Tracking F plasmid TraI relaxase processing reactions provides insight into F plasmid transfer

Early in F plasmid conjugative transfer, the F relaxase, TraI, cleaves one plasmid strand at a site within the origin of transfer called nic. The reaction covalently links TraI Tyr16 to the 5′-ssDNA phosphate. Ultimately, TraI reverses the cleavage reaction to circularize the plasmid strand. The joining reaction requires a ssDNA 3′-hydroxyl; a second cleavage reaction at nic, regenerated by extension from the plasmid cleavage site, may generate this hydroxyl. Here we confirm that TraI is transported to the recipient during transfer. We track the secondary cleavage reaction and provide evidence it occurs in the donor and F ssDNA is transferred to the recipient with a free 3′-hydroxyl. Phe substitutions for four Tyr within the TraI active site implicate only Tyr16 in the two cleavage reactions required for transfer. Therefore, two TraI molecules are required for F plasmid transfer. Analysis of TraI translocation on various linear and circular ssDNA substrates supports the assertion that TraI slowly dissociates from the 3′-end of cleaved F plasmid, likely a characteristic essential for plasmid re-circularization

Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM

The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM–sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM’s N-terminal ribbon–helix–helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore

Bioengineering approaches to simulate human colon microbiome ecosystem

Background Several diseases associated to colon microbial imbalance (dysbiosis), such as obesity, diabetes, inflammatory bowel disease, cardiovascular disease and cancer, are being reverted by modulation of gut microbiota composition through treatment with prebiotics and probiotics. Multiple in vitro models have been developed over the past three decades, with several experimental configurations, as they provide a quick, easy, and cost-effective approach to study the gut microbiome, as compared to troublesome and time-consuming in vivo studies. Scope and approach This review aims to provide an overview of the most relevant available in vitro models used to mimic the human colon microbiome dynamics, including macro-scale and microfluidic-based models. Main characteristics, functionalities, current applications and advantages or disadvantages of the models are discussed in order to provide useful information for end users (namely food and pharmaceutical researchers), when selecting the most appropriated model for assessing health claims and safety of novel functional food and drugs. Finally, the use of these colon models as a tool to study prebiotic and probiotic response in host-microbiota interaction is reviewed. Key findings and conclusions A wide range of in vitro models representing specific colon parts have been developed. However, none of these models can simultaneously cover all the key conditions found in the human colon (namely anatomical, physical, biochemical, and biological characteristics), as well as the complex microbiome-host interaction. Thus, there is a significant opportunity for further improvement of the models experimental setups towards more realistic operating systems, including mucosal surfaces, intestinal cells and tissues allowing microbiomehost crosstalk representation.SFRH/BD/139884/2018 from the Portuguese Foundation for Science and Technology (FCT). This study was supported by the FCT under the scope of the strategic funding of UIDB/04469/2020 unit and the Project ColOsH PTDC/BTM–SAL/30071/2017 (POCI–01–0145–FEDER–030071)info:eu-repo/semantics/publishedVersio

Atorvastatin Improves Survival in Septic Rats: Effect on Tissue Inflammatory Pathway and on Insulin Signaling

The aim of the present study was to investigate whether the survival-improving effect of atorvastatin in sepsis is accompanied by a reduction in tissue activation of inflammatory pathways and, in parallel, an improvement in tissue insulin signaling in rats. Diffuse sepsis was induced by cecal ligation and puncture surgery (CLP) in male Wistar rats. Serum glucose and inflammatory cytokines levels were assessed 24 h after CLP. The effect of atorvastatin on survival of septic animals was investigated in parallel with insulin signaling and its modulators in liver, muscle and adipose tissue. Atorvastatin improves survival in septic rats and this improvement is accompanied by a marked improvement in insulin sensitivity, characterized by an increase in glucose disappearance rate during the insulin tolerance test. Sepsis induced an increase in the expression/activation of TLR4 and its downstream signaling JNK and IKK/NF-κB activation, and blunted insulin-induced insulin signaling in liver, muscle and adipose tissue; atorvastatin reversed all these alterations in parallel with a decrease in circulating levels of TNF-α and IL-6. In summary, this study demonstrates that atorvastatin treatment increased survival, with a significant effect upon insulin sensitivity, improving insulin signaling in peripheral tissues of rats during peritoneal-induced sepsis. The effect of atorvastatin on the suppression of the TLR-dependent inflammatory pathway may play a central role in regulation of insulin signaling and survival in sepsis insult

Towards a new image processing system at Wendelstein 7-X: From spatial calibration to characterization of thermal events

Wendelstein 7-X (W7-X) is the most advanced fusion experiment in the stellarator line and is aimed at proving that the stellarator concept is suitable for a fusion reactor. One of the most important issues for fusion reactors is the monitoring of plasma facing components when exposed to very high heat loads, through the use of visible and infrared (IR) cameras. In this paper, a new image processing system for the analysis of the strike lines on the inboard limiters from the first W7-X experimental campaign is presented. This system builds a model of the IR cameras through the use of spatial calibration techniques, helping to characterize the strike lines by using the information given by real spatial coordinates of each pixel. The characterization of the strike lines is made in terms of position, size, and shape, after projecting the camera image in a 2D grid which tries to preserve the curvilinear surface distances between points. The description of the strike-line shape is made by means of the Fourier Descriptors

Forward modeling of collective Thomson scattering for Wendelstein 7-X plasmas: Electrostatic approximation

In this paper, we present a method for numerical computation of collective Thomson scattering (CTS). We developed a forward model, eCTS, in the electrostatic approximation and benchmarked it against a full electromagnetic model. Differences between the electrostatic and the electromagnetic models are discussed. The sensitivity of the results to the ion temperature and the plasma composition is demonstrated. We integrated the model into the Bayesian data analysis framework Minerva and used it for the analysis of noisy synthetic data sets produced by a full electromagnetic model. It is shown that eCTS can be used for the inference of the bulk ion temperature. The model has been used to infer the bulk ion temperature from the first CTS measurements on Wendelstein 7-X

Temporal changes in the epidemiology, management, and outcome from acute respiratory distress syndrome in European intensive care units: a comparison of two large cohorts

Background: Mortality rates for patients with ARDS remain high. We assessed temporal changes in the epidemiology and management of ARDS patients requiring invasive mechanical ventilation in European ICUs. We also investigated the association between ventilatory settings and outcome in these patients. Methods: This was a post hoc analysis of two cohorts of adult ICU patients admitted between May 1–15, 2002 (SOAP study, n = 3147), and May 8–18, 2012 (ICON audit, n = 4601 admitted to ICUs in the same 24 countries as the SOAP study). ARDS was defined retrospectively using the Berlin definitions. Values of tidal volume, PEEP, plateau pressure, and FiO2 corresponding to the most abnormal value of arterial PO2 were recorded prospectively every 24 h. In both studies, patients were followed for outcome until death, hospital discharge or for 60 days. Results: The frequency of ARDS requiring mechanical ventilation during the ICU stay was similar in SOAP and ICON (327[10.4%] vs. 494[10.7%], p = 0.793). The diagnosis of ARDS was established at a median of 3 (IQ: 1–7) days after admission in SOAP and 2 (1–6) days in ICON. Within 24 h of diagnosis, ARDS was mild in 244 (29.7%), moderate in 388 (47.3%), and severe in 189 (23.0%) patients. In patients with ARDS, tidal volumes were lower in the later (ICON) than in the earlier (SOAP) cohort. Plateau and driving pressures were also lower in ICON than in SOAP. ICU (134[41.1%] vs 179[36.9%]) and hospital (151[46.2%] vs 212[44.4%]) mortality rates in patients with ARDS were similar in SOAP and ICON. High plateau pressure (> 29 cmH2O) and driving pressure (> 14 cmH2O) on the first day of mechanical ventilation but not tidal volume (> 8 ml/kg predicted body weight [PBW]) were independently associated with a higher risk of in-hospital death. Conclusion: The frequency of and outcome from ARDS remained relatively stable between 2002 and 2012. Plateau pressure > 29 cmH2O and driving pressure > 14 cmH2O on the first day of mechanical ventilation but not tidal volume > 8 ml/kg PBW were independently associated with a higher risk of death. These data highlight the continued burden of ARDS and provide hypothesis-generating data for the design of future studies

The clinical relevance of oliguria in the critically ill patient : Analysis of a large observational database

Funding Information: Marc Leone reports receiving consulting fees from Amomed and Aguettant; lecture fees from MSD, Pfizer, Octapharma, 3 M, Aspen, Orion; travel support from LFB; and grant support from PHRC IR and his institution. JLV is the Editor-in-Chief of Critical Care. The other authors declare that they have no relevant financial interests. Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Urine output is widely used as one of the criteria for the diagnosis and staging of acute renal failure, but few studies have specifically assessed the role of oliguria as a marker of acute renal failure or outcomes in general intensive care unit (ICU) patients. Using a large multinational database, we therefore evaluated the occurrence of oliguria (defined as a urine output 16 years) patients in the ICON audit who had a urine output measurement on the day of admission were included. To investigate the association between oliguria and mortality, we used a multilevel analysis. Results: Of the 8292 patients included, 2050 (24.7%) were oliguric during the first 24 h of admission. Patients with oliguria on admission who had at least one additional 24-h urine output recorded during their ICU stay (n = 1349) were divided into three groups: transient - oliguria resolved within 48 h after the admission day (n = 390 [28.9%]), prolonged - oliguria resolved > 48 h after the admission day (n = 141 [10.5%]), and permanent - oliguria persisting for the whole ICU stay or again present at the end of the ICU stay (n = 818 [60.6%]). ICU and hospital mortality rates were higher in patients with oliguria than in those without, except for patients with transient oliguria who had significantly lower mortality rates than non-oliguric patients. In multilevel analysis, the need for RRT was associated with a significantly higher risk of death (OR = 1.51 [95% CI 1.19-1.91], p = 0.001), but the presence of oliguria on admission was not (OR = 1.14 [95% CI 0.97-1.34], p = 0.103). Conclusions: Oliguria is common in ICU patients and may have a relatively benign nature if only transient. The duration of oliguria and need for RRT are associated with worse outcome.publishersversionPeer reviewe