Enzymatic glycosylation of the topical antibiotic mupirocin

Mupirocin is a commercially available antibiotic that acts on bacterial isoleucyl-tRNA synthetase, thereby inhibiting protein synthesis and preventing bacterial infection. An in vitro glycosylation approach was applied to synthesize glycoside derivatives of mupirocin using different NDP-sugars and glycosyltransferase from Bacillus licheniformis. Ultra pressure liquid chromatography-photo diode array analyses of the reaction mixtures revealed the generation of product peak(s). The results were further supported by high-resolution quadruple time of flight electrospray ionization mass spectrometry analyses. The product purified from the reaction mixture with UDP-D-glucose was subjected to NMR analysis, and the structure was determined to be mupirocin 6-O-β-D-glucoside. Other glycoside analogs of mupirocin were determined based on high-resolution mass analyses. Antibacterial activity assays against Staphylococcus aureus demonstrated complete loss of antibacterial activity after glucosylation of mupirocin at the 6-hydroxyl position.     

Overexpression of NAC gene from Lepidium latifolium L. enhances biomass,shortens life cycle and induces cold stress tolerance in tobacco: potential for engineering fourth generation biofuel crops

We report elevated biomass and altered growth characteristics of tobacco plants up on transformation with a NAC (NAM, ATAF1/2,CUC2) gene (GenBank Accession FJ754254) isolated from Lepidium latifolium L. (LlaNAC). Transgenic plants showed significant differences in fresh weight, midrib length of longest leaf, leaf area, height of the plant, root and shoot weights, etc. during vegetative phase. On 100th day after sowing (DAS), plants of transgenic lines were 2–3 times taller than the wild type plants, though no significant difference was recorded in moisture contents of any of the plant tissues. Over-expression of NAC gene up to 2,000 fold was recorded in leaves of transgenic plants on 100th DAS. Interestingly, transgenic plants showed significantly shortened (P(t) = 0.02–0.04) life cycle, as they showed a completely altered growth behaviour. Transgenic plants entered reproductive phase earlier by 60 days, with lines NC2 and NC7b entering first, followed by line NC10. However, the time period spent in the reproductive phase by the plant was nearly twice in case of transgenic lines NC2, NC7b and NC10, as compared to the wild type plants. Despite that, these lines completed their life cycle in 45–60 days lesser than the time taken by wild-type tobacco plants. No difference was recorded in fruit and seed yield of transgenic or wild type plants. To the best of our knowledge, this is the first report on over-expression of NAC gene causing altered growth and biomass patterns. We expect this study to become an important reference towards future engineering of plants for fuel and fodder purposes.     

Neuron Specific Enolase and C-reactive Protein Levels in Stroke and Its Subtypes: Correlation with Degree of Disability

Stroke is an emergency which threatens life and third leading cause of death and long term disability in developed countries. The use of biomarkers in diagnosing stroke and assessing prognosis is an emerging and rapidly evolving field. The study aimed to investigate the predictive value of biochemical marker of brain damage neuron-specific enolase (NSE) and systemic inflammatory marker C-reactive protein (CRP) with respect to degree of disability at the time of admission and short term in stroke patients. We investigated 120 patients with cerebrovascular stroke who were admitted within 72 h of onset of stroke in the Department of Neurology at Sri Aurobindo Institute of Medical Sciences, Indore, India. NSE and CRP were analyzed by solid enzyme linked immunosorbent assay using analyzer and micro plate reader from Biorad 680. In all patients, the neurological status was evaluated by a standardized neurological examination and the National Institutes of Health Stroke Scale on admission and on day 7. Serum NSE and CRP concentration were found significantly increased in acute stroke cases as compared to control in present study (<0.05 and <0.001 respectively). The maximum serum NSE and CRP levels within 72 h of admission were significantly higher in patients with greater degree of disability at the time of admission. Both biomarkers were found significantly correlated with neurological disability and short term outcome. Our study showed that serum biomarkers NSE and CRP have high predictive value for determining severity and early neurobehavioral outcome after acute stroke.     

Population structure and genetic diversity in bottle gourd [Lagenaria siceraria (Mol.) Standl.] germplasm from India assessed by ISSR markers

Genetic diversity analysis was undertaken in 42 geographically distant genotypes accessions of bottle gourd (Lagenaria siceraria) from India northeastern (14) and northern region (28) using inter-simple sequence repeat (ISSR) markers. A total of 209 amplified bands were obtained from 20 ISSR primers used in this study, of which 186 were polymorphic with 89.00 % band polymorphism. Various parameters namely, observed number of alleles, effective number of alleles, Nei’s gene diversity/heterozygosity, resolving power, Shannon’s information index and gene flow were estimated under experiment. Jaccard’s similarity coefficient matrix was generated for pairwise comparisons between individual ISSR profiles and UPGMA cluster analysis based on this matrix showed clustering into six groups. Jaccard’s coefficient of similarity values ranged from 0.409 to 0.847, with a mean of 0.628 revealing a moderate level of genetic diversity. The Bayesian model-based approach to infer hidden genetic population structures using the multilocus ISSR markers revealed two populations among the 42 genotypes. This is the first report on the assessment of genetic variation using ISSR markers in this medicinal vegetable plant, and this study of diversity analysis will be helpful in analyzing future hybrid breeding strategy and devising effective germplasm exploration and conservation strategy.     

Genomics and molecular breeding in lesser explored pulse crops: Current trends and future opportunities

Pulses are multipurpose crops for providing income, employment and food security in the underprivileged regions, notably the FAO-defined low-income food-deficit countries. Owing to their intrinsic ability to endure environmental adversities and the least input/management requirements, these crops remain central to subsistence farming. Given their pivotal role in rain-fed agriculture, substantial research has been invested to boost the productivity of these pulse crops. To this end, genomic tools and technologies have appeared as the compelling supplement to the conventional breeding. However, the progress in minor pulse crops including dry beans (Vigna spp.), lupins, lablab, lathyrus and vetches has remained unsatisfactory, hence these crops are often labeled as low profile or lesser researched. Nevertheless, recent scientific and technological breakthroughs particularly the next generation sequencing (NGS) are radically transforming the scenario of genomics and molecular breeding in these minor crops. NGS techniques have allowed de novo assembly of whole genomes in these orphan crops. Moreover, the availability of a reference genome sequence would promote re-sequencing of diverse genotypes to unlock allelic diversity at a genome-wide scale. In parallel, NGS has offered high-resolution genetic maps or more precisely, a robust genetic framework to implement whole-genome strategies for crop improvement. As has already been demonstrated in lupin, sequencing-based genotyping of the representative sample provided access to a number of functionally-relevant markers that could be deployed straight away in crop breeding programs. This article attempts to outline the recent progress made in genomics of these lesser explored pulse crops, and examines the prospects of genomics assisted integrated breeding to enhance and stabilize crop yields.     

KINETIC ASSESSMENT AND EFFECT ON DEVELOPMENTAL PHYSIOLOGY OF A TRYPSIN INHIBITOR FROM Eugenia jambolana (JAMBUL) SEEDS ON Helicoverpa armigera (HÜBNER)

A trypsin inhibitor was purified from the seeds of Eugenia jambolana (Jambul) with a fold purification of 14.28 and a yield recovery of 2.8%. Electrophoretic analysis of E. jambolana trypsin inhibitor (EjTI) revealed a molecular weight of approximately 17.4 kDa on 12% denaturing polyacrylamide gel electrophoresis with or without reduction. EjTI exhibited high stability over a wide range of temperatures (4–80 °C for 30 min) and pH (3.0–10.0) and inhibited trypsin‐like activities of the midgut proteinases of fourth instar Helicoverpa armigera larvae by approximately 86%. Feeding assays containing 0.05, 0.15, and 0.45 (% w/w) EjTI on functionally important fourth‐instar larvae indicated a dose‐dependent downfall in the larval body weight as well as on extent of survival. The nutritional analysis suggests that EjTI exerts toxic effects on H. armigera. Dixon plot analysis revealed competitive inhibition of larval midgut proteinases by EjTI, with an inhibition constant (K_i) of approximately 3.1 × 10^?9 M. However, inhibitor kinetics using double reciprocal plots for trypsin inhibition demonstrated a mixed inhibition pattern. These observations suggest the potential of E. jambolana trypsin inhibitor protein in insect pest management.     

Glucosylation of Isoflavonoids in Engineered Escherichia coli

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-α-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4′ and 7 hydroxyl groups, but not at the 5^th hydroxyl group of the A-ring, resulting in the production of genistein 4′-O-β-D-glucoside, genistein 7-O-β-D-glucoside (genistin), genistein 4′,7-O-β-D-diglucoside, biochanin A-7-O-β-D-glucoside (sissotrin), daidzein 4′-O-β-D-glucoside, daidzein 7-O-β-D-glucoside (daidzin), daidzein 4′, 7-O-β-D-diglucoside, and formononetin 7-O-β-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOFESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli BL21(DE3)/ΔpgiΔzwfΔushA overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of 200 μM of supplemented isoflavonoids, without any additional UDP-α-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.     

Hyaluronic Acid Receptor for Endocytosis (HARE)-mediated Endocytosis of Hyaluronan,Heparin, Dermatan Sulfate,and Acetylated Low Density Lipoprotein (AcLDL), but Not Chondroitin Sulfate Types A,C, D,or E,Activates NF-κB-regulated Gene Expression

The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638–648). Unique intermediate size Select-HA^TM, heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10–25 nm. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by ∼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.     

An Iron–Sulfur Cluster in the Polymerase Domain of Yeast DNA Polymerase ε

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits—Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1–1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2^ins) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.