Decreased Rhes mRNA levels in the brain of patients with Parkinson's disease and MPTP-treated macaques

In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinsonâ\u80\u99s disease (PD), Schizophrenia (SCZ), and Bipolar Disorder (BD), when compared to healthy controls (about 200 post-mortem samples). Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the putamen of patients suffering from PD showed a significant 35% reduction of this transcript, compared to healthy subjects. Interestingly, in line with observations obtained in humans, we found 27% decrease in Rhes mRNA levels in the putamen of MPTP-treated primates. Based on the established inhibitory influence of Rhes on dopamine-related responses, we hypothesize that its striatal downregulation in PD patients and animal models of PD might represent an adaptive event of the dopaminergic system to functionally counteract the reduced nigrostriatal innervation

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Mitochondrial DNA depletion by ethidium bromide decreases neuronal mitochondrial creatine kinase: Implications for striatal energy metabolism

<div><p>Mitochondrial DNA (mtDNA), the discrete genome which encodes subunits of the mitochondrial respiratory chain, is present at highly variable copy numbers across cell types. Though severe mtDNA depletion dramatically reduces mitochondrial function, the impact of tissue-specific mtDNA reduction remains debated. Previously, our lab identified reduced mtDNA quantity in the putamen of Parkinson’s Disease (PD) patients who had developed L-DOPA Induced Dyskinesia (LID), compared to PD patients who had not developed LID and healthy subjects. Here, we present the consequences of mtDNA depletion by ethidium bromide (EtBr) treatment on the bioenergetic function of primary cultured neurons, astrocytes and neuron-enriched cocultures from rat striatum. We report that EtBr inhibition of mtDNA replication and transcription consistently reduces mitochondrial oxygen consumption, and that neurons are significantly more sensitive to EtBr than astrocytes. EtBr also increases glycolytic activity in astrocytes, whereas in neurons it reduces the expression of mitochondrial creatine kinase mRNA and levels of phosphocreatine. Further, we show that mitochondrial creatine kinase mRNA is similarly downregulated in dyskinetic PD patients, compared to both non-dyskinetic PD patients and healthy subjects. Our data support a hypothesis that reduced striatal mtDNA contributes to energetic dysregulation in the dyskinetic striatum by destabilizing the energy buffering system of the phosphocreatine/creatine shuttle.</p></div

Impaired mitochondrial respiration increases astrocytic, but not neuronal, rate of glycolysis.

<p>(A) Basal extracellular acidification rate (ECAR) of NECos, neurons, and astrocytes, in the presence and absence of EtBr. (B) Relative quantity of electron donors (i.e. NAD(P)H, FADH₂) across culture conditions, measured by MTS absorbance and normalized to cell number. (C) Relative ADP plotted against relative ATP. Arrows show the direction of change from control to EtBr treatment for each culture condition. RLU, relative luminescence units. Error bars reflect +/-SEM. N for each group is included in figure legends. (A) *** = p < 0.001; * = p < 0.05, following Dunn’s post-hoc test. (B), (C) *** = p < 0.001, ** = p < 0.01 compared to respective controls.</p

EtBr treatment reduces oxygen consumption and increases mitochondrial spare capacity in astrocytes.

<p>(A) Diagram of the procedure and measurements of the Seahorse assay. The sequential addition of mitochondrial toxins permits the measurement of different respiratory states. After recording basal respiration, oligomycin (OGM) is added to block complex V and to eliminate ATP production-linked oxygen consumption. Addition of FCCP allows the free flux of protons through the mitochondrial inner membrane and maximum oxygen consumption. Rotenone and antimycin A (ROT+AMA) inhibit complex I and III, and prevent proton pumping. Non-mitochondrial residual oxygen consumption is subtracted from all measurements. (B) Oxygen consumption rate (OCR) of NECos, neurons, and glia, in the presence and absence of EtBr. (C) Basal, leak, max, and spare capacity OCR of NECos, neurons, and astrocytes, in the presence and absence of EtBr. N for each group is included in figure legends. Error bars reflect +/-SEM. *** = p < 0.001; ** = p < 0.01; * = p < 0.05, after Dunn’s post-hoc test.</p

Exposure to EtBr decreases mtRNA in rat striatal co-cultures, purified neuronal cultures, and astrocytes.

<p>(A-C) Log2-fold decrease in mtRNA expression after EtBr treatment assayed with RNASeq, in (A) NECos, (B), pure neuronal cultures, and (C) astrocytes. Each bar is a comparison of three control samples to three EtBr-treated samples, each pooled from two independently dissected culture experiments. MultiRankSeq analysis with false discovery rate–adjusted p-values taken from the DESeq comparison. mtRNAs were the most significantly changed RNA transcripts in neurons with a p-value of 0. (D-F) Log2-fold decrease in mtRNA expression after EtBr treatment assayed with qPCR. Error bars reflect delta-method propagated +/-SEM, with level of significance determined following Dunn’s post-hoc test. 2D[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190456#pone.0190456.ref005" target="_blank">5</a>] = 5ng/ml EtBr for 2 days, 4D[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190456#pone.0190456.ref005" target="_blank">5</a>] = 5ng/ml EtBr for 4 days, 2D[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190456#pone.0190456.ref050" target="_blank">50</a>] = 50ng/ml EtBr for 2 days, 4D[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190456#pone.0190456.ref050" target="_blank">50</a>] = 50ng/ml EtBr for 4 days. N for each group is included in figure legends. *** = p < 0.001, ** = p < 0.01, relative to controls. Transcripts are arranged according to their distance from HSP2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190456#pone.0190456.g001" target="_blank">Fig 1A</a>). The ND6 gene, the only mRNA on the light chain, is shown last.</p

The mitochondrial genome (mtDNA).

<p>rRNA-encoding and protein-encoding genes are shown; tRNA-encoding genes are withheld for clarity. Teal arrows depict the direction of mtDNA replication from the heavy strand or light strand origins of replication (O_H, O_L); red arrows depict the direction of polycistronic transcription from the heavy strand or light strand promoters (HSP1/2, LSP). *—ND6 is the only protein-encoding gene on the light chain. Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190456#pone.0190456.ref029" target="_blank">29</a>].</p