Translational coupling in Escherichia coli of a heterologous Bacillus subtilis-Escherichia coli gene fusion.

The efficient expression in Escherichia coli of the Tn9-derived chloramphenicol acetyltransferase (EC 2.3.1.28) gene fused distal to the promoter and N terminus of the Bacillus subtilis aprA gene was dependent on the initiation of translation from the ribosome-binding site in the aprA gene.     

Influence of sowing windows and genotypes on growth,radiation interception,conversion efficiency and yield of guar

Crop growth largely depends on radiation. Radiation is the main impetus for photosynthesis and movement of photosynthates from source to sink. Therefore, identification of the optimum sowing windows and suitable cultivars for efficient utilization of radiation is of prime importance. A field study was conducted in red clay soil during 2014 and 2015 Kharif season and the treatments consisted of three genotypes and three sowing windows by using randomized complete block design with three replications. The effect of genotypes and sowing windows was found significant with respect to number of trifoliate leaves, leaf area ratio, dry matter production, grain numbers, pod length, test weight, grain yield, and stover yield of guar during 2014 as compared to 2015 sown crop. Statistically significant plant height, number of trifoliate leaves, number of branches, leaf area ratio, absolute growth rate, leaf area index, dry matter, grain number, pod length, grain yield, stover yield and a higher cumulative radiation interception were recorded with 15th August sown crop as compared to other sowing windows. The plant height, number of trifoliate leaves, number of branches, leaf area ratio, absolute growth rate, leaf area index, dry matter, grain number, pod length, grain yield, stover yield and maximum cumulative interception of radiation were significant with RGC-1003 as compared to RGC-936 and HG-365. It is observed that the incident PAR to dry matter accumulation conversion efficiency was varied with cultivars and different sowing windows which ranges from 0.74 g MJ⁻¹ to 0.79 g MJ⁻¹.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

Soil seed bank contributes significantly to genetic variation of Hypericum sinaicum in a changing environment

The contribution of soil seed bank of a desert endemic plant species in maintaining genetic diversity has been addressed in this paper through investigating the differences in genetic diversity and structure (using AFLP markers) between plants grown from soil seed bank and standing crop plants within and among five populations of H. sinaicum growing at St. Katherine Protectorate, southern Sinai, Egypt. Standard genetic diversity measures showed that the molecular variation within and among populations was highly significantly different between standing crop and soil seed bank. While soil seed bank had lower genetic diversity than standing crop populations, pooling soil seed bank with standing crop samples resulted in higher diversity. The results revealed also that soil seed bank had lower differentiation (7 %) than among populations of the standing crop (18 %). Results of neighbor-joining, Bayesian clustering and principal coordinate analysis showed that soil seed banks had a separate gene pool different from standing crop. The study came to the conclusion that the genetic variation of the soil seed bank contributes significantly to the genetic variation of the species. This also stresses the importance of elucidating the genetic diversity and structure of the soil seed bank for any sound and long-term conservation efforts for desert species. These have been growing in small-size populations for a long time that any estimates gained only from aboveground sampling of populations may be ambiguous.     

Designing an immunoinformatic vaccine for peri-implantitis using a structural biology approach

ObjectivesPeri-implantitis is a destructive inflammatory process that affects the soft and hard tissues around dental implants. porphyromonas gingivalis, an anaerobic gram-negative bacterium, appears to be the main culprit. Since there is no efficient and specific vaccine to treat peri-implantitis, the goal of our research has been to develop a multi-epitope vaccination utilizing an immunoinformatics approach that targeted P. gingivalis type I fim A.Materials and methodsP. gingivalis peptides 6JKZ and 6KMF are suitable for vaccine development. B- and T-cell epitopes from 6KMF and 6JKZ were detected and evaluated based on critical factors to produce a multi-epitope vaccine construct. It was assessed based on allergenicity, antigenicity, stability. The vaccine's dual major histocompatibility complex (MHC-I and MHC-II) binding epitopes allowed it to reach a larger population. P. gingivalis fimbriae induce immune subversion through TLR -CXCR4 receptor complex pathway. The ClusPro 2.0 server was used to do the molecular docking using TLR2 - CXCR4 and vaccine epitopes as receptor and ligand respectively.ResultsThe designed vaccine was non-allergenic and had a high antigenicity, solubility, and stability. The 3D structure of the vaccine revealed strong interaction with CXCR4(TLR2) using molecular docking. The vaccine-CXCR4 interface was more consistent, possibly because the vaccination has a higher affinity for the CXCR4-TLR2 complex.ConclusionThis study details the vaccine's distinct and sustained interaction with the CXCR4(TLR2) immunological receptor and its consistent and effective utterance in the bacterial system. As a result, our vaccine formulation will evoke a significant memory response and induce an adaptive immune response against P. gingivalis.     

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.     

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V_max = 2.31 pmol min^?1 U^?1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.     

Osmotic shock: a mechanosensitive channel blocker can prevent release of cytoplasmic but not periplasmic proteins

When over-expressed in the cytoplasm of Escherichia coli, carboxylesterase Est55 of Geobacillus stearothermophilus was found to be released from cells upon osmotic shock. Comparing two osmotic shock protocols showed that release of Est55 was abolished in the absence of mechanosensitive channel MscL by one method but not the other. The discrepancy extended to several previously reported cytoplasmic proteins released by osmotic shock, including: EF-Tu, thioredoxin, and DnaK in E. coli. Stepwise analyses of parameters between these two protocols revealed that the use of mechanical pipetting instead of gentle dilution of cells prior to exposure to hypotonic solution abolished the effect of MscL. Furthermore, while this phenomenon of release of certain cytoplasmic proteins was sustained in all three wild type strains of E. coli, presence of gadolinium was able to serve as an MscL channel blocker and prevented release of Est55 and EF-Tu in the process. An optimized protocol of osmotic shock was developed from this study to provide a more reliable assessment of location of proteins in E. coli. This method allowed release of authentic periplasmic MalE and beta-lactamase proteins comparable to that by EDTA-lysozyme treatment.