Wolbachia and DNA barcoding insects: patterns, potential and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein – wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor – for which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region

Mammalian Genes Preferentially Co-Retained in Radiation Hybrid Panels Tend to Avoid Coexpression

Coexpression has been frequently used to explore modules of functionally related genes in eukaryotic genomes. However, we found that genetically interacting mammalian genes identified through radiation hybrid (RH) genotypes tend not to be coexpressed across tissues. This pattern remained unchanged after controlling for potential confounding factors, including chromosomal linkage, chromosomal distance, and gene duplication. Because >99.9% of the genetically interacting genes were identified according to the higher co-retention frequencies, our observation implies that coexpression is not necessarily an indication of the need for the co-presence of two genes in the genome, which is a prerequisite for cofunctionality of their coding proteins in the cell. Therefore, coexpression information must be applied cautiously to the exploration of the functional relatedness of genes in a genome

Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection

Human blood Vc9/Vd2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vc9/Vd2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vc9/Vd2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-c and tumor necrosis factor (TNF)-a. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vc9/Vd2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-a dependent proliferation of Vc9/Vd2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting cd T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vc9/Vd2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The cd T celldriven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of cd T cells and TNF-a and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive cd T cells in early infection and suggest novel diagnostic and therapeutic approaches.Martin S. Davey, Chan-Yu Lin, Gareth W. Roberts, Sinéad Heuston, Amanda C. Brown, James A. Chess, Mark A. Toleman, Cormac G.M. Gahan, Colin Hill, Tanya Parish, John D. Williams, Simon J. Davies, David W. Johnson, Nicholas Topley, Bernhard Moser and Matthias Eber

Optimal functional outcome measures for assessing treatment for Dupuytren's disease: A systematic review and recommendations for future practice

This article is available through the Brunel Open Access Publishing Fund. Copyright © 2013 Ball et al.; licensee BioMed Central Ltd.Background: Dupuytren's disease of the hand is a common condition affecting the palmar fascia, resulting in progressive flexion deformities of the digits and hence limitation of hand function. The optimal treatment remains unclear as outcomes studies have used a variety of measures for assessment. Methods: A literature search was performed for all publications describing surgical treatment, percutaneous needle aponeurotomy or collagenase injection for primary or recurrent Dupuytren’s disease where outcomes had been monitored using functional measures. Results: Ninety-one studies met the inclusion criteria. Twenty-two studies reported outcomes using patient reported outcome measures (PROMs) ranging from validated questionnaires to self-reported measures for return to work and self-rated disability. The Disability of Arm, Shoulder and Hand (DASH) score was the most utilised patient-reported function measure (n=11). Patient satisfaction was reported by eighteen studies but no single method was used consistently. Range of movement was the most frequent physical measure and was reported in all 91 studies. However, the methods of measurement and reporting varied, with seventeen different techniques being used. Other physical measures included grip and pinch strength and sensibility, again with variations in measurement protocols. The mean follow-up time ranged from 2 weeks to 17 years. Conclusions: There is little consistency in the reporting of outcomes for interventions in patients with Dupuytren’s disease, making it impossible to compare the efficacy of different treatment modalities. Although there are limitations to the existing generic patient reported outcomes measures, a combination of these together with a disease-specific questionnaire, and physical measures of active and passive individual joint Range of movement (ROM), grip and sensibility using standardised protocols should be used for future outcomes studies. As Dupuytren’s disease tends to recur following treatment as well as extend to involve other areas of the hand, follow-up times should be standardised and designed to capture both short and long term outcomes

Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice

Handling can stimulate stress and anxiety in laboratory animals that negatively impacts welfare and introduces a confounding factor in many areas of research. Picking up mice by the tail is a major source of handling stress that results in strong aversion to the handler, while mice familiarised with being picked up in a tunnel or cupped on the open hand show low stress and anxiety, and actively seek interaction with their handlers. Here we investigate the duration and frequency of handling required for effective familiarisation with these non-aversive handling methods, and test whether this is sufficient to prevent aversion and anxiety when animals then experience immobilisation and a mild procedure (subcutaneous injection). Very brief handling (2 s) was sufficient to familiarise mice with tunnel handling, even when experienced only during cage cleaning. Brief but more frequent handling was needed for familiarisation with cup handling, while pick up by tail induced strong aversion even when handling was brief and infrequent. Experience of repeated immobilisation and subcutaneous injection did not reverse the positive effects of tunnel handling. Our findings demonstrate that replacing tail with tunnel handling during routine cage cleaning and procedures provides a major refinement with little if any cost for familiarisation

Genetic Variations in the Regulator of G-Protein Signaling Genes Are Associated with Survival in Late-Stage Non-Small Cell Lung Cancer

The regulator of G-protein signaling (RGS) pathway plays an important role in signaling transduction, cellular activities, and carcinogenesis. We hypothesized that genetic variations in RGS gene family may be associated with the response of late-stage non-small cell lung cancer (NSCLC) patients to chemotherapy or chemoradiotherapy. We selected 95 tagging single nucleotide polymorphisms (SNPs) in 17 RGS genes and genotyped them in 598 late-stage NSCLC patients. Thirteen SNPs were significantly associated with overall survival. Among them, rs2749786 of RGS12 was most significant. Stratified analysis by chemotherapy or chemoradiation further identified SNPs that were associated with overall survival in subgroups. Rs2816312 of RGS1 and rs6689169 of RGS7 were most significant in chemotherapy group and chemoradiotherapy group, respectively. A significant cumulative effect was observed when these SNPs were combined. Survival tree analyses identified potential interactions between rs944343, rs2816312, and rs1122794 in affecting survival time in patients treated with chemotherapy, while the genotype of rs6429264 affected survival in chemoradiation-treated patients. To our knowledge, this is the first study to reveal the importance of RGS gene family in the survival of late-stage NSCLC patients

Overexpression of the Lung Cancer-Prognostic miR-146b MicroRNAs Has a Minimal and Negative Effect on the Malignant Phenotype of A549 Lung Cancer Cells

INTRODUCTION:Expression levels of miR-146b-5p and -3p microRNAs in human non-small cell lung cancer (NSCLC) are associated with recurrence of the disease after surgery. To understand this, the effect of miR-146b overexpression was studied in A549 human lung cancer cells. METHODS:A549 cells, engineered with lentiviruses to overexpress the human pre-miR-146b precursor microRNA, were examined for proliferation, colony formation on plastic surface and in soft agar, migration and invasiveness in cell culture and in vivo in mice, chemosensitivity to cisplatin and doxorubicin, and global gene expression. miR-146b expressions were assessed in microdissected stroma and epithelia of human NSCLC tumors. Association of miR-146b-5p and -3p expression in early stage NSCLC with recurrence was analyzed. PRINCIPAL FINDINGS:A549 pre-miR-146b-overexpressors had 3-8-fold higher levels of both miR-146b microRNAs than control cells. Overexpression did not alter cellular proliferation, chemosensitivity, migration, or invasiveness; affected only 0.3% of the mRNA transcriptome; and, reduced the ability to form colonies in vitro by 25%. In human NSCLC tumors, expression of both miR-146b microRNAs was 7-10-fold higher in stroma than in cancerous epithelia, and higher miR-146b-5p but lower -3p levels were predictive of recurrence. CONCLUSIONS:Only a minimal effect of pre-miR-146b overexpression on the malignant phenotype was seen in A549 cells. This could be because of opposing effects of miR-146b-5p and -3p overexpression as suggested by the conflicting recurrence-predictive values of the two microRNAs, or because miR-146b expression changes in non-cancerous stroma and not cancerous epithelia of tumors are responsible for the prognostic value of miR-146b

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma

Background Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. Methods We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Results Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Conclusions Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer