non-ionic surfactant, followed by 7 days of biological treatment resulted in 80–85% removal of PAHs (100 ppm). Electronic Publication     

Mapping of porcine genetic markers generated by representational difference analysis

Representational difference analysis (RDA) was performed using pig genomic DNA from a Landrace non-selected control population and a Landrace population selected for increased loin muscle area (LMA) for five generations. Pigs used for the analysis differed phenotypically for various carcass traits and were divergent in genotype at the skeletal muscle ryanodine receptor 1 locus. Two RDA experiments were performed using BamHI and BglII. Fourteen BamHI and 37 BglII difference products were cloned and sequenced. Oligonucleotide primers were designed to amplify RDA difference products and sequence-tagged sites (STS) were developed for 16 RDA fragments (two BamHI and 14 BglII). These 16 STS were mapped using the INRA-Minnesota porcine Radiation Hybrid panel. Polymorphisms identified in nine of the STS were used to place these markers on the PiGMaP genetic linkage map. Sequence-tagged sites were localized to 11 different chromosomes including three markers on chromosome 11 and four markers on chromosome 14. Development of RDA markers increases the resolution of the pig genome maps and markers located within putative quantitative trait locus (QTL) regions can be used to refine QTL positions.     

Prevalence of Myopia and Its Risk Factors in Urban School Children in Delhi: The North India Myopia Study (NIM Study)

PurposeAssess prevalence of myopia and identify associated risk factors in urban school children.MethodsThis was a cross-sectional study screening children for sub-normal vision and refractive errors in Delhi. Vision was tested by trained health workers using ETDRS charts. Risk factor questionnaire was filled for children with vision <6/9.5, wearing spectacles and for a subset (10%) of randomly selected children with normal vision. All children with vision <6/9.5 underwent cycloplegic refraction. The prevalence of myopia <-0.5 diopters was assessed. Association of risk factors and prevalence of myopia was analyzed for children with myopia and randomly selected non myopic children and adjusted odds ratio values for all risk factors were estimated.ResultsA total number of 9884 children were screened with mean age of 11.6 + 2.2 years and 66.8% boys. Prevalence of myopia was 13.1% with only 320 children (24.7%) wearing appropriate spectacles. Mean myopic spherical error was -1.86 + 1.4 diopters. Prevalence of myopia was higher in private schools compared to government schools (p<0.001), in girls vs. boys (p = 0.004) and among older (> 11 years) children (p<0.001). There was a positive association of myopia with studying in private schools vs. government schools (p<0.001), positive family history (p< 0.001) and higher socio-economic status (p = 0.037). Positive association of presence of myopia was observed with children studying/reading > 5 hours per day (p < 0.001), watching television > 2 hours / day (p < 0.001) and with playing computer/video/mobile games (p < 0.001). An inverse association with outdoor activities/playing was observed with children playing > 2 hours in a day.ConclusionMyopia is a major health problem in Indian school children. It is important to identify modifiable risk factors associated with its development and try to develop cost effective intervention strategies.