Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue

Identification and characterization of EhCaBP2: a second member of the calcium-binding protein family of the protozoan parasite entamoeba histolytica

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca2+ and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca2+ binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca2+ binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca2+-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca2+ concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

A TCR derived from a patient with relapsing-remitting multiple sclerosis engages the self-peptide myelin basic protein in the context of HLA-DQ1 in a very unusual way

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis

The purification, identification, crystallization and preliminary crystallographic studies of an allergy-related protein, Pru du amandin, from P. dulcis nuts are reported

Outcome of perioperative 24-hour infusion of intravenous lignocaine on pain and QoR-15 scores after breast cancer surgery-A randomised controlled trial

Background and Aims: Despite many analgesic modalities available, postoperative pain management after breast cancer surgery remains a challenge, which translates into poor quality of recovery, if untreated. Intravenous lignocaine with its anti-inflammatory, antihyperalgesic, and analgesic properties could provide a good option for these patients. The aim of this study was to evaluate the effect of intravenous lignocaine on postoperative pain relief and quality of recovery in patients undergoing surgery for breast cancer. Methods: In this prospective double-blind placebo-controlled randomised study, sixty-six patients undergoing breast cancer surgery were assigned 1:1 to placebo or intravenous lignocaine (Group L). Group L received an intravenous 1.5 mg/kg of lignocaine bolus at induction, followed by an intravenous infusion of 1 mg/kg/h for 24 hours intravenously, while the control group was given equal volume of normal saline. Pain scores, opioid utilisation, and quality of recovery (QoR-15) at 24 hours and on the day of suture removal were compared. Results: Statistically significant reduction was observed in both static (P = 0.01, 6 hours) and dynamic postoperative pain (P = 0.030, 24 hours), with consequential delay in the need for the first dose of opioid (P = 0.014) as well as decreased 24-hour postoperative opioid consumption (P < 0.001) and decreased post-operative nausea and vomiting (PONV) (P < 0.05) in the lignocaine group. Global QoR-15 was significantly better at 24 -hours in group L on postoperative day 1 (P < 0.001), albeit there was no significant difference at suture removal. No lignocaine related side effects were observed. Conclusion: Intravenous lignocaine can be safely used as an alternative perioperative non-opioid analgesic for early postoperative pain and recovery

Purification, identification and preliminary crystallographic studies of an allergenic protein from Lathyrus sativus

A 24 kDa protein was purified from the seeds of L. sativus by ammonium sulfate fractionation and ion-exchange chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method

Absence of two N-terminal peptide residues (P-2, P-1) causes no major conformational change of peptide-binding groove.

<p>(A) Top view of peptide-binding groove with pHA*^{P1-P11 [P1V]} linked at αC65 (DRα, ribbon, cyan; DR1β, ribbon, blue; peptide, stick, green). (B) Close-up view of the peptide N-terminus of the structure shown in (A). Hydrogen bonds between DR1 residues and the peptide N-terminus are shown with dotted lines. A water molecule coordinating a hydrogen bond from DR1β H81 to the peptide N-terminus is shown as a blue sphere. (C) Top view of peptide-binding groove with full-length pHA^P-2-P11 (ribbon and stick, gray; 1DLH). (D) Close-up view of the peptide N-terminus of the structure shown in (C). Hydrogen bonds between DR1 residues and the peptide N-terminus are shown with dotted lines. (E) Superimposition of truncated (green) and full-length (gray) peptide.</p

DM-catalyzed peptide release is enhanced by mutations that weaken interactions between the peptide N-terminus and DR1.

<p>(A, B) DR1β mutants H81A, V85S and V85D and DRα mutant S53A were compared with wild type DR1 complexes in peptide dissociation assays. Dissociation at pH 5.3 and 25 ^oC of Alexa-488-labeled full-length pHA^{P-2-P11 [P5Alexa]} peptide from preloaded DR1 complexes (100 nM) in the presence of 50 µM unlabeled pHA^P-2-P11 competitor peptide was measured by FP. Dissociation curves were preformed in the absence (A) or presence (B) of 100 nM DM. FP values are presented in millipolarization units (mP).</p

Structural comparison of DR1 carrying truncated and full-length peptide.

<p>(A–D) Overlay of DR1 carrying a truncated HA peptide variant (pHA*^{P1-P11 [P1V]}) missing N-terminal residues P-2 and P-1 (DRα cyan, DR1β blue, peptide green) and DR1 carrying full-length HA peptide (pHA^P-2-P11, gray, 1DLH). (A) Side view of overlaid DR1 ectodomains. (B) Top view of overlaid peptide-binding grooves with side chains near the peptide N-terminus shown in stick representation. (C) Close-up view of selected DRα chain residues shown in stick model near the P1 anchor residue. (D) Close-up view of selected DR1β chain residues shown in stick model near the P1 anchor.</p