Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene

BACKGROUND: Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing. RESULTS: A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results. CONCLUSION: This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification

Development of a Multiple-Locus Variable number of tandem repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia

BACKGROUND: Leptospirosis is a zoonotic disease caused by the genus, Leptospira. Leptospira interrogans is the most common genomospecies implicated in the disease. Epidemiological investigations are needed to distinguish outbreak situations or to trace reservoirs of the organisms. Current methodologies used for typing Leptospira have significant drawbacks. The development of an easy to perform yet high resolution method is needed for this organism. METHODS: In this study we have searched the available genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 for the presence of tandem repeats [1]. These repeats were evaluated against reference strains for diversity. Six loci were selected to create a Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) to explore the genetic diversity within L. interrogans serovar Australis clinical isolates from Far North Queensland. RESULTS: The 39 reference strains used for the development of the method displayed 39 distinct patterns. Diversity Indexes for the loci varied between 0.80 and 0.93 and the number of repeat units at each locus varied between less than one to 52 repeats. When the MLVA was applied to serovar Australis isolates three large clusters were distinguishable, each comprising various hosts including Rattus species, human and canines. CONCLUSION: The MLVA described in this report, was easy to perform, analyse and was reproducible. The loci selected had high diversity allowing discrimination between serovars and also between strains within a serovar. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made

Comparative analysis of the human serine hydrolase OVCA2 to the model serine hydrolase homolog FSH1 from S. cerevisiae

Over 100 metabolic serine hydrolases are present in humans with confirmed functions in metabolism, immune response, and neurotransmission. Among potentially clinically relevant but uncharacterized human serine hydrolases is OVCA2, a serine hydrolase that has been linked with a variety of cancer-related processes. Herein, we developed a heterologous expression system for OVCA2 and determined the comprehensive substrate specificity of OVCA2 against two ester substrate libraries. Based on this analysis, OVCA2 was confirmed as a serine hydrolase with a strong preference for long-chain alkyl ester substrates (\u3e10-carbons) and high selectivity against a variety of short, branched, and substituted esters. Substitutional analysis was used to identify the catalytic residues of OVCA2 with a Ser117-His206-Asp179 classic catalytic triad. Comparison of the substrate specificity of OVCA2 to the model homologue FSH1 from Saccharomyces cerevisiae illustrated the tighter substrate selectivity of OVCA2, but their overlapping substrate preference for extended straight-chain alkyl esters. Conformation of the overlapping biochemical properties of OVCA2 and FSH1 was used to model structural information about OVCA2. Together our analysis provides detailed substrate specificity information about a previously, uncharacterized human serine hydrolase and begins to define the biological properties of OVCA2

Switchable mirrors based on nickel-magnesium films

A new type of electrochromic mirror electrode based on reversible uptake of hydrogen in nickel magnesium alloy films is reported. Thin,magnesium-rich Ni-Mg films prepared on glass substrates by cosputtering from Ni and Mg targets are mirror-like in appearance and have low visible transmittance. Upon exposure to hydrogen gas or on reduction in alkaline electrolyte, the films take up hydrogen and become transparent. When hydrogen is removed, the mirror properties are recovered. The transition is believed to result from reversible formation of Mg2NiH4 and MgH2. A thin overlayer of palladium was found to enhance the kinetics of hydrogen insertion and extraction,and to protect the metal surface against oxidation

Effects of the nicotinic agonist varenicline, nicotinic antagonist r-bPiDI, and DAT inhibitor R-modafinil on co-use of ethanol and nicotine in female P rats.

Rationale: Co-users of alcohol and nicotine are the largest group of polysubstance users worldwide. Commonalities in mechanisms of action for ethanol (EtOH) and nicotine proposes the possibility of developing a single pharmacotherapeutic to treat co-use. Objectives: Toward developing a preclinical model of co-use, female alcohol-preferring (P) rats were trained for voluntary EtOH drinking and i.v. nicotine self-administration in three phases: (1) EtOH alone (0 vs. 15%, 2-bottle choice); (2) nicotine alone (0.03 mg/kg/infusion, active vs. inactive lever); and (3) concurrent access to both EtOH and nicotine. Using this model, we examined the effects of (1) varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist with high affinity for the α4β2 subtype; (2) r-bPiDI, a subtype-selective antagonist at α6β2* nAChRs; and (3) (R)-modafinil, an atypical inhibitor of the dopamine transporter (DAT). Results: In Phases 1 and 2, pharmacologically relevant intake of EtOH and nicotine was achieved. In the concurrent access phase (Phase 3), EtOH consumption decreased while nicotine intake increased relative to Phases 1 and 2. For drug pretreatments, in the EtOH access phase (Phase 1), (R)-modafinil (100 mg/kg) decreased EtOH consumption, with no effect on water consumption. In the concurrent access phase, varenicline (3 mg/kg), r-bPiDI (20 mg/kg), and (R)-modafinil (100 mg/kg) decreased nicotine self-administration, but did not alter EtOH consumption, water consumption, or inactive lever pressing. Conclusions: These results indicate that therapeutics which may be useful for smoking cessation via selective inhibition of α4β2 or α6β2* nAChRs, or DAT inhibition, may not be sufficient to treat EtOH and nicotine co-use

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

Activation of the Ras–MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane–proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf–MEK1–MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK

Systems-level analyses identify extensive coupling among gene expression machines

Here, we develop computational methods to assess and consolidate large, diverse protein interaction data sets, with the objective of identifying proteins involved in the coupling of multicomponent complexes within the yeast gene expression pathway. From among ∼43 000 total interactions and 2100 proteins, our methods identify known structural complexes, such as the spliceosome and SAGA, and functional modules, such as the DEAD-box helicases, within the interaction network of proteins involved in gene expression. Our process identifies and ranks instances of three distinct, biologically motivated motifs, or patterns of coupling among distinct machineries involved in different subprocesses of gene expression. Our results confirm known coupling among transcription, RNA processing, and export, and predict further coupling with translation and nonsense-mediated decay. We systematically corroborate our analysis with two independent, comprehensive experimental data sets. The methods presented here may be generalized to other biological processes and organisms to generate principled, systems-level network models that provide experimentally testable hypotheses for coupling among biological machines

Impact of electrostatic crosstalk on spin qubits in dense CMOS quantum dot arrays

Quantum processors based on integrated nanoscale silicon spin qubits are a promising platform for highly scalable quantum computation. Current CMOS spin qubit processors consist of dense gate arrays to define the quantum dots, making them susceptible to crosstalk from capacitive coupling between a dot and its neighbouring gates. Small but sizeable spin-orbit interactions can transfer this electrostatic crosstalk to the spin g-factors, creating a dependence of the Larmor frequency on the electric field created by gate electrodes positioned even tens of nanometers apart. By studying the Stark shift from tens of spin qubits measured in nine different CMOS devices, we developed a theoretical frawework that explains how electric fields couple to the spin of the electrons in increasingly complex arrays, including those electric fluctuations that limit qubit dephasing times $T_2^*$. The results will aid in the design of robust strategies to scale CMOS quantum technology.Comment: 9 pages, 4 figure

Long-Distance Signals Are Required for Morphogenesis of the Regenerating Xenopus Tadpole Tail, as Shown by Femtosecond-Laser Ablation

tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches

Hnf4α is a key gene that can generate columnar metaplasia in oesophageal epithelium

AbstractBarrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium