Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli

Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, double-stranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide-mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase-poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids

Novel parent-of-origin-specific differentially methylated loci on chromosome 16

BACKGROUND: Congenital malformations associated with maternal uniparental disomy of chromosome 16, upd(16)mat, resemble those observed in newborns with the lethal developmental lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interestingly, ACDMPV-causative deletions, involving FOXF1 or its lung-specific upstream enhancer at 16q24.1, arise almost exclusively on the maternally inherited chromosome 16. Given the phenotypic similarities between upd(16)mat and ACDMPV, together with parental allelic bias in ACDMPV, we hypothesized that there may be unknown imprinted loci mapping to chromosome 16 that become functionally unmasked by chromosomal structural variants. RESULTS: To identify parent-of-origin biased DNA methylation, we performed high-resolution bisulfite sequencing of chromosome 16 on peripheral blood and cultured skin fibroblasts from individuals with maternal or paternal upd(16) as well as lung tissue from patients with ACDMPV-causative 16q24.1 deletions and a normal control. We identified 22 differentially methylated regions (DMRs) with ≥ 5 consecutive CpG methylation sites and varying tissue-specificity, including the known DMRs associated with the established imprinted gene ZNF597 and DMRs supporting maternal methylation of PRR25, thought to be paternally expressed in lymphoblastoid cells. Lastly, we found evidence of paternal methylation on 16q24.1 near LINC01082 mapping to the FOXF1 enhancer. CONCLUSIONS: Using high-resolution bisulfite sequencing to evaluate DNA methylation across chromosome 16, we found evidence for novel candidate imprinted loci on chromosome 16 that would not be evident in array-based assays and could contribute to the birth defects observed in patients with upd(16)mat or in ACDMPV

Comparing the Consistency of Atom Probe Tomography Measurements of Small-Scale Segregation and Clustering between the LEAP 3000 and LEAP 5000 Instruments

The local electrode atom probe (LEAP) has become the primary instrument used for atom probe tomography measurements. Recent advances in detector and laser design, together with updated hit detection algorithms, have been incorporated into the latest LEAP 5000 instrument, but the implications of these changes on measurements, particularly the size and chemistry of small clusters and elemental segregations, have not been explored. In this study, we compare data sets from a variety of materials with small-scale chemical heterogeneity using both a LEAP 3000 instrument with 37% detector efficiency and a 532-nm green laser and a new LEAP 5000 instrument with a manufacturer estimated increase to 52% detector efficiency, and a 355-nm ultraviolet laser. In general, it was found that the number of atoms within small clusters or surface segregation increased in the LEAP 5000, as would be expected by the reported increase in detector efficiency from the LEAP 3000 architecture, but subtle differences in chemistry were observed which are attributed to changes in the way multiple hit detection is calculated using the LEAP 5000

Pathways through the criminal justice system for prisoners with acute and serious mental illness

Purpose: To evaluate pathways through the criminal justice system for 63 prisoners under the care of prison mental health services. Results: Only a small number (3%) were acutely mentally ill on reception to prison, and this may reflect the successful operation of liaison and diversion services at earlier stages in the pathway. However, a third (33%) went onto display acute symptoms at later stages. Cases displaying suicide risk at arrest, with a history of in-patient care, were at increased risk of acute deterioration in the first weeks of imprisonment, with a general lack of health assessments for these cases prior to their imprisonment. Inconsistencies in the transfer of mental health information to health files may result in at-risk cases being overlooked, and a lack of standardisation at the court stage resulted in difficulties determining onward service provision and outcomes. Foreign national prisoners were under-represented in the sample. Conclusions: Greater consistency in access to pre-prison health services in the criminal justice system is needed, especially for those with pre-existing vulnerabilities, and it may have a role in preventing subsequent deterioration. A single system for health information flow across the whole pathway would be beneficial. Summary points: 1. Only 3% of cases were acutely mentally ill at prison reception. 2. Cases identified at arrest as presenting a high risk of suicide, with a history of in-patient care, may be at increased risk of acute deterioration in the first weeks of imprisonment; but they were over-looked for assessment prior to prison. 3. Inconsistencies in the transfer of mental health information into prison health files may result in overlooked at-risk cases

Megakaryocyte NLRP3 hyperactivation induces mild anemia and potentiates inflammatory response in mice

BackgroundThe NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been described in both immune cells and platelets, but its role in the megakaryocyte (MK) lineage remains elusive.ObjectiveThe aim of this study was to explore the role of NLRP3 inflammasome in megakaryocytes and platelets.MethodsWe generated Nlrp3A350V/+/Gp1ba-CreKI/+ mice carrying a mutation genetically similar to the one observed in human Muckle–Wells syndrome, which leads to hyperactivity of NLRP3 specifically in MK and platelets.ResultsPlatelets from the mutant mice expressed elevated levels of both precursor and active form of caspase-1, suggesting hyperactivity of NLRP3 inflammasome. Nlrp3A350V/+/Gp1ba-CreKI/+ mice developed normally and had normal platelet counts. Expression of major platelet receptors, platelet aggregation, platelet deposition on collagen under shear, and deep vein thrombosis were unchanged. Nlrp3A350V/+/Gp1ba-CreKI/+ mice had mild anemia, reduced Ter119+ cells in the bone marrow, and splenomegaly. A mild increase in MK TGF-β1 might be involved in the anemic phenotype. Intraperitoneal injection of zymosan in Nlrp3A350V/+/Gp1ba-CreKI/+ mice induced increased neutrophil egression and elevated levels of a set of proinflammatory cytokines, alongside IL-10 and G-CSF, in the peritoneal fluid as compared with control animals.ConclusionMK/platelet NLRP3 inflammasome promotes the acute inflammatory response and its hyperactivation in mice leads to mild anemia and increased extramedullary erythropoiesis

Experiences and impact of psychiatric inpatient admissions far away from home: a qualitative study with young people, parents/carers and healthcare professionals

Background: There are significant clinical, policy and societal concerns about the impact on young people (YP), from admission to psychiatric wards far from home. However, research evidence is scarce. Aims: To investigate the impact of at-distance admissions to general adolescent units, from the perspectives of YP, parents/carers and healthcare professionals (HCPs) including service commissioners, to inform clinical practice, service development and policy. Method: Semistructured interviews with purposive samples of YP aged 13–17 years (n=28) and parents/carers (n=19) across five large regions in England, and a national sample of HCPs (n=51), were analysed using a framework approach. Results: There was considerable agreement between YP, parents/carers and HCPs on the challenges of at-distance admissions. YP and parents/carers had limited or no involvement in decision-making processes around admission and highlighted a lack of available information about individual units. Being far from home posed challenges with maintaining home contact and practical/financial challenges for families visiting. HCPs struggled with ensuring continuity of care, particularly around maintaining access to local clinical teams and educational support. However, some YP perceived separation from their local environment as beneficial because it removed them from unhelpful environments. At-distance admissions provided respite for some families struggling to support their child. Conclusions: At-distance admissions lead to additional distress, uncertainty, compromised continuity of care and educational, financial and other practical difficulties, some of which could be better mitigated. For a minority, there are some benefits from such admissions. Clinical implications: Standardised online information, accessible prior to admission, is needed for all Child and Adolescent Mental Health Services units. Additional practical and financial burden placed on families needs greater recognition and consideration of potential sources of support. Policy changes should incorporate findings that at-distance or adult ward admissions may be preferable in certain circumstances

Genomic attributes of airway commensal bacteria and mucosa

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance