Identifying conformational changes with site-directed spin labeling reveals that the GTPase domain of HydF is a molecular switch

[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases

Age-Specific 18F-FDG Image Processing Pipelines and Analysis Are Essential for Individual Mapping of Seizure Foci in Paediatric Patients with Intractable Epilepsy

Fluoro-18-deoxyglucose positron emission tomography (FDG-PET) is an important tool for the pre-surgical assessment of children with drug-resistant epilepsy. Standard assessment is carried out visually and this is often subjective and highly user-dependent. Voxel-wise statistics can be used to remove user-dependent biases by automatically identifying areas of significant hypo/hyper-metabolism, associated to the epileptogenic area. In the clinical settings, this analysis is carried out using commercially available software. These software packages suffer from two main limitations when applied to paediatric PET data: 1) paediatric scans are spatially normalised to an adult standard template and 2) statistical comparisons use an adult control dataset. The aim of this work is to provide a reliable observer-independent pipeline for the analysis of paediatric FDG-PET scans, as part of pre-surgical planning in epilepsy. METHODS: A pseudo-control dataset (n = 19 for 6-9y, n = 93 for 10-20y) was used to create two age-specific FDG-PET paediatric templates in standard paediatric space. The FDG-PET scans of 46 epilepsy patients (n = 16 for 6-9y, n = 30 for 10-17y) were retrospectively collated and analysed using voxel-wise statistics. This was implemented with the standard pipeline available in the commercial software Scenium and an in-house Statistical Parametric Mapping v.8 (SPM8) pipeline (including age-specific paediatric templates and normal database). A kappa test was used to assess the level of agreement between findings of voxel-wise analyses and the clinical diagnosis of each patient. The SPM8 pipeline was further validated using post-surgical seizure-free patients. RESULTS: Improved agreement with the clinical diagnosis was reported using SPM8, in terms of focus localisation, especially for the younger patient group: kScenium=0.489 versus kSPM=0.805. The proposed pipeline also showed a sensitivity of ~70% in both age ranges, for the localisation of hypo-metabolic areas on paediatric FDG-PET scans in post-surgical seizure-free patients. CONCLUSION: We show that by creating age-specific templates and using paediatric control databases, our pipeline provides an accurate and sensitive semi-quantitative method for assessing FDG-PET scans of patients under 18y

The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells

Pipeline Comparison for the Pre-Processing of Resting-State Data in Epilepsy

Noise removal is a critical step to recover the signal of interest from resting-state fMRI data. Several pre-processing pipelines have been developed mainly based on nuisance regression or independent component analysis. The aim of this work was to evaluate the ability in removing spurious non-BO LD signals of different cleaning pipelines when applied to a dataset of healthy controls and temporal lobe epilepsy patients. Increased tSNR and power spectral density in the resting-state frequency range (0.01-0.1 Hz) were found for all pre-processing pipelines with respect to the minimally pre-processed data, suggesting a positive gain in terms of temporal properties when optimal cleaning procedures are applied to the acquired fMRI data. All the pre-processing pipelines considered were able to recover the DMN through group ICA. By visually comparing this network across all the pipelines and groups, we found that AROMA, SPM12, FIX and FIXMC were able to better delineate the posterior cingulate cortex

Destination shapes antibiotic resistance gene acquisitions, abundance increases, and diversity changes in Dutch travelers

BACKGROUND: Antimicrobial-resistant bacteria and their antimicrobial resistance (AMR) genes can spread by hitchhiking in human guts. International travel can exacerbate this public health threat when travelers acquire AMR genes endemic to their destinations and bring them back to their home countries. Prior studies have demonstrated travel-related acquisition of specific opportunistic pathogens and AMR genes, but the extent and magnitude of travel\u27s effects on the gut resistome remain largely unknown. METHODS: Using whole metagenomic shotgun sequencing, functional metagenomics, and Dirichlet multinomial mixture models, we investigated the abundance, diversity, function, resistome architecture, and context of AMR genes in the fecal microbiomes of 190 Dutch individuals, before and after travel to diverse international locations. RESULTS: Travel markedly increased the abundance and α-diversity of AMR genes in the travelers\u27 gut resistome, and we determined that 56 unique AMR genes showed significant acquisition following international travel. These acquisition events were biased towards AMR genes with efflux, inactivation, and target replacement resistance mechanisms. Travel-induced shaping of the gut resistome had distinct correlations with geographical destination, so individuals returning to The Netherlands from the same destination country were more likely to have similar resistome features. Finally, we identified and detailed specific acquisition events of high-risk, mobile genetic element-associated AMR genes including qnr fluoroquinolone resistance genes, bla CONCLUSIONS: Our results show that travel shapes the architecture of the human gut resistome and results in AMR gene acquisition against a variety of antimicrobial drug classes. These broad acquisitions highlight the putative risks that international travel poses to public health by gut resistome perturbation and the global spread of locally endemic AMR genes

Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens

© 2015 Brabec et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. The attached file is the published version of the article

Arterial Spin Labeling Reveals Disrupted Brain Networks and Functional Connectivity in Drug-Resistant Temporal Epilepsy

Resting-state networks (RSNs) and functional connectivity (FC) have been increasingly exploited for mapping brain activity and identifying abnormalities in pathologies, including epilepsy. The majority of studies currently available are based on bloodoxygenation- level-dependent (BOLD) contrast in combination with either independent component analysis (ICA) or pairwise region of interest (ROI) correlations. Despite its success, this approach has several shortcomings as BOLD is only an indirect and non-quantitative measure of brain activity. Conversely, promising results have recently been achieved by arterial spin labeling (ASL) MRI, primarily developed to quantify brain perfusion. However, the wide application of ASL-based FC has been hampered by its complexity and relatively low robustness to noise, leaving several aspects of this approach still largely unexplored. In this study, we firstly aimed at evaluating the effect of noise reduction on spatio-temporal ASL analyses and quantifying the impact of two ad-hoc processing pipelines (basic and advanced) on connectivity measures. Once the optimal strategy had been defined, we investigated the applicability of ASL for connectivity mapping in patients with drug-resistant temporal epilepsy vs. controls (10 per group), aiming at revealing between-group voxel-wise differences in each RSN and ROI-wise FC changes. We first found ASL was able to identify the main network (DMN) along with all the others generally detected with BOLD but never previously reported from ASL. For all RSNs, ICA-based denoising (advanced pipeline) allowed to increase their similarity with the corresponding BOLD template. ASL-based RSNs were visibly consistent with literature findings; however, group differences could be identified in the structure of some networks. Indeed, statistics revealed areas of significant FC decrease in patients within different RSNs, such as DMN and cerebellum (CER), while significant increases were found in some cases, such as the visual networks. Finally, the ROI-based analyses identified several inter-hemispheric dysfunctional links (controls > patients) mainly between areas belonging to the DMN, right-left thalamus and right-left temporal lobe. Conversely, fewer connections, predominantly intra-hemispheric, showed the opposite pattern (controls < patients). All these elements provide novel insights into the pathological modulations characterizing a “network disease” as epilepsy, shading light on the importance of perfusion-based approaches for identifying the disrupted areas and communications between brain regions

Testing Biochemistry Revisited: How In Vivo Metabolism Can Be Understood from In Vitro Enzyme Kinetics

A decade ago, a team of biochemists including two of us, modeled yeast glycolysis and showed that one of the most studied biochemical pathways could not be quite understood in terms of the kinetic properties of the constituent enzymes as measured in cell extract. Moreover, when the same model was later applied to different experimental steady-state conditions, it often exhibited unrestrained metabolite accumulation