Association of Acinetobacter baumannii EF-Tu with Cell Surface, Outer Membrane Vesicles, and Fibronectin

A conundrum has long lingered over association of cytosol elongation factor Tu (EF-Tu) with bacterial surface. Here we investigated it with Acinetobacter baumannii, an emerging opportunistic pathogen associated with a wide spectrum of infectious diseases. The gene for A. baumannii EF-Tu was sequenced, and recombinant EF-Tu was purified for antibody development. EF-Tu on the bacterial surface and the outer membrane vesicles (OMVs) was revealed by immune electron microscopy, and its presence in the outer membrane (OM) and the OMV subproteomes was verified by Western blotting with the EF-Tu antibodies and confirmed by proteomic analyses. EF-Tu in the OM and the OMV subproteomes bound to fibronectin as detected by Western blot and confirmed by a label-free real-time optical sensor. The sensor that originates from photonic crystal structure in a total-Internal-reflection (PC-TIR) configuration was functionalized with fibronectin for characterizing EF-Tu binding. Altogether, with a novel combination of immunological, proteomical, and biophysical assays, these results suggest association of A. baumannii EF-Tu with the bacterial cell surface, OMVs, and fibronectin

Fast Liquid Chromatography Coupled with Tandem Mass Spectrometry for the Analysis of Vanillic and Syringic Acids in Ice Cores

The development of new analytical systems and the improvement of the existing ones to obtain high-resolution measurements of chemical markers in samples from ice cores, is one of the main challenges the paleoclimatic scientific community is facing. Different chemical species can be used as markers for tracking emission sources or specific environmental processes. Although some markers, such as methane sulfonic acid (a proxy of marine productivity), are commonly used, there is a lack of data on other organic tracers in ice cores, making their continuous analysis analytically challenging. Here, we present an innovative combination of fast liquid chromatography coupled with tandem mass spectrometry (FLC-MS/MS) to continuously determine organic markers in ice cores. After specific optimization, this approach was applied to the quantification of vanillic and syringic acids, two specific markers for biomass burning. Using the validated method, detection limits of 3.6 and 4.6 pg mL-1for vanillic and syringic acids, respectively, were achieved. Thanks to the coupling of FLC-MS/MS with the continuous flow analytical system, we obtained one measurement every 30 s, which corresponds to a sampling resolution of a sample every 1.5 cm with a melting rate of 3.0 cm min-1. To check the robustness of the method, we analyzed two parallel sticks of an alpine ice core over more than 5 h. Vanillic acid was found with concentrations in the range of picograms per milliliter, suggesting the combustion of coniferous trees, which are found throughout the Italian Alps

Vesiculation from Pseudomonas aeruginosa under SOS

Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from the Pseudomonas aeruginosa wild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in the lexAN strains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity

ALPAGE: towards the setting-up of a collaborative work tool

Alpage program, based on a collaboration between historians, geographers and computer researchers, aims to build a historical GIS of Paris. First, we reconstructed the pre-Haussmannian plan of Paris by georeferencing and vectorizing the survey of the cadastre made by P. Vasserot (1810-1836). Then, on these fundamental layers, historical information layers, like medieval and modern ones, will be built by different researchers according to their specific interests (churches, town walls, fiefs, parishes, etc.). Since this tool is technologically complex and since it is intended to be a reference work for further historical studies about Paris, we must immediately take into consideration the organization of further collaborative work. Accessing the GIS data, both to share them with different types of users and to edit new data in it, is an essential question, although it is often considered a trivial one. The issue of the use of a tool by the researchers seems nevertheless to be decisive and intrinsically linked to the success of a GIS. Meeting the needs and expectations of the users, webmapping might be a good solution for editing the geometric and attribute data in a GIS. But today, setting up this kind of platform for collaborative work is still difficult and time consuming. That is why, for Alpage, a temporary solution was found, revealing what organization theorists call a “community of practice”: it combines a centralized management of the references and object identifiers via the web and an independent edition of the objects by thematicians on their own computers. For this reason the DBMS ALPAGE-References has been adapted and posted on the website of the LAMOP

Pulsed Radiofrequency Rhizotomy of the Genicular Nerves of the Knee Guided by Radioscopy and Ultrasonography: Step-By-Step Technique

Osteoarthritis (OA) of the knee is highly prevalent and causes pain, stiffness, and harms the quality of life of millions of patients. Scientific evidence about radiofrequency ablation or rhizotomy of genicular nerves has been presented with increasing frequency in the literature for the treatment of chronic pain related to knee OA as an alternative to total knee arthroplasty. The main indication for this procedure is symptomatic OA unresponsive to conservative treatment, regardless of the disease evolution, although more common indications are in Kellgren-Lawrence grade III or IV, in post-total knee arthroplasty residual pain without an identified cause, in patients with comorbidities and high surgical risk, and those who do not want to undergo surgery. The aim of this study is to describe the step-by-step rhizotomy technique with pulsed radiofrequency of the 3 genicular nerves, guided by radioscopy and ultrasonography.info:eu-repo/semantics/publishedVersio

Calibration and assessment of electrochemical low-cost sensors in remote alpine harsh environments

This work presents results from an original open-source low-cost sensor (LCS) system developed to measure tropospheric O3 in a remote high altitude alpine site. Our study was conducted at the Col Margherita Observatory (2543 m above sea level), in the Italian Eastern Alps. The sensor system mounts three commercial low-cost O3/NO2 sensors that have been calibrated before field deployment against a laboratory standard (Thermo Scientific; 49i-PS), calibrated against the standard reference photometer no. 15 calibration scale of the World Meteorological Organization (WMO). Intra- and intercomparison between the sensors and a reference instrument (Thermo Scientific; 49c) have been conducted for 7 months from May to December 2018. The sensors required an individual calibration, both in laboratory and in the field. The sensor's dependence on the environmental meteorological variables has been considered and discussed. We showed that it is possible to reduce the bias of one LCS by using the average coefficient values of another LCS working in tandem, suggesting a way forward for the development of remote field calibration techniques. We showed that it is possible reconstruct the environmental ozone concentration during the loss of reference instrument data in situations caused by power outages. The evaluation of the analytical performances of this sensing system provides a limit of detection (LOD) <5 ppb (parts per billion), limit of quantification (LOQ) <17 ppb, linear dynamic range (LDR) up to 250 ppb, intra-Pearson correlation coefficient (PCC) up to 0.96, inter-PCC >0.8, bias >3.5 ppb and ±8.5 at 95 % confidence. This first implementation of a LCS system in an alpine remote location demonstrated how to obtain valuable data from a low-cost instrument in a remote environment, opening new perspectives for the adoption of low-cost sensor networks in atmospheric sciences

T-Cell Immune Responses Against Env from CRF12_BF and Subtype B HIV-1 Show High Clade-Specificity that Can Be Overridden by Multiclade Immunizations

BACKGROUND: The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors. METHODOLOGY/PRINCIPAL FINDINGS: As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B. CONCLUSIONS/SIGNIFICANCE: This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate

Ss-Sl2, a Novel Cell Wall Protein with PAN Modules, Is Essential for Sclerotial Development and Cellular Integrity of Sclerotinia sclerotiorum

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum