Non-thermal emission processes in massive binaries

In this paper, I present a general discussion of several astrophysical processes likely to play a role in the production of non-thermal emission in massive stars, with emphasis on massive binaries. Even though the discussion will start in the radio domain where the non-thermal emission was first detected, the census of physical processes involved in the non-thermal emission from massive stars shows that many spectral domains are concerned, from the radio to the very high energies. First, the theoretical aspects of the non-thermal emission from early-type stars will be addressed. The main topics that will be discussed are respectively the physics of individual stellar winds and their interaction in binary systems, the acceleration of relativistic electrons, the magnetic field of massive stars, and finally the non-thermal emission processes relevant to the case of massive stars. Second, this general qualitative discussion will be followed by a more quantitative one, devoted to the most probable scenario where non-thermal radio emitters are massive binaries. I will show how several stellar, wind and orbital parameters can be combined in order to make some semi-quantitative predictions on the high-energy counterpart to the non-thermal emission detected in the radio domain. These theoretical considerations will be followed by a census of results obtained so far, and related to this topic... (see paper for full abstract)Comment: 47 pages, 5 postscript figures, accepted for publication in Astronomy and Astrophysics Review. Astronomy and Astrophysics Review, in pres

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery

Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

The Second Type VI Secretion System of Pseudomonas aeruginosa Strain PAO1 Is Regulated by Quorum Sensing and Fur and Modulates Internalization in Epithelial Cells

The genome of Pseudomonas aeruginosa PAO1 contains three type VI secretion systems (T6SSs) called H1-, H2-, and H3-T6SS. The H1-T6SS secretes three identified toxins that target other bacteria, providing a fitness advantage for P. aeruginosa, and likely contributes to bacterial pathogenesis in chronic infections. However, no specific substrates or defined roles have been described for the two other systems. Here, we demonstrate that the expression of H2-T6SS genes of strain PAO1 is up-regulated during the transition from exponential to stationary phase growth and regulated by the Las and Rhl quorum sensing systems. In addition, we identify two putative Fur boxes in the promoter region and find that H2-T6SS transcription is negatively regulated by iron. We also show that the H2-T6SS system enhances bacterial uptake into HeLa cells (75% decrease in internalization with a H2-T6SS mutant) and into lung epithelial cells through a phosphatidylinositol 3-kinase-dependent pathway that induces Akt activation in the host cell (50% decrease in Akt phosphorylation). Finally, we show that H2-T6SS plays a role in P. aeruginosa virulence in the worm model. Thus, in contrast to H1-T6SS, H2-T6SS modulates interaction with eukaryotic host cells. Together, T6SS can carry out different functions that may be important in establishing chronic P. aeruginosa infections in the human host

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC

Differential modulation of Quorum-Sensing signaling through QslA in Pseudomonas Running title: Differential QS modulation by QslA

International audienceTwo clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite being closely related, PA14 exhibits increased virulence compared to PAO1. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected for a transcriptomic approach through RNA sequencing. We found 2029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by Quorum-Sensing (QS), known to control most of the virulence factors in P. aeruginosa. Among them, we chose to focus our study on QslA, an anti-activator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hyper-virulence phenotype. After confirming Qs lA protein was highly produced in PAO1 but not in the PA14 strain, we provided evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics compared to PA14. Moreover, known virulence factors activated by QS such as (i) pyocyanin production, (ii) H2-T6SS(Type VI Secretion System) gene expression, and (iii) Xcp-T2SS (Type II Secretion System) machinery production and secretion were all lower in PAO1 compared to PA14 strain, due to higher qslAexpression. However, biofilm formation and cytotoxicity towards macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Altogether, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginos astrains PAO1 and PA14