Sterol glycosyltransferases required for adaptation of Withania somnifera at high temperature

Heat is a major environmental stress factor that confines growth, productivity, and metabolism of plants. Plants respond to such unfavorable conditions through changes in their physiological, biochemical and developmental processes. Withania somnifera, an important medicinal plant, grows in hot and dry conditions, however, molecular mechanisms related to such adaptive properties are not known. Here, we elucidated that members of the sterol glycosyltransferases (SGT) gene family play important roles in the survival of W. somnifera under adverse conditions through maintaining the integrity of the membrane. SGTs are enzymes involved in sterol modifications and participate in metabolic flexibility during stress. Silencing of WsSGT members, for instance WsSGTL1, WsSGTL2 and WsSGTL4, was inimical for important physiological parameters, such as electron transport rate, photochemical quantum yield, acceptor side limitation, non‐photochemical quenching (NPQ), Fv/Fm and net photosynthetic rate, whereas stomatal conductance, transpiration rate and dark respiration rates (Rds) were increased. Decreased NPQ and increased Rds helped to generate significant amount of ROS in the Wsamisgt lines. After heat stress, H₂O₂, lipid peroxidation and nitric oxide production increased in the Wsamisgt lines due to high ROS generation. The expression of HSPs in Wsamisgt lines might be involved in regulation of physiological processes during stress. We have also observed increased proline accumulation which might be involved in restricting water loss in the Wsamisgt lines. Taken together, our observations revealed that SGTL enzyme activity is required to maintain the internal damages of the cell against high temperature by maintaining the sterol vs sterol glycosides ratio in the membranes of W. somnifera.     

Selenium supplementation influences growth performance, antioxidant status and immune response in lambs

To investigate and compare the effect of inorganic and organic Se supplementation, 18 male lambs (24.68 ± 2.89 kg mean body weight, about 8–9 months of age) were divided into three groups of six animals in each, following randomized block design. While animals in the control group (Gr I) were fed a standard TMR containing 195 g/kg crushed maize grain, 175.5 g/kg soybean meal, 260 g/kg wheat bran, 13 g/kg mineral mixture (without Se), 6.5 g/kg common salt and 350 g/kg wheat straw, animals in Gr II and Gr III were additionally supplemented with 0.15 mg Se/kg of diet through sodium selenite (inorganic Se) and Jevsel-101 (organic Se), respectively. Experimental feeding was done for a period of 90 days. To assess the humoral immune response, all the lambs were intramuscularly inoculated with a single dose (2 mL) of Haemorrhagic septicaemia oil adjuvant vaccine on day 0; and blood samples were collected on day 0, 30, 60 and 90. Supplementation of Se had no effect on serum total cholesterol, total protein, albumin, globulin, albumin:globulin ratio, T₃, T₄, T₄:T₃ ratio; serum Ca and P levels and SGOT and SGPT activity. However, there was a significant increase in the serum Se level, RBC GSH-Px activity and humoral immune response in both the Se supplemented groups as compared to control group. Average daily gain (g) was highest (110) in Gr III, followed by Gr II (98.2) and lowest in Gr I (89.1). Thus, supplementation of organic as well as inorganic Se was found to improve the growth rate, humoral immune response and antioxidant status of the lambs; and between two sources, organic Se was more effective than inorganic Se.     

Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli

Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. We show that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, we show that a compromise in the RRF activity affords increased initiation with a mutant tRNA^fMet wherein the three consecutive G-C base pairs (₂₉GGG₃₁:₃₉CCC₄₁), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNA^Met (₂₉UCA₃₁:₃₉ψGA₄₁). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. We discuss these and earlier findings to propose that RRF plays a crucial role during all the steps of protein synthesis.     

Synthesis and antibacterial evaluation of isoxazolinyl oxazolidinones: Search for potent antibacterial

A series of (5S) N-(3-{3-fluoro-4-[4-(3-aryl-4,5-dihydro-isoxazole-5-carbonyl)-piperazin-1-yl]-phenyl}-2-oxo-oxazolidin-5-ylmethyl)-acetamide(6a–o) were synthesized and their in vitro antibacterial activity against various resistant Gram-positive and Gram-negative bacteria were evaluated. Most of the synthesized compounds showed 2 to 10 fold lower MIC values compared to linezolid against Staphylococcus aureus ATCC 25923, ATCC 70069, ATCC 29213, Bacillus cereus MTCC 430, Enterococcus faecalis MTCC439, Klebsiella pneumoniae ATCC 27736, and Streptococcus pyogens.     

Cross-transferability and Polymorphic Potential of Genomic STMS Markers of Brassica Species

The present study was carried out with the objective of evaluating genomic STMS markers developed earlier in Brassica napus, B. oleracea, B. rapa and B. nigra for their use in Brassica juncea and B. carinata. Ninety-six of the 100 STMS markers used under standardized annealing temperatures and gel concentrations produced clear reproducible amplification pattern. For majority of the markers 60 °C annealing temperature and 3.5% metaphor agarose gel were found suitable. High cross-transferability of STMS markers to related Brassica species including B. carinata (91.6%) and B. juncea (87.5%) suggested the possibility of utilizing these markers for genome analysis in the species where no such markers are available. The ‘B’ genome derived markers showed lower level of transferability to the ‘A’ and ‘C’ genome Brassica species. The potential of STMS markers to detect polymorphism among Brassica species and genera was 98.9%. The level of inter-specific polymorphism was much higher than the intea-specific polymorphism. The markers capable of revealing polymorphism among Brassica species and genera would be useful in Brassica introgression breeding programme. The polymorphic markers were found efficient in establishing the expected evolutionary relationships among the six different Brassica species and two related genera. Low level of intra-specific polymorphism revealed by these markers suggested use of a large set of such markers for various applications in Brassica genetics, genomics and breeding.     

Multilocus variable number tandem repeat analysis as a tool to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A

Aims:  To identify variable number tandem repeat (VNTR)-containing loci, and to use multilocus VNTR (MLVA) to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A isolated from diverse sources.
Methods and Results:  The whole genome sequence of Y. enterocolitica 8081 was analysed and eight VNTR loci with repeat sizes between 4 and 9 bp, and each containing more than four repeat copies were selected for MLVA typing of 88 strains of Y. enterocolitica . Of these, four loci were polymorphic and generated 26 MLVA genotypes among 81 strains of Y. enterocolitica biovar 1A. MLVA was found to be quite discriminatory (DI = 0·87). Cluster analysis and population modelling using minimum spanning tree (MST) clearly clustered Y. enterocolitica biovar 1A into two major groups.
Conclusions:  The MLVA is easy to perform and can be used to discern clonal relationship among strains of Y. enterocolitica . Also the phylogenetic relationships obtained with MLVA genotypes were in good agreement with those established by other typing methods.
Significance and Impact of the Study:  The MLVA method reported is relatively more discriminatory than the other genotyping methods and has the potential to be used as an epidemiological tool for the study of strains of Y. enterocolitica biovar 1A.     

Regional stiffening of the mitral valve anterior leaflet in the beating ovine heart

Left atrial muscle extends into the proximal third of the mitral valve (MV) anterior leaflet and transient tensing of this muscle has been proposed as a mechanism aiding valve closure. If such tensing occurs, regional stiffness in the proximal anterior mitral leaflet will be greater during isovolumic contraction (IVC) than isovolumic relaxation (IVR) and this regional stiffness difference will be selectively abolished by β-receptor blockade. We tested this hypothesis in the beating ovine heart. Radiopaque markers were sewn around the MV annulus and on the anterior MV leaflet in 10 sheep hearts. Four-dimensional marker coordinates were obtained from biplane videofluoroscopy before (CRTL) and after administration of esmolol (ESML). Heterogeneous finite element models of each anterior leaflet were developed using marker coordinates over matched pressures during IVC and IVR for CRTL and ESML. Leaflet displacements were simulated using measured left ventricular and atrial pressures and a response function was computed as the difference between simulated and measured displacements. Circumferential and radial elastic moduli for ANNULAR, BELLY and EDGE leaflet regions were iteratively varied until the response function reached a minimum. The stiffness values at this minimum were interpreted as the in vivo regional material properties of the anterior leaflet. For all regions and all CTRL beats IVC stiffness was 40–58% greater than IVR stiffness. ESML reduced ANNULAR IVC stiffness to ANNULAR IVR stiffness values. These results strongly implicate transient tensing of leaflet atrial muscle during IVC as the basis of the ANNULAR IVC–IVR stiffness difference.     

Development and Characterization of 99mTc-timolol Maleate for Evaluating Efficacy of In Situ Ocular Drug Delivery System

In situ gel-forming systems have drawn much attention of current researchers to overcome the poor bioavailability from the conventional eye drops. The present work described formulation and pharmacoscintigraphic evaluation of timolol-maleate-loaded chitosan/hydroxy propyl methyl cellulose (HPMC)-based polymer matrix for enhanced ocular retention. Chitosan and HPMC ratio was optimized and formulation was characterized for various in vitro parameters. The ocular retention was studied on New Zealand rabbits by gamma scintigraphy, which is a very simple and noninvasive technique. For scintigraphy study, the drug timolol maleate was radiolabeled ^99mTc by direct labeling method using SnCl₂·2H₂O as reducing agent. The labeling procedure was optimized to get maximum labeling efficiency (>98%). In vitro stability of the radiolabeled drug (^99mTc-timolol maleate complex) was checked and it was found to be stable for up to 24 h. Plain drug eliminates rapidly as significant activity was recorded in kidney and bladder after 2 h of ocular administration. It was evident from the scintigraphic images and the time–activity curve plotted from the data that the plain drug solution cleared very rapidly from the corneal region and reached into systemic circulation via nasolachrymal drainage system, as significant activity was recorded in kidney and bladder after 2 h of ocular administration. Developed formulation cleared at a slow rate and remained at corneal surface for longer time duration. No radioactivity was observed in systemic circulation after 2 h. Ocular irritation of the developed formulation was also checked by hen’s egg chorioallantoic membrane test and formulation was found to be practically nonirritant. The study signified the potential of gamma scintigraphy in evaluation of novel drug delivery systems in a noninvasive manner.     

Features of SNP and SSR diversity in a set of ICARDA barley germplasm collection

Detection and utilization of genetic variation available in the germplasm collection for crop improvement have been the prime activities of breeders. Here a set of ICARDA barley germplasm collection comprising of 185 cultivated (Hordeum vulgare L.) and 38 wild (H. spontaneum L.) genotypes originated from 30 countries of four continents was genotyped with 68 single nucleotide polymorphism (SNP) and 45 microsatellite or simple sequence repeat (SSR) markers derived from genes (expressed sequence tags, ESTs). As two SNP markers provided 2 and 3 datapoints, a total of 71 SNPs were surveyed that yielded a total of 143 alleles. The number of SSR alleles per locus ranged from 3 to 22 with an average of 7.9 per marker. Average PIC (polymorphism information content) value for SSR and SNP markers were recorded as 0.63 and 0.38, respectively. Heterogeneity was recorded at both SNP and SSR loci in an average of 5.72 and 12.42% accessions, respectively. Genetic similarity matrices for SSR and SNP allelic data were highly correlated (r = 0.75, P < 0.005) and therefore allelic data for both markers were combined and analyzed for understanding the genetic relationships among the germplasm surveyed. Majority of clusters/subclusters were found to contain genotypes from the same geographic origins. While comparing the genetic diversity, the accessions coming from Middle East Asia and North East Asia showed more diversity as compared to that of other geographic regions. Majority of countries representing Africa, Middle East Asia, North East Asia and Arabian Peninsula included the genotypes that contained rare alleles. As expected, spontaneum accessions, as compared to vulgare accessions, showed a higher number of total alleles, higher number of alleles per locus, higher effective number of alleles and higher allelic richness and a higher number of rare alleles were observed. In summary, the examined ICARDA germplasm set showed ample natural genetic variation that can be harnessed for future breeding of barley as climate change and sustainability have become important throughout all growing areas of the world, drought/heat tolerance being the most important ones.