DOCK11 deficiency in patients with X-linked actinopathy and autoimmunity.

Dedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho-guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients' autoimmune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients' lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity.

Treatment of two or more metatarsal fractures.

INTRODUCTION: Metatarsal fractures are the most common type of foot fracture. When surgical treatment is needed, pinning is typically used, either percutaneous or open. However, this fixation method has been criticized by some authors who lament residual malunion and prefer to use plate and/or screws. The primary objective of our study was to compare the outcomes of K-wire versus plate and/or screw fixation for the surgical treatment of two or more metatarsal fractures. The secondary objective was to evaluate the factors that contribute to poor outcomes and complications. We hypothesized that plate and/or screw fixation will produce better functional outcomes than K-wire fixation. MATERIALS AND METHODS: This was a prospective and retrospective multicenter study carried out between 1 January 2010 and 1 June 2018 with a minimum follow-up of 12 months. Three functional scores were determined (AOFAS, FAAM and SF12 physical and mental) preoperatively, postoperatively and at the final assessment. We evaluated the outcomes in the entire study population and in four injury type subgroups, including one with isolated metatarsal fractures to control analysis bias. RESULTS: Our analysis compiled data from 165 patients (123 men, 42 women) who had a mean age of 38 years (16-82). The mean follow-up time was 27.9months (10-120). There were no complications in 130 patients (79%). Skin necrosis occurred in 25 patients (15%). The FAAM score was significantly higher in the plate and/or screw group 70.2 (17-84) versus 60.3 (31-84) in the K-wire group (P=0.033). The 78 (19-100) AOFAS was higher, but not significantly, in the plate and/or screw group versus 70 (12-100) in the K-wire group (P=0.144). CONCLUSION: Trauma to the foot that causes a fracture in two or more metatarsals often occurs due to a crush injury (39%). The frequency of associated bone lesions means that a preoperative CT scan should be done routinely to analyze the injury pattern and determine the best treatment. The fixation method should be adapted to the local conditions; when possible, it is preferable to use rigid fixation with plates and/or screws as it yields better functional outcomes. LEVEL OF EVIDENCE: IV; study with retrospective component.

Peptidomics of enteroendocrine cells and characterisation of potential effects of a novel preprogastrin derived-peptide on glucose tolerance in lean mice.

OBJECTIVES: To analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides. METHODS: High resolution nano-flow liquid chromatography mass spectrometry (LC-MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance. RESULTS: A large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretion in vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435-462a) had no measurable effect, but a progastrin-derived peptide (Gast p59-79), modestly improved glucose tolerance in lean mice. CONCLUSION: LC-MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59-79, with minor effects on glucose tolerance.

Time is money: quantifying savings in outpatient appendectomy.

BACKGROUND: Laparoscopic appendectomy can be performed on a fast-track, short-stay, or outpatient basis with high success rates, low morbidity, low readmission rates, and shorter length of hospital stay. Cost savings from outpatient appendectomy have not been well described. We hypothesize that outpatient laparoscopic appendectomy is associated with cost savings. METHODS: We performed an original retrospective cohort analysis of patients undergoing laparoscopic appendectomy between June 2013 and April 2017 at our academic medical center before and after implementation of an outpatient protocol which began on January 1, 2016. We assessed appendicitis grade, length of stay (LOS), cost, net revenue, and profit margin. RESULTS: After protocol implementation, the percentage of patients discharged from the the postanesthesia care unit (PACU) increased from 3.7% to 29.7% (χ2 p<0.001). The proportion of inpatient admissions and admissions to observation decreased by 5.7% and 20.3%, respectively. On average, PACU-to-home patients had a total hospital cost of $4734 compared with $5781 in patients admitted to observation, for an estimated savings of $1047 per patient (p<0.001). Comparing the time periods, the mean LOS decreased for all groups (p<0.001). Appendicitis grade was higher in those who required inpatient admission, but could not distinguish which patients required an observation bed. DISCUSSION: Outpatient appendectomy saves approximately $1000 per patient. Adoption of an outpatient appendectomy pathway is likely to be gradual, but will result in incremental improvement in resource utilization immediately. Grade does not predict which patients should be observed. Considering established safety, our data support widespread implementation of this protocol. LEVEL OF EVIDENCE: III.

Subunit disassembly and inhibition of TNFα by a semi-synthetic bicyclic peptide.

Macrocyclic peptides are potentially a source of powerful drugs, but their de novo discovery remains challenging. Here we describe the discovery of a high-affinity (Kd = 10 nM) peptide macrocycle (M21) against human tumor necrosis factor-alpha (hTNFα), a key drug target in the treatment of inflammatory disorders, directly from diverse semi-synthetic phage peptide repertoires. The bicyclic peptide M21 (ACPPCLWQVLC) comprises two loops covalently anchored to a 2,4,6-trimethyl-mesitylene core and upon binding induces disassembly of the trimeric TNFα cytokine into dimers and monomers. A 2.9 Å crystal structure of the M21/hTNFα complex reveals the peptide bound to a hTNFα dimer at a normally buried epitope in the trimer interface overlapping the binding site of a previously discovered small molecule ligand (SPD304), which also induces TNF trimer dissociation and synergizes with M21 in the inhibition of TNFα cytotoxicity. The discovery of M21 underlines the potential of semi-synthetic bicyclic peptides as ligands for the discovery of cryptic epitopes, some of which are poorly accessible to antibodies.

Inhibition of platelet adhesion by peptidomimetics mimicking the interactive ß-hairpin of glycoprotein Ibα.

ß-Hairpin peptidomimetics mimicking the interaction sites of the platelet receptor glycoprotein (GP)Ibα with von Willebrand factor (vWF) were synthesised and evaluated for their ability to increase platelet velocity under high shear conditions and to inhibit shear-induced platelet aggregation. A cyclic and bridged dodecapeptide 2e containing a heterochiral diproline motif was identified as a lead compound for the generation of a novel class of potential antiplatelet agents.

CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

Ligand switching in cell-permeable peptides: manipulation of the alpha-integrin signature motif.

A synthetic cell-permeable peptide corresponding to the highly conserved alpha-integrin signature motif, Palmityl-K(989)VGFFKR(995) (Pal-FF), induces integrin activation and aggregation in human platelets. Systematic replacement of the F(992)-F(993) with amino acids of greater or lesser hydrophobicity to create Pal-KVGxxKR peptides demonstrate that hydrophobic amino acids (isoleucine, phenylalanine, tyrosine, tryptophan) are essential for agonist potency. In marked contrast, substitution with small and/or hydrophilic amino acids (glycine, alanine, serine) causes a switch in the biological activity resulting in inhibition of platelet aggregation, adhesion, ADP secretion, and thromboxane synthesis. These substituted, hydrophilic peptides are not true pharmacological antagonists, as they actively induce a phosphotyrosine signaling cascade in platelets. Singly substituted peptides (Pal-AF and Pal-FA) cause preferential retention of pro- or anti-thrombotic properties, respectively. Because the alpha-integrin signature motif is an established docking site for a number of diverse cytoplasmic proteins, we conclude that eliminating critical protein-protein interactions mediated through the hydrophobic amino acids, especially F(993), favors an anti-thrombotic pathway in platelets. Agents derived from the inhibitory peptides described in this study may represent a new therapeutic strategy for anti-platelet or anti-integrin drug development.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

Development of lipopeptides for inhibiting 20S proteasomes.

Proteasomes are responsible for the cytoplasmic turnover of the vast majority of proteins including regulatory proteins. We have synthesized lipopeptides a new class of non-covalent inhibitors of the 20S proteasome and assayed their inhibitory capacities. Their ability to inhibit at micromolar concentrations chymotrypsin-like and post-acid activities depends on peptide length (3 or 6 amino acids), sequence (presence of a positively or negatively charged amino acid), and alkyl chain length (C6-C18). These structural features could be varied to selectively inhibit one or more of the three proteasome activities.