Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.     

Perfluoroalkyl Acid Concentrations in Blood Samples Subjected to Transportation and Processing Delay

Background

In studies of perfluoroalkyl acids, the validity and comparability of measured concentrations may be affected by differences in the handling of biospecimens. We aimed to investigate whether measured plasma levels of perfluoroalkyl acids differed between blood samples subjected to delay and transportation prior to processing and samples with immediate processing and freezing.

Methods

Pregnant women recruited at Aarhus University Hospital, Denmark, (n = 88) provided paired blood samples. For each pair of samples, one was immediately processed and plasma was frozen, and the other was delayed and transported as whole blood before processing and freezing of plasma (similar to the Danish National Birth Cohort). We measured 12 perfluoroalkyl acids and present results for compounds with more than 50% of samples above the lower limit of quantification.

Results

For samples taken in the winter, relative differences between the paired samples ranged between -77 and +38% for individual perfluoroalkyl acids. In most cases concentrations were lower in the delayed and transported samples, e.g. the relative difference was -29% (95% confidence interval -30; -27) for perfluorooctane sulfonate. For perfluorooctanoate there was no difference between the two setups [corresponding estimate 1% (0, 3)]. Differences were negligible in the summer for all compounds.

Conclusions

Transport of blood samples and processing delay, similar to conditions applied in some large, population-based studies, may affect measured perfluoroalkyl acid concentrations, mainly when outdoor temperatures are low. Attention to processing conditions is needed in studies of perfluoroalkyl acid exposure in humans.     

Performance of Serum microRNAs -122, -192 and -21 as Biomarkers in Patients with Non-Alcoholic Steatohepatitis

Objectives

Liver biopsies are the current gold standard in non-alcoholic steatohepatitis (NASH) diagnosis. Their invasive nature, however, still carries an increased risk for patients’ health. The development of non-invasive diagnostic tools to differentiate between bland steatosis (NAFL) and NASH remains crucial. The aim of this study is the evaluation of investigated circulating microRNAs in combination with new targets in order to optimize the discrimination of NASH patients by non-invasive serum biomarkers.

Methods

Serum profiles of four microRNAs were evaluated in two cohorts consisting of 137 NAFLD patients and 61 healthy controls. In a binary logistic regression model microRNAs of relevance were detected. Correlation of microRNA appearance with known biomarkers like ALT and CK18-Asp396 was evaluated. A simplified scoring model was developed, combining the levels of microRNA in circulation and CK18-Asp396 fragments. Receiver operating characteristics were used to evaluate the potential of discriminating NASH.

Results

The new finding of our study is the different profile of circulating miR-21 in NASH patients (p<0.0001). Also, it validates recently published results of miR-122 and miR-192 to be differentially regulated in NAFL and NASH. Combined microRNA expression profiles with CK18-Asp396 fragment level scoring model had a higher potential of NASH prediction compared to other risk biomarkers (AUROC = 0.83, 95% CI = 0.754–0.908; p<0.001). Evaluation of score model for NAFL (Score = 0) and NASH (Score = 4) had shown high rates of sensitivity (91%) and specificity (83%).

Conclusions

Our study defines candidates for a combined model of miRNAs and CK18-Asp396 levels relevant as a promising expansion for diagnosis and in turn treatment of NASH.     

The Glasgow consensus on the delineation between pesticide emission inventory and impact assessment for LCA

Purpose

Pesticides are applied to agricultural fields to optimise crop yield and their global use is substantial. Their consideration in life cycle assessment (LCA) is affected by important inconsistencies between the emission inventory and impact assessment phases of LCA. A clear definition of the delineation between the product system model (life cycle inventory—LCI, technosphere) and the natural environment (life cycle impact assessment—LCIA, ecosphere) is missing and could be established via consensus building.

Methods

A workshop held in 2013 in Glasgow, UK, had the goal of establishing consensus and creating clear guidelines in the following topics: (1) boundary between emission inventory and impact characterisation model, (2) spatial dimensions and the time periods assumed for the application of substances to open agricultural fields or in greenhouses and (3) emissions to the natural environment and their potential impacts. More than 30 specialists in agrifood LCI, LCIA, risk assessment and ecotoxicology, representing industry, government and academia from 15 countries and four continents, met to discuss and reach consensus. The resulting guidelines target LCA practitioners, data (base) and characterisation method developers, and decision makers.

Results and discussion

The focus was on defining a clear interface between LCI and LCIA, capable of supporting any goal and scope requirements while avoiding double counting or exclusion of important emission flows/impacts. Consensus was reached accordingly on distinct sets of recommendations for LCI and LCIA, respectively, recommending, for example, that buffer zones should be considered as part of the crop production system and the change in yield be considered. While the spatial dimensions of the field were not fixed, the temporal boundary between dynamic LCI fate modelling and steady-state LCIA fate modelling needs to be defined.

Conclusions and recommendations

For pesticide application, the inventory should report pesticide identification, crop, mass applied per active ingredient, application method or formulation type, presence of buffer zones, location/country, application time before harvest and crop growth stage during application, adherence with Good Agricultural Practice, and whether the field is considered part of the technosphere or the ecosphere. Additionally, emission fractions to environmental media on-field and off-field should be reported. For LCIA, the directly concerned impact categories and a list of relevant fate and exposure processes were identified. Next steps were identified: (1) establishing default emission fractions to environmental media for integration into LCI databases and (2) interaction among impact model developers to extend current methods with new elements/processes mentioned in the recommendations.

     

Efficient introduction of phosphorothioates into RNA oligonucleotides by 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH).

We recently reported on the use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as effective sulfurizing reagents for the preparation of phosphorothioate-containing oligodeoxyribo-nucleotides [Xu et al. (1996) Nucleic Acids Res., 24, 1602-1607]. One challenge in automated solid-phase synthesis of phosphorothioate-containing RNA is to develop sulfurization reagents that are effective in the presence of bulky 2'-OH protecting groups. The present study demonstrates that EDITH is exceedingly effective at low concentrations (0.05 M) and short reaction times (2 min) for the automated synthesis of oligoribonucleotides.     

Practical approach to solid-phase synthesis of C-terminal peptide amides under mild conditions based on a photolysable anchoring linkage

Several 3-nitro-4-(N-protected aminomethyl)benzoic acids; with protection provided by tert.-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl (Tfa), dithiasuccinoyl (Dts), or phthaloyl (Phth), have been prepared by reproducible routes. Synthesis of Dts-handle 6 illustrates some particularly novel and efficient chemistry, and is preferred over more intricate routes to Boc-handle 3 and Fmoc-handle 4. The five handles were each evaluated for their application to the synthesis of peptide amides. Coupling onto amino-functionalized supports provided a general starting point for peptide chain assembly. The handle amino function was deblocked (Boc, Fmoc, Dts), the C-terminal residue was coupled as its N alpha-protected free acid, and ultimately the ortho-nitrobenzylamide anchorage linkage was cleaved photolytically to give the corresponding amide. Starting with handles 3, 4, and 6, several free and protected peptide amides were synthesized.     

Analysis of SCAMP1 function in secretory vesicle exocytosis by means of gene targeting in mice.

Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTPgammaS in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores.     

Microexplosion of a hot point in an X-pinch constriction

The dynamics of an X-pinch in the diode of a high-power nanosecond current generator is studied experimentally and theoretically. The X-ray backlighting technique with subnanosecond time resolution and micron space resolution made it possible to trace both the formation of the constriction before the X-ray burst and the subsequent breaking and decay of the constriction. The radiative MHD model allowed simulation of the main characteristics of the process, including the formation of a minidiode and constriction, microexplosion of the hot point, and the generation of shock waves, followed by breaking of the constriction. __________ Translated from Fizika Plazmy, Vol. 26, No. 10, 2000, pp. 927–933. Original Russian Text Copyright /sC 2000 by Ivanenkov, Pikuz, Sinars, Stepnievski, Hammer, Shelkovenko.