Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.     

Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance

Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion–cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation.     

Microbial calcium carbonate precipitation with high affinity to fill the concrete pore space: nanobiotechnological approach

Bioprocess and Biosystems Engineering - Despite the advantages of concrete, it has a pore structure and is susceptible to cracking. The initiated cracks as well as pores and their connectivity...     

Development of sensitive benzofurazan‐based spectrometric methods for analysis of spectinomycin in vials and human biological samples

Spectinomycin hydrochloride (SPEC) is an aminoglycoside antibiotic that has a broad spectrum against several bacterial strains of humans and veterinary infections. However, SPEC lacks a fluorophore or chromophore and this lack makes its analysis a challenge. Our study provides a simple, sensitive and low‐cost spectrofluorimetric/spectrophotometric method based on the reaction between secondary amine groups and a benzofurazan reagent using borate buffer (pH 9.2). The yielding compound was measured fluorimetrically at 530 nm (excitation at 460 nm) with colorimetry at 410 nm. The calibration curves ranged from 30 to 400 ng ml^?1 and from 0.2 to 6 μg ml^?1 for spectrofluorimetric and spectrophotometric analyses, respectively. The limits of detection were calculated to be 4.15 ng ml^?1 and 0.05 μg ml^?1 for spectrofluorimetric and spectrophotometric processes, respectively. The ultra‐affectability and high selectivity of the proposed method permitted analysis of SPEC in the dosage form and in human plasma samples, with good recoveries of about 101.19 and 97.11%, respectively, without any matrix interference. The proposed strategy was validated using International Conference on Harmonization standards and validated bioanalytically using USFDA recommendations.     

Family Ismaridae Thomson (Hymenoptera,Diaprioidea), new to fauna of Iran

Occurrence of the family Ismaridae Thomson is recorded for the first time from Iran, represented by a single species, Ismarus rugulosus Förster, 1850. A series of diagnostic characters and morphometric ratios for this species is presented, as well as a distribution map throughout the Holarctic region.     

Design and total synthesis of (-)-codonopsinine, (-)-codonopsine and codonopsinine analogues by O-(2-oxopyrrolidin-5-yl)trichloroacetimidate as amidoalkylating agent with improved antimicrobial activity via solid lipid nanoparticle formulations

A general strategy towards total synthesis of (-)-codonopsinine, (-)-codonopsine and codonopsinine analogues has been developed from (D)-tartaric acid via the intermediate (3S,4R)-1-methyl-2-oxo-5-(2,2,2-trichloroacetamido)pyrrolidinediacetate (7). α-amidoalkylation studies of 7 with electron rich benzene derivative 8a-g as C-nucleophiles afforded (aryl derivatives) 9a-g. The target compounds 1, 2 and 13c-g were readily obtained from 10a-g via Grignard addition to the homochiral lactam which was produced by deoxygenation using Lewis-acid followed by deacetylation. The synthesized compounds were loaded onto solid lipid nanoparticle formulations (SLNs) prepared by hot emulsification-ultrasonication technique using Compritol as solid lipid and Pluronic f68 as surfactant. SLNs were fully evaluated and the permeation of synthesized compound from SLNs was assayed against non-formulated compounds through dialysis membranes using Franz cell. The data indicated good physical characteristics of the prepared SLNs, sustaining of release profiles and significant improvement of permeation ability when compared to the non-formulated compounds. The antibacterial and antifungal activities of 1, 2 and 13c-g were determined by disc diffusion and microbroth dilution method to determine the minimum inhibitory concentrations (MIC) against seven microorganisms (Staphyloccus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii and Candida albicans). The most active compounds against the Gram positive S. aureus were 1, 13C, 13d, and 13g. Also, 13c, 13d, and 13e had antibacterial activity but not 13f against some Gram negative organisms (E. coli, and P. mirabilis). MIC concentrations against P. aeruginosa, and K. pneumoniae were?≥512?μg/ml, while that against A. baumannii was?≥128?μg/ml except for nanoformulae of 13e and 13f that were 16 and 64?μg/ml, respectively. No antifungal activity against Candida albicans was recorded for all compounds and their nanoformulae (MIC?>?1024?μg/ml). SLNs were found to decrease the MIC values for some of the compounds with no effect on the antifungal activity. In conclusion, we demonstrated a novel, straight-forward and economical procedure for the total synthesis of (-)-codonopsinine 1, (-)-codonopsine 2 and codonopsinine analogues 13c-g from simple and commercially available starting materials; d-tartaric acid; with antimicrobial activities against Gram positive and Gram-negative organisms that were improved by SLNs formulations.     

Whole-genome sequencing and assembly with high-throughput, short-read technologies

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology.