Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

<p>Abstract</p> <p>Background</p> <p>SXT is an integrating conjugative element (ICE) originally isolated from <it>Vibrio cholerae</it>, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.</p> <p>Results</p> <p>SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn²⁺or Mg²⁺ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in <it>E. coli </it>cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.</p> <p>Conclusions</p> <p>The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected <it>V. cholerae </it>cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.</p

The role of circulating tumour cells and nucleic acids in blood for the detection of bladder cancer: A systematic review

BACKGROUND: Blood-based biomarkers are a neglected resource in bladder cancer, where the mainstay of focus has been on urinary biomarkers. However, blood-based biomarkers are gaining popularity in other solid cancers, particularly circulating tumour cells (CTCs) and circulating nucleic acids. In this systematic review, we identify and discuss the diagnostic value of CTC, cell-free DNA and RNA based biomarkers in bladder cancer. METHODS: A MEDLINE/Pubmed systematic search was performed using the following keywords: (bladder cancer) AND (blood OR plasma OR serum) AND biomarker AND (DNA OR RNA OR cfDNA OR cell-free DNA OR RNA OR CTC). All studies including blood-based biomarkers based on DNA, RNA and CTCs were reviewed. Of the included studies, studies reporting sensitivity, specificity and/or AUC/ROC values were further described. RESULTS: Systematic searched yielded 47 studies that were eligible, of which 21, 19 and 3 studies reported DNA, RNA and CTC biomarkers respectively. 15 of these studies included sensitivity, specificity and/or AUC/ROC values. Biomarkers sensitivity and specificity ranged widely at 2.4-97.6% and 43.3-100% respectively. Median number of patients recruited in the studies was 56 (IQR 41-90). Only 3 studies included an independent validation cohort. The highest sensitivity and specificity pairing achieved in the validation cohort was 80.0% and 89.1% respectively. CONCLUSIONS: This systematic review provides a comprehensive overview of the blood-based CTC and nucleic acid biomarkers that have been investigated. An overlap in interest of targets between studies suggests that these could be promising biomarkers, but few biomarkers achieve high sensitivity and specificity, and fewer still have been validated independently

Novel urinary biomarkers for the detection of bladder cancer: A systematic review

BACKGROUND: Urinary biomarkers for the diagnosis of bladder cancer represents an area of considerable research which has been tested in both patients presenting with haematuria and non-muscle invasive bladder cancer patients requiring surveillance cystoscopy. In this systematic review, we identify and appraise the diagnostic sensitive and specificity of reported novel biomarkers of different 'omic' class and highlight promising biomarkers investigated to date. METHODS: A MEDLINE/Pubmed systematic search was performed between January 2013 and July 2017 using the following keywords: (bladder cancer OR transitional cell carcinoma OR urothelial cell carcinoma) AND (detection OR diagnosis) AND urine AND (biomarker OR assay). All studies had a minimum of 20 patients in both bladder cancer and control arms and reported sensitivity and/or specificity and/or receiver operating characteristics (ROC) curve. QUADAS-2 tool was used to assess risk of bias and applicability of studies. The search protocol was registered in the PROSPERO database (CRD42016049918). RESULTS: Systematic search yielded 115 reports were included for analysis. In single target biomarkers had a sensitivity of 2-94%, specificity of 46-100%, positive predictive value (PPV) of 47-100% and negative predictive value (NPV) of 21-94%. Multi-target biomarkers achieved a sensitivity of 24-100%, specificity of 48-100%, PPV of 42-95% and NPV of 32-100%. 50 studies achieved a sensitivity and specificity of ≥80%. Protein (n = 59) and transcriptomic (n = 21) biomarkers represents the most studied biomarkers. Multi-target biomarker panels had a better diagnostic accuracy compared to single biomarker targets. Urinary cytology with urinary biomarkers improved the diagnostic ability of the biomarker. The sensitivity and specificity of biomarkers were higher for primary diagnosis compared to patients in the surveillance setting. Most studies were case control studies and did not have a predefined threshold to determine a positive test result indicating a possible risk of bias. CONCLUSION: This comprehensive systematic review provides an update on urinary biomarkers of different 'omic' class and highlights promising biomarkers. Few biomarkers achieve a high sensitivity and negative predictive value. Such biomarkers will require external validation in a prospective observational setting before adoption in clinical practice

Investigation on the competing effects of clay dispersion and matrix plasticisation for polypropylene/clay nanocomposites. Part I: morphology and mechanical properties

The key compatibiliser role of maleated polypropylene (MAPP) to improve the clay dispersability has been explicitly addressed in the fabrication process and material characterisation of polypropylene (PP)/clay nanocomposites. However, its matrix plasticiser role, which has been rarely mentioned, could adversely influence the excellent mechanical properties of such nanocomposites, resulting from the homogeneous clay dispersion. PP/clay nanocomposites in the presence of MAPP were prepared by twin screw extrusion and subsequently injection moulded with three typical material formulations in fixed parametric settings: (1) weight ratio (WR) of clay and MAPP, WR = 1:2; (2) MAPP content of 6 wt% and (3) clay content of 5 wt%. The morphological structures and mechanical properties of PP/clay nanocomposites were examined by using X-ray diffraction (XRD) analysis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and universal mechanical testing. The further improvement of mechanical properties was evidently hindered with very inconsiderable alteration of morphological structures in terms of the clay dispersion level. This observation could be ascribed to the change of MAPP role from a compatibiliser to a plasticiser because of its excessive amount used above a certain saturation level, which was found in the range of 3–6 wt% in MAPP contents for the enhancements of tensile and flexural properties of PP/clay nanocomposites

A Tale of Two Catchments: Causality Analysis and Isotope Systematics Reveal Mountainous Watershed Traits That Regulate the Retention and Release of Nitrogen

Mountainous watersheds are characterized by variability in functional traits, including vegetation, topography, geology, and geomorphology, which determine nitrogen (N) retention, and release. Coal Creek and East River are two contrasting catchments within the Upper Colorado River Basin that differ markedly in total nitrate (NO3−) export. The East River has a diverse vegetation cover, and sinuous floodplains, and is underlain by N-rich marine shale. At 0.21&nbsp;±&nbsp;0.14&nbsp;kg&nbsp;ha−1&nbsp;yr−1, the East River exports ∼3.5 times more NO3− relative to the conifer-dominated Coal Creek (0.06&nbsp;±&nbsp;0.02&nbsp;kg&nbsp;ha−1&nbsp;yr−1). While this can partly be explained by the larger size of the East River, the distinct watershed traits of these two catchments imply different mechanisms controlling the aggregate N-export signal. A causality analysis shows physical and biogenic processes were critical in determining NO3− export from the East River catchment. Stable isotope ratios of NO3− (δ15NNO3 and δ18ONO3) show the East River catchment is a strong hotspot for biogeochemical processing of NO3− at the hillslope soil-saprolite. By contrast, the conifer-dominated Coal Creek retained nearly all atmospherically deposited NO3−, and its export was controlled by catchment hydrological traits (i.e., snowmelt periods and water table depth). The conservative N-cycle within Coal Creek is likely due to the abundance of conifer trees, and smaller riparian regions, retaining more NO3− overall and reduced processing prior to export. This study highlights the value of integrating isotope systematics to link watershed functional traits to mechanisms of watershed element retention and release

Subfamily specific conservation profiles for proteins based on n-gram patterns

<p>Abstract</p> <p>Background</p> <p>A new algorithm has been developed for generating conservation profiles that reflect the evolutionary history of the subfamily associated with a query sequence. It is based on n-gram patterns (NP{<it>n,m</it>}) which are sets of <it>n </it>residues and <it>m </it>wildcards in windows of size <it>n+m</it>. The generation of conservation profiles is treated as a signal-to-noise problem where the signal is the count of n-gram patterns in target sequences that are similar to the query sequence and the noise is the count over all target sequences. The signal is differentiated from the noise by applying singular value decomposition to sets of target sequences rank ordered by similarity with respect to the query.</p> <p>Results</p> <p>The new algorithm was used to construct 4,248 profiles from 120 randomly selected Pfam-A families. These were compared to profiles generated from multiple alignments using the consensus approach. The two profiles were similar whenever the subfamily associated with the query sequence was well represented in the multiple alignment. It was possible to construct subfamily specific conservation profiles using the new algorithm for subfamilies with as few as five members. The speed of the new algorithm was comparable to the multiple alignment approach.</p> <p>Conclusion</p> <p>Subfamily specific conservation profiles can be generated by the new algorithm without aprioi knowledge of family relationships or domain architecture. This is useful when the subfamily contains multiple domains with different levels of representation in protein databases. It may also be applicable when the subfamily sample size is too small for the multiple alignment approach.</p

Effect of Heat Treatment on Fracture Toughness ( K IC ) and Microstructure of a Fluorcanasite-Based Glass-Ceramic

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73358/1/j.1532-849X.2007.00233.x.pd

Dzyaloshinskii-Moriya Interaction and Spiral Order in Spin-orbit Coupled Optical Lattices

We show that the recent experimental realization of spin-orbit coupling in ultracold atomic gases can be used to study different types of spin spiral order and resulting multiferroic effects. Spin-orbit coupling in optical lattices can give rise to the Dzyaloshinskii-Moriya (DM) spin interaction which is essential for spin spiral order. By taking into account spin-orbit coupling and an external Zeeman field, we derive an effective spin model in the Mott insulator regime at half filling and demonstrate that the DM interaction in optical lattices can be made extremely strong with realistic experimental parameters. The rich finite temperature phase diagrams of the effective spin models for fermions and bosons are obtained via classical Monte Carlo simulations.Comment: 7 pages, 5 figure

In Situ Synthesis of Reduced Graphene Oxide and Gold Nanocomposites for Nanoelectronics and Biosensing

In this study, an in situ chemical synthesis approach has been developed to prepare graphene–Au nanocomposites from chemically reduced graphene oxide (rGO) in aqueous media. UV–Vis absorption, atomic force microscopy, scanning electron microscopy, transmission electron microscopy, and Raman spectroscopy were used to demonstrate the successful attachment of Au nanoparticles to graphene sheets. Configured as field-effect transistors (FETs), the as-synthesized single-layered rGO-Au nanocomposites exhibit higher hole mobility and conductance when compared to the rGO sheets, promising its applications in nanoelectronics. Furthermore, we demonstrate that the rGO-Au FETs are able to label-freely detect DNA hybridization with high sensitivity, indicating its potentials in nanoelectronic biosensing

A discriminative method for protein remote homology detection and fold recognition combining Top-n-grams and latent semantic analysis

<p>Abstract</p> <p>Background</p> <p>Protein remote homology detection and fold recognition are central problems in bioinformatics. Currently, discriminative methods based on support vector machine (SVM) are the most effective and accurate methods for solving these problems. A key step to improve the performance of the SVM-based methods is to find a suitable representation of protein sequences.</p> <p>Results</p> <p>In this paper, a novel building block of proteins called Top-<it>n</it>-grams is presented, which contains the evolutionary information extracted from the protein sequence frequency profiles. The protein sequence frequency profiles are calculated from the multiple sequence alignments outputted by PSI-BLAST and converted into Top-<it>n</it>-grams. The protein sequences are transformed into fixed-dimension feature vectors by the occurrence times of each Top-<it>n</it>-gram. The training vectors are evaluated by SVM to train classifiers which are then used to classify the test protein sequences. We demonstrate that the prediction performance of remote homology detection and fold recognition can be improved by combining Top-<it>n</it>-grams and latent semantic analysis (LSA), which is an efficient feature extraction technique from natural language processing. When tested on superfamily and fold benchmarks, the method combining Top-<it>n</it>-grams and LSA gives significantly better results compared to related methods.</p> <p>Conclusion</p> <p>The method based on Top-<it>n</it>-grams significantly outperforms the methods based on many other building blocks including N-grams, patterns, motifs and binary profiles. Therefore, Top-<it>n</it>-gram is a good building block of the protein sequences and can be widely used in many tasks of the computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the prediction of protein binding sites.</p