Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5′-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH

Pharmacological assessment of ibuprofen arginate on platelet aggregation and colon cancer cell killing

This work was funded in part by grants from the Wellcome Trust (0852551Z108/Z, to J.A.M.) and British Heart Foundation (FS/16/1/31699, to NSK). SM is a recipient of a PhD award from the King of Saud University, AT is a recipient of a MRC PhD studentship

Antimicrobial resistance of Campylobacter isolates from small scale and backyard chicken in Kenya

Background Thermophilic Campylobacter species are a major cause of bacterial foodborne diarrhoea in humans worldwide. Poultry and their products are the predominant source for human campylobacteriosis. Resistance of Campylobacter to antibiotics is increasing worldwide, but little is known about the antibiotic resistance in Campylobacter isolated from chicken in Kenya. In this study, 35 suspected Campylobacter strains isolated from faeces and cloacal swabs of chicken were tested for their susceptibility to seven antibiotics using a broth microdilution assay and molecular biological investigations. Results Overall, DNA of thermophilic Campylobacter was identified in 53 samples by PCR (34 C. jejuni, 18 C. coli and one mix of both species) but only 35 Campylobacter isolates (31 C. jejuni and 4 C. coli) could be re-cultivated after transportation to Germany. Isolates were tested for their susceptibility to antibiotics using a broth microdilution assay. Additionally, molecular biological detection of antibiotic resistance genes was carried out. C. jejuni isolates showed a high rate of resistance to nalidixic acid, tetracycline and ciprofloxacin of 77.4, 71.0 and 71.0 %, respectively. Low resistance (25.8 %) was detected for gentamicin and chloramphenicol. Multidrug resistance in C. jejuni could be detected in 19 (61.3 %) isolates. Resistance pattern of C. coli isolates was comparable. Resistance to ciprofloxacin was confirmed by MAMA–PCR and PCR–RFLP in all phenotypically resistant isolates. The tet(O) gene was detected only in 54.5 % of tetracycline resistant C. jejuni isolates. The tet(A) gene, which is also responsible for tetracycline resistance, was found in 90.3 % of C. jejuni and in all C. coli isolates. Thirteen phenotypically erythromycin-resistant isolates could not be characterised by using PCR–RFLP and MAMA–PCR. Conclusions To the best of our knowledge, this study is the first report about resistance to antibiotics in thermophilic Campylobacter originating from chicken in Kenya. Campylobacter spp. show a high level of resistance to ciprofloxacin, nalidixic acid and tetracycline but also a remarkable one to chloramphenicol and gentamicin and they are multidrug resistant. Resistance to antibiotics is a global public health concern. In Kenya, resistance surveillance needs further attention in the future. Efforts to establish at least a National Laboratory with facilities for performing phenotypic and genotypic characterization of thermophilic Campylobacter is highly recommended

Genotyping and antibiotic resistance of thermophilic Campylobacter isolated from chicken and pig meat in Vietnam

Background Campylobacter species are recognized as the most common cause of foodborne bacterial gastroenteritis in humans. In this study nine Campylobacter strains isolated from chicken meat and pork in Hanoi, Vietnam, were characterized using molecular methods and tested for antibiotic resistance. Results The nine isolates (eight C. jejuni and one C. coli) were identified by multiplex PCR, and tested for the presence or absence of 29 gene loci associated with virulence, lipooligosaccharide (LOS) biosynthesis and further functions. flaA typing, multilocus sequence typing and microarray assay investigation showed a high degree of genetic diversity among these isolates. In all isolates motility genes (flaA, flaB, flhA, fliM), colonization associated genes (cadF, docB), toxin production genes (cdtA, cdtB, secD, secF), and the LOS biosynthesis gene pglB were detected. Eight gene loci (fliY, virB11, Cje1278, Cj1434c, Cj1138, Cj1438c, Cj1440c, Cj1136) could not be detected by PCR. A differing presence of the gene loci ciaB (22.2 %), Cje1280 (77.8 %), docC (66.7 %), and cgtB (55.6 %) was found. iamA, cdtC, and the type 6 secretion system were present in all C. jejuni isolates but not in C. coli. flaA typing resulted in five different genotypes within C. jejuni, MLST classified the isolates into seven sequence types (ST-5155, ST-6736, ST-2837, ST-4395, ST-5799, ST-4099 and ST-860). The microarray assay analysis showed a high genetic diversity within Vietnamese Campylobacter isolates which resulted in eight different types for C. jejuni. Antibiotic susceptibility profiles showed that all isolates were sensitive to gentamicin and most isolates (88.8 %) were sensitive to chloramphenicol, erythromycin and streptomycin. Resistance rates to nalidixic acid, tetracycline and ciprofloxacin were 88.9, 77.8 and 66.7 %, respectively. Conclusions To the best of our knowledge, this study is the first report that shows high genetic diversity and remarkable antibiotic resistance of Campylobacter strains isolated from meat in Vietnam which can be considered of high public health significance. These preliminary data show that large scale screenings are justified to assess the relevance of Campylobacter infections on human health in Vietnam

Ratiometric Near-Infrared Fluorescent Probes Based On Through-Bond Energy Transfer and π-Conjugation Modulation between Tetraphenylethene and Hemicyanine Moieties for Sensitive Detection of pH Changes in Live Cells

© 2018 American Chemical Society. In this paper, we present three ratiometric near-infrared fluorescent probes (A-C) for accurate, ratiometric detection of intracellular pH changes in live cells. Probe A consists of a tetraphenylethene (TPE) donor and near-infrared hemicyanine acceptor in a through-bond energy transfer (TBET) strategy, while probes B and C are composed of TPE and hemicyanine moieties through single and double sp 2 carbon-carbon bond connections in a π-conjugation modulation strategy. The specific targeting of the probes to lysosomes in live cells was achieved by introducing morpholine residues to the hemicyanine moieties to form closed spirolactam ring structures. Probe A shows aggregation-induced emission (AIE) property at neutral or basic pH, while probes B and C lack AIE properties. At basic or neutral pH, the probes only show fluorescence of TPE moieties with closed spirolactam forms of hemicyanine moieties, and effectively avoid blind fluorescence imaging spots, an issue which typical intensity-based pH fluorescent probes encounter. Three probes show ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with TPE fluorescence decreases and hemicyanine fluorescence increases, because acidic pH makes the spirolactam rings open to enhance π-conjugation of hemicyanine moieties. However, probe A shows much more sensitive ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with remarkable ratio increase of TPE fluorescence to hemicyanine fluorescence up to 238-fold than probes B and C because of its high efficiency of energy transfer from TPE donor to the hemicyanine acceptor in the TBET strategy. The probe offers dual Stokes shifts with a large pseudo-Stokes shift of 361 nm and well-defined dual emissions, and allows for colocalization of the imaging readouts of visible and near-infrared fluorescence channels to achieve more precisely double-checked ratiometric fluorescence imaging. These platforms could be employed to develop a variety of novel ratiometric fluorescent probes for accurate detection of different analytes in applications of chemical and biological sensing, imaging, and diagnostics by introducing appropriate sensing ligands to hemicyanine moieties to form on-off spirolactam switches

Application performance and flexibility on Exokernel systems

The exokernel operating system architecture safely gives untrusted software efficient control over hardware and software resources by separating management from protection. This paper describes an exokernel system that allows specialized applications to achieve high performance without sacrificing the performance of unmodified UNIX programs. It evaluates the exokernel architecture by measuring end-to-end application performance on Xok, an exokernel for Intel x86-based computers, and by comparing Xok’s performance to the performance of two widely-used 4.4BSD UNIX systems (Free-BSD and OpenBSD). The results show that common unmodified UNIX applications can enjoy the benefits of exokernels: applications either perform comparably on Xok/ExOS and the BSD UNIXes, or perform significantly better. In addition, the results show that customized applications can benefit substantially from control over their resources (e.g., a factor of eight for a Web server). This paper also describes insights about the exokernel approach gained through building three different exokernel systems, and presents novel approaches to resource multiplexing.