Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Retracing the evolution of enzyme regulation.

α-Isopropylmalate synthase (IPMS) catalyses the first committed step in the leucine biosynthesis pathway in microorganisms and some plants. It catalyses the condensation of ketoisovalerate (KIV) and acetyl-coenzyme A (AcCoA) to form isopropylmalate and coenzyme A (CoA). IPMS is allosterically inhibited by the product of the pathway, L-leucine. Structurally, IPMS is a homodimer, and each chain consists of a N-terminal (α/β)8 barrel where the active site is located, a catalytic accessory unit formed of subdomain I and subdomain II, and a C-terminal regulatory domain that binds L-leucine. Truncation of IPMS that removes subdomain II or part of subdomain II abolishes catalysis.¹,² Of particular interest in this thesis is IPMS from Neisseria meningitidis (NmeIPMS). Although this enzyme has been extensively studied, there is no full-length crystal structure available, although there are several of a related enzyme, IPMS from Mycobacterium tuberculosis (MtuIPMS), with KIV, and with L-leucine, bound. There is no substantial conformational change observed when the substrate-bound crystal structure is compared to the structure with L-leucine bound. Untangling the dynamic nature of allostery, and how it has evolved, in these proteins is a particular focus of this thesis. There are also structurally similar proteins that catalyse similar reactions that are also of interest. Citramalate synthase (CMS) is structurally similar to IPMS but catalyses the reaction of pyruvate and AcCoA to form citramalate and CoA in an isoleucine biosynthesis pathway and is inhibited by L-isoleucine. Homocitrate synthase (HCS) functions in a lysine biosynthesis pathway in some organisms and utilises ketoglutarate and AcCoA to form homocitrate and CoA. HCS contains a homologous catalytic domain and catalytic accessory unit as IPMS but lacks a regulatory domain and is competitively inhibited by lysine. The similarities, differences, and modularity of these proteins is explored using computational methods and also by the construction of truncated and fusion proteins. Chapter 2 utilises a computational method, statistical coupling analysis, to identify a potential network in NmeIPMS-like IPMS proteins. Subsequent alanine mutations in NmeIPMS demonstrated that mutating charged residues in this proposed network can abolish or attenuate the allosteric signal, suggesting that the network identified may represent a way the allosteric signal is transferred from the allosteric site to the active site. Isothermal titration calorimetry is also used to explore the thermodynamics of L-leucine binding to the wild-type NmeIPMS and to the L-leucine insensitive alanine mutants. Chapter 3 broadens the scope of statistical coupling analysis (SCA) and also uses another computational method, mutual information (MI), to investigate how structurally similar subdomains facilitate catalysis in the presence and absence of a regulatory domain. A population of proteins that contain a regulatory domain, and a population that do not, were assessed using SCA and MI to determine whether there were differences, particularly in the subdomains, that may provide information about maintenance of the balance of flexibility and stability that is crucial to catalysis in these proteins. Chapter 4 uses an active, truncated, form of NmeIPMS to compare and contrast with the wild-type protein. The kinetics of both the truncated NmeIPMS and the wild-type NmeIPMS are investigated under crowded conditions to explore the impact that viscosity has on these dynamic proteins. Alanine mutations are also made in subdomains I and II to investigate the role of particular residues in catalysis and allostery, and these allow comparison with previous work done on MtuIPMS that highlights the difference between two structurally similar groups of IPMS proteins. Chapter 5 describes the cloning, expression, and partial purification of an HCS, SsoHCS, from Sulfolobus solfataricus that appears to have a different type of regulatory domain to the canonical IPMS/CMS regulatory domain. The partial characterisation of this protein suggests that an allosterically regulated HCS has been identified. Chapter 5 also describes the construction of several fusion proteins, where parts of IPMS, HCS, and CMS, are fused to together to explore the modularity of these proteins. Catalysis was preserved in some of the fusions although allostery was not preserved in any so far investigated. The final chapter includes a broad summary of the work in this thesis as well as ideas for future research. This chapter also contains a discussion about the considerable differences between some IPMS enzymes that, although they catalyse the same reaction, are considerably different taxonomically. Additionally, the important role of networks of residues that facilitate catalysis and allostery is analysed

DeltaPCA: A Statistically Robust Method for Detecting Protein Analyte Binding to Aptamer-Functionalised Nanoparticles using Surface-Enhanced Raman Spectroscopy

In this work, we introduce a novel joint experimental design and computational analysis procedure to reliably and reproducibly quantify protein analyte binding to DNA aptamer-functionalised silver nanoparticles using slippery surface-enhanced Raman spectroscopy. We employ an indirect detection approach, based upon monitoring spectral changes in the covalent bond-stretching region as intermolecular bonds are formed between the surface-immobilized probe biomolecule and its target analyte. Sample variability is minimized by preparing aptamer-only and aptamer-plus-analyte samples under the same conditions, and then analysing difference spectra. To account for technical variability, multiple spectra are recorded from the same sample. Our new DeltaPCA analysis procedure takes into account technical variability within each spectral data set while also extracting statistically robust difference spectra between data sets. Proof of principle experiments using thiolated aptamers to detect CoV-SARS-2 spike protein reveal that analyte binding is mediated through the formation of N-H...X and C-H...X hydrogen bonds between the aptamer (H-bond donor) and protein (H-bond acceptor). Our computational analysis code can be freely downloaded from https://github.com/dlc62/DeltaPCA

Independent catalysis of the short form HisG from Lactococcus lactis

ATP-phosphoribosyltransferase (ATP-PRT) catalyses the first step of histidine biosynthesis. Two different forms of ATP-PRT have been described; the homo-hexameric long form, and the hetero-octameric short form. Lactococcus lactis possesses the short form ATP-PRT comprising four subunits of HisG, the catalytic subunit, and four subunits of HisZ, a histidyl-tRNA synthetase paralogue. Previous studies have suggested that HisG requires HisZ for catalysis. Here, we reveal that the dimeric HisG does display ATP-PRT activity in the absence of HisZ. This result reflects the evolutionary relationship between the long and short form ATP-PRT, which acquired allosteric inhibition and enhanced catalysis via two divergent strategies

The Histone Deacetylase HDAC4 Regulates Long-Term Memory in <i>Drosophila</i>

<div><p>A growing body of research indicates that pharmacological inhibition of histone deacetylases (HDACs) correlates with enhancement of long-term memory and current research is concentrated on determining the roles that individual HDACs play in cognitive function. Here, we investigate the role of HDAC4 in long-term memory formation in <i>Drosophila</i>. We show that overexpression of HDAC4 in the adult mushroom body, an important structure for memory formation, resulted in a specific impairment in long-term courtship memory, but had no affect on short-term memory. Overexpression of an HDAC4 catalytic mutant also abolished LTM, suggesting a mode of action independent of catalytic activity. We found that overexpression of HDAC4 resulted in a redistribution of the transcription factor MEF2 from a relatively uniform distribution through the nucleus into punctate nuclear bodies, where it colocalized with HDAC4. As MEF2 has also been implicated in regulation of long-term memory, these data suggest that the repressive effects of HDAC4 on long-term memory may be through interaction with MEF2. In the same genetic background, we also found that RNAi-mediated knockdown of HDAC4 impairs long-term memory, therefore we demonstrate that HDAC4 is not only a repressor of long-term memory, but also modulates normal memory formation.</p> </div

Emotion Regulation Is Associated with Anxiety, Depression and Stress in Adults with Cerebral Palsy

Emotion regulation difficulties are associated with many neurological conditions and negatively impact daily function. Yet little is known about emotion regulation in adults with cerebral palsy (CP). Our aim was to investigate emotion regulation in adults with CP and its relationship with condition-related and/or socio-demographic factors. In a cross-sectional study of adults with CP, participants completed a survey containing the Difficulties in Emotion Regulation Scale (DERS), Depression Anxiety and Stress Scale-21 (DASS-21), and socio-demographic and condition-related questions. Descriptive statistics, chi-squared and Mann–Whitney tests were performed. Of the 42 adults with CP (x31.5 years, SD13.5) that were tested, 38 had within normal limits DERS total scores; however, a significantly higher proportion of participants experienced elevated scores (i.e., more difficulties with emotion regulation) than would be expected in the general population across five of the six DERs subdomains. Moderate–extremely severe depression and anxiety symptoms were reported by 33% and 60% of participants, respectively. The DERS total scores for participants with elevated depression, anxiety, and stress scores were significantly higher than the DERS totals score for those without elevated depression, anxiety, and stress scores. DERS and DASS-21 scores did not differ significantly by condition-related nor socio-demographic characteristics. In conclusion, emotion regulation difficulties were associated with elevated symptoms of depression and anxiety, which were overrepresented in the adults with CP participating in this study

Histories, trends, futures: round table on the Australian internet

This round table was conducted online in February-March 2004, and has been edited for publication. Participants were Roger Clarke, Fiona Martin, Andrew Garton, Jock Given, Gerard Goggin, Lelia Green and Geert Lovink

Domain organisation of human and Drosophila HDAC4 proteins.

<p>A. One isoform of each protein is shown (Human HDAC4 GenBank accession NP_006028, 1084 amino acids; Drosophila HDAC4, isoform D, GenBank accession NP_572868, 1252 amino acids). Regions of human HDAC4 essential for function and corresponding regions of Drosophila HDAC4 are depicted. The deacetylase domain is shown in blue, MEF2 binding motif in yellow, 14-3-3 association sites in red, and the nuclear import signal in brown. B-C. Sequence alignment of Drosophila HDAC4 with regions of human HDAC4 essential for function. B. MEF2 binding motif. C. Conserved serine residues required for 14-3-3ζ binding are shown. D. The residues identified to mediate nuclear localization of vertebrate HDAC4 are also highly conserved. </p