Composite of Relevant Endpoints for Sjogren's Syndrome (CRESS):development and validation of a novel outcome measure

Background Recent randomised controlled trials (RCTs) in primary Sjogren's syndrome used the European League Against Rheumatism (EULAR) Sjogren's Syndrome Disease Activity Index (ESSDAI) as their primary endpoint. Given the heterogeneous and complex nature of primary Sjogren's syndrome, it might be more appropriate to also assess other clinically relevant disease features. We aimed to develop a novel composite endpoint for assessing treatment efficacy in patients with primary Sjogren's syndrome: the Composite of Relevant Endpoints for Sjogren's Syndrome (CRESS). Methods A multidisciplinary expert team selected clinically relevant items and candidate measurements for inclusion in the composite score. For each measurement, cutoff points for response to treatment were chosen based on expert opinion, previously published data on minimal clinically important improvements, and trial data, primarily the week-24 data of the single-centre ASAP-III trial of abatacept versus placebo. CRESS was validated using data from three independent RCTs: one trial of rituximab (TRACTISS), one of abatacept (multinational trial), and one of tocilizumab (ETAP). We calculated the number and percentage of patients who were responders in the separate CRESS items, and the percentage of responders based on the total CRESS at the primary endpoint visits (week 48 for TRACTISS, week 24 for the other two trials). Patients with fewer than three items available for evaluating CRESS response were imputed as non-responders. Findings Based on expert opinion, five complementary items were selected to assess response: (1) systemic disease activity by Clinical ESSDAI (less than 5 points); (2) patient-reported symptoms by EULAR Sjogren's Syndrome Patient Reported Index, assessed by a decrease of at least 1 point or at least 15% from baseline; (3) tear gland item by Schirmer's test and ocular staining score, assessed by an increase of at least 5 mm or decrease of at least 2 points, respectively, in patients with abnormal Schirmer's test or ocular staining score findings at baseline, or, in patients with normal baseline values, assessed by no change to abnormal for both; (4) salivary gland item, assessed by unstimulated whole saliva secretion (increase of at least 25%) and salivary gland ultrasonography (decrease of at least 25%); and (5) serology, assessed by rheumatoid factor (decrease of at least 25%) and IgG (decrease of at least 10%). Total CRESS response is defined as response on at least three of five items. Post-hoc assessment of phase 3 trial data showed that CRESS response rates at the primary endpoint visits were 60% (24 of 40) for abatacept versus 18% (seven of 39) for placebo (p Interpretation The CRESS is a feasible, well-balanced, composite endpoint for use in trials of primary Sjogren's syndrome. As a next step, the CRESS will require validation in a prospective RCT. Copyright (C) 2021 Elsevier Ltd. All rights reserved

Collagen-Like Proteins in Pathogenic E. coli Strains

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages

Endogenous Retinoic Acid Activity in Principal Cells and Intercalated Cells of Mouse Collecting Duct System

Background: Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in postnatal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.Methodology/Principal Findings: RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, beta-galactosidase. Double immunostaining was performed for beta-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle's loop and distal tubules.Conclusions/Significance: Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the collecting duct system

Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop.

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites

Collagen-like proteins from the genomes of <i>E. coli</i> O157:H7 EDL933 and Sakai strains, and their corresponding prophage locations.

*<p>Defective protein sequence, with frame-loss between domains. The original gene sequence may correspond to two open reading frames.</p

Collagen-like proteins from prophages embedded in the genomes of <i>E. coli</i> O157:H7 and other EHEC strains, referred here as EPclA to EPclD (EHEC Prophage collagen-like A to D).

<p>(<b>A</b>) Domain architectures. The collagen triple helical domains are labelled “Col”, and domains predicted to adopt an α-helical coiled-coil conformation (see text) are labelled “PCoil” (for phage coiled-coils). Key to other domain labels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037872#pone-0037872-t001" target="_blank">Table 1</a>): PfN, phage fibre N-terminal domain; PfC, phage fibre C-terminal domain; PfC2, phage fibre C-terminal domain, variant 2; Pf2, phage fibre repeat 2. (<b>B</b>) Sequence of a representative collagen-like protein with EPclA architecture (<u>ECs2717</u>), from the genome of <i>E. coli</i> O157:H7 Sakai. (<b>C</b>) Sequence of a representative collagen-like protein with EPclB architecture (<u>Z1483</u>), from the genome of <i>E. coli</i> O157:H7 EDL933. Amino acid sequences corresponding to the different predicted domains are colour-coded as in (<b>A</b>).</p

Molecular Investigations of Protriptyline as a Multi-Target Directed Ligand in Alzheimer's Disease

<div><p>Alzheimer's disease (AD) is a complex neurodegenerative disorder involving multiple cellular and molecular processes. The discovery of drug molecules capable of targeting multiple factors involved in AD pathogenesis would greatly facilitate in improving therapeutic strategies. The repositioning of existing non-toxic drugs could dramatically reduce the time and costs involved in developmental and clinical trial stages. In this study, preliminary screening of 140 FDA approved nervous system drugs by docking suggested the viability of the tricyclic group of antidepressants against three major AD targets, viz. Acetylcholinesterase (AChE), β-secretase (BACE-1), and amyloid β (Aβ) aggregation, with one member, protriptyline, showing highest inhibitory activity. Detailed biophysical assays, together with isothermal calorimetry, fluorescence quenching experiments, kinetic studies and atomic force microscopy established the strong inhibitory activity of protriptyline against all three major targets. The molecular basis of inhibition was supported with comprehensive molecular dynamics simulations. Further, the drug inhibited glycation induced amyloid aggregation, another important causal factor in AD progression. This study has led to the discovery of protriptyline as a potent multi target directed ligand and established its viability as a promising candidate for AD treatment.</p></div

Destabilization of amyloid dimer by protriptyline.

<p><b>A</b>. Evolution of monomer-monomer interaction strength over time for free dimer (broken line) and Protriptylline-bound dimer (solid line). <i>Inset</i>. Distributions of the interactions from multiple trajectories, and the dimer interactions with Protriptylline (in brown) <b>B</b>. Distributions of the asphericity for free (in broken line) and Protriptylline-bound (solid line) dimer <b>C</b>. Representative snapshot of most populated cluster of free, and <b>D</b>. Protriptylline-bound dimer [16–20 region in blue colour with 19–20 showed in line representation; protriptyline in red colour and two Aβ peptides are in cyan and limon colour respectively] <b>E</b>. Residue-residue contact probabilities for free dimer, and <b>F</b>. Protriptylline-bound dimer <b>G</b>. Residue-wise Beta sheet percentages for free dimer (in red) and Protriptylline-bound dimer (in blue) <b>H</b>. Residue-wise helical percentages for free dimer (in red) and Pro-bound dimer (in blue).</p

Protriptyline as MTDL.

<p>The scheme represents that protriptyline (at the center) is able to inhibit key targets of AD pathogenesis such as AChE, BACE-1, Amyloid aggregation and glycation induced amyloid aggregation.</p