Identification of human exTreg cells as CD16+CD56+ cytotoxic CD4+ T cells.

In atherosclerosis, some regulatory T (Treg) cells become exTreg cells. We crossed inducible Treg and exTreg cell lineage-tracker mice (FoxP3eGFP-Cre-ERT2ROSA26CAG-fl-stop-fl-tdTomato) to atherosclerosis-prone Apoe-/- mice, sorted Treg cells and exTreg cells and determined their transcriptomes by bulk RNA sequencing (RNA-seq). Genes that were differentially expressed between mouse Treg cells and exTreg cells and filtered for their presence in a human single-cell RNA-sequencing (scRNA-seq) panel identified exTreg cell signature genes as CST7, NKG7, GZMA, PRF1, TBX21 and CCL4. Projecting these genes onto the human scRNA-seq with CITE-seq data identified human exTreg cells as CD3+CD4+CD16+CD56+, which was validated by flow cytometry. Bulk RNA-seq of sorted human exTreg cells identified them as inflammatory and cytotoxic CD4+T cells that were significantly distinct from both natural killer and Treg cells. DNA sequencing for T cell receptor-ß showed clonal expansion of Treg cell CDR3 sequences in exTreg cells. Cytotoxicity was functionally demonstrated in cell killing and CD107a degranulation assays, which identifies human exTreg cells as cytotoxic CD4+T cells.

Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor.

The Hippo pathway, which plays a critical role in organ size control and cancer, features numerous WW domain-based protein-protein interactions. However, ~100 WW domains and 2,000 PY motif-containing peptide ligands are found in the human proteome, raising a "WW-PY" binding specificity issue in the Hippo pathway. In this study, we have established the WW domain binding specificity for Hippo pathway components and uncovered a unique amino acid sequence required for it. By using this criterion, we have identified a WW domain-containing protein, STXBP4, as a negative regulator of YAP. Mechanistically, STXBP4 assembles a protein complex comprising α-catenin and a group of Hippo PY motif-containing components/regulators to inhibit YAP, a process that is regulated by actin cytoskeleton tension. Interestingly, STXBP4 is a potential tumor suppressor for human kidney cancer, whose downregulation is correlated with YAP activation in clear cell renal cell carcinoma. Taken together, our study not only elucidates the WW domain binding specificity for the Hippo pathway, but also reveals STXBP4 as a player in actin cytoskeleton tension-mediated Hippo pathway regulation.

Intramolecular activity regulation of adhesion GPCRs in light of recent structural and evolutionary information.

The class B2 of GPCRs known as adhesion G protein-coupled receptors (aGPCRs) has come under increasing academic and nonacademic research focus over the past decade due to their physiological importance as mechano-sensors in cell-cell and cell-matrix contexts. A major advance in understanding signal transduction of aGPCRs was achieved by the identification of the so-called Stachel sequence, which acts as an intramolecular agonist at the interface between the N terminus (Nt) and the seven-transmembrane helix domain (7TMD). Distinct extracellular signals received by the Nt are integrated at the Stachel into structural changes of the 7TMD towards an active state conformation. Until recently, little information was available on how the activation process of aGPCRs is realized at the molecular level. In the past three years several structures of the 7TMD plus the Stachel in complex with G proteins have been determined, which provide new insights into the architecture and molecular function of this receptor class. Herein, we review this structural information to extract common and distinct aGPCR features with particular focus on the Stachel binding site within the 7TMD. Our analysis extends the current view of aGPCR activation and exposes similarities and differences not only between diverse aGPCR members, but also compared to other GPCR classes.

Partnering for Integrated Care: A Learning Collaborative for Primary Care and Oral Health Teams.

PURPOSE: The Teaming and Integrating for Smiles and Health (TISH) Learning Collaborative was developed to help health care organizations accelerate progress in integrating delivery of oral and primary care. By providing expert support and a structure for testing change, the project aimed to improve the early detection of hypertension in the dental setting and of gingivitis in the primary care setting, and to increase the rate of bidirectional referrals between oral and primary care partners. We report its outcomes. METHODS: A total of 17 primary and oral health care teams were recruited to participate in biweekly virtual calls over 3 months. Participants tested changes to their models of care through Plan-Do-Study-Act cycles between calls. Sites tracked the percentages of patients screened and referred, completed the TeamSTEPPS (Team Strategies and Tools to Enhance Performance and Patient Safety) and Interprofessional Assessment questionnaires, and provided qualitative feedback and updates in storyboard presentations. RESULTS: On average, with implementation of the TISH Learning Collaborative, sites displayed a nonrandom improvement in the percentages of patients screened for hypertension, referred for hypertension, referred to primary care, and referred for gingivitis. Gingivitis screening and referral to oral health care were not markedly improved. Qualitative responses indicated that teams made progress in screening and referral workflows, improved communication between medical and dental partners, and furthered understanding of the connection between primary care and oral care among staff and patients. CONCLUSIONS: The TISH project is evidence that a virtual Learning Collaborative is an accessible and productive avenue to improve interprofessional education, further primary care and oral partnerships, and achieve practical progress in integrated care.

Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B100-Reactive CD4+ T-Regulatory Cells.

BACKGROUND: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4+ T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (TH1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4+ T cells with an atheroprotective, regulatory T cell (Treg) phenotype in healthy individuals. Yet, the function of apoB-reactive Tregs and their relationship with pathogenic TH1 cells remain unknown. METHODS: To interrogate the function of autoreactive CD4+ T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B978-993 (apoB+) at the single-cell level. RESULTS: We found that apoB+ T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a Treg-like transcriptome, although only 21% of all apoB+ T cells expressed the Treg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB+ T cells formed several clusters with mixed TH signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of TH1, T helper cell type 2 (TH2), and T helper cell type 17 (TH17), and of follicular-helper T cells. ApoB+ T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic TH1/TH17-like cells with proinflammatory properties and only a residual Treg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed TH1/TH17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB+ Tregs in lineage tracing of hyperlipidemic Apoe-/- mice. In adoptive transfer experiments, converting apoB+ Tregs failed to protect from atherosclerosis. CONCLUSIONS: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive Tregs as a novel cellular target in atherosclerosis.

Proteome-level assessment of origin, prevalence and function of leucine-aspartic acid (LD) motifs.

MOTIVATION: Leucine-aspartic acid (LD) motifs are short linear interaction motifs (SLiMs) that link paxillin family proteins to factors controlling cell adhesion, motility and survival. The existence and importance of LD motifs beyond the paxillin family is poorly understood. RESULTS: To enable a proteome-wide assessment of LD motifs, we developed an active learning based framework (LD motif finder; LDMF) that iteratively integrates computational predictions with experimental validation. Our analysis of the human proteome revealed a dozen new proteins containing LD motifs. We found that LD motif signalling evolved in unicellular eukaryotes more than 800 Myr ago, with paxillin and vinculin as core constituents, and nuclear export signal as a likely source of de novo LD motifs. We show that LD motif proteins form a functionally homogenous group, all being involved in cell morphogenesis and adhesion. This functional focus is recapitulated in cells by GFP-fused LD motifs, suggesting that it is intrinsic to the LD motif sequence, possibly through their effect on binding partners. Our approach elucidated the origin and dynamic adaptations of an ancestral SLiM, and can serve as a guide for the identification of other SLiMs for which only few representatives are known. AVAILABILITY AND IMPLEMENTATION: LDMF is freely available online at www.cbrc.kaust.edu.sa/ldmf; Source code is available at https://github.com/tanviralambd/LD/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Aesthetic monitoring-based assessment of oncological safety of oncoplastic management of breast cancer: a multi-center research study.

BACKGROUND: Oncoplastic Breast surgeries (OBS) in breast cancer have evolved to preserve the cancerous breast rather than its amputation to improve postoperative cosmetic results. The lack of evidence to support the oncological safety and benefits of OBS is questionable. In this study, we evaluate various aspects of oncoplastic surgeries with a focused monitoring of aesthetic results and oncological safety. METHODS: This was a multi-center observational study focused on the statistics of data collected from cases who underwent oncoplastic surgeries from the cohort of breast cancer candidates at Mansoura University Hospitals/Egypt and King Faisal Medical Complex/KSA from January 2015 to June 2018. All data were analyzed carefully using SPSS v-26. RESULTS: Eighty cases who underwent different oncoplastic surgeries were included and reviewed for the aesthetic outcome and oncological safety. The recurrence rate was found to be 2.5%. The breast impact treatment scale assessment method was used to analyze the aesthetic outcomes, and average scores were accepted in 90% of patients. CONCLUSIONS: The oncoplastic breast surgeries are feasible and they had a high rate of oncological safety with the maintenance of good aesthetic outcomes and patient satisfaction.

Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy.

The parallel plate flow chamber assay is widely utilized to study physiological cell-cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin-ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.

Breast desmoid tumor management in France: toward a new strategy.

PURPOSE: Desmoid tumors (DTs) are rare tumors that originate from myofibroblastic tissue. Recently, initial wait and see was recommended (ESMO guidelines Ann Oncol 2017) in the most frequent locations. This study investigates the outcome of breast desmoid tumor (BDT) according to the initial strategy. METHOD: Data from all consecutive patients treated from a BDT in four referral centers were collected. Only intra-mammary desmoid tumors were included. A pathological review and a molecular analysis (CTNNB1 gene mutation) were performed (National re-reading network of sarcomas-RRePS). Patients were grouped according to initial strategy: surgery group (SG) and active surveillance group (ASG). RESULTS: A total of 63 patients (61 women, 2 men) met the inclusion criteria. Median age was 50 years (16-86). CTNNB1 mutation was found in 61% (n = 36). SG included 46 patients (73%) (41 partial mastectomies, 2 mastectomies, and 3 mastectomies associated to parietectomies). Surgical margins were positive in 15 patients (33.3%). Median follow-up of SG was 24.9 (0.5-209) months; and 4 patients (8.7%) developed recurrence. ASG included 17 patients (27%). Their median follow-up was 42.2 (0-214) months, and 15 patients (88.2%) did not require any additional treatment. Six patients (35%) had a spontaneous regression, 9 patients (52%) were stable, and 2 patients presented a significant progression that was treated by partial mastectomy. CONCLUSION: This study supports an initial nonsurgical approach to BDTs followed by surgery based on tumor growth in select cases, which is consistent with current ESMO recommendations.

Rapidly growing breast desmoid tumor with intra-thoracic involvement after reconstructive surgery for breast cancer.

Desmoid tumors are very rare soft tissue neoplasia that are slow growing and locally aggressive. They grow anywhere in the body and are rarely develop in the breast . Histopathologic examination confirms diagnosis. Recurrence rate is very high even after complete resection. We report the management of a rare case of rapidly growing breast desmoid with intra-thoracic involvement causing cardiac compression.

Ovarian hormones and antioxidant biomarkers in dromedary camels synchronized with new and re-used controlled intravaginal drug release (CIDR)/GPG (Ovsynch) program during breeding season.

This study aimed to investigate the effect of CIDR, re-used wCIDR, and Ovsynch protocols for the synchronization of follicular waves on ovarian hormones, oxidative stress, and antioxidant biomarkers during the breeding season. Dromedary camels (N = 18) were divided into three equal groups. The first group received CIDR. The second group received previously used wCIDR after thorough cleaning and disinfection. The third group was subjected to GPG protocol. Progesterone (P4), estradiol (E2) l, total antioxidant capacity (TAC), catalase (CAT), lipid peroxide product (MDA), nitric oxide (NO), and glutathione reduced (GSH) were measured. Days during CIDR affected P4 (P = 0.0001), E2 (P = 0.047), TAC (P = 0.01), NO (P = 0.028), and GSH (P = 0.005). Days during re-used wCIDR effected P4, TAC, CAT, NO, GSH, and MDA (P ≤ 0.001). Days during GPG effected P4, E2, TAC, GSH (P = 0.0001), MDA (P = 0.036), and NO (P = 0.02). CIDR-treated camels had high P4 (P = 0.0001), E2 (P = 0.0001), TAC (P = 0.012), and NO (P = 0.0001), with low GSH (P = 0.001), and MDA (P = 0.003). Exogenous progesterone improved ovarian hormones and the antioxidant capacity and minimized the oxidative stress than the GPG treatment and is recommended for future reproductive management of camels.

WITHDRAWN: Incidence of methicillin resistant Staphylococcus aureus (MRSA) in microbial community of cancer patients and evaluation of their resistant pattern.

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Isolation, Characterization and Bioactivities of an Extracellular Polysaccharide Produced from Streptomyces sp. MOE6.

A Streptomyces strain was isolated from soil and the sequence of 1471 nucleotides of its 16S rDNA showed 99% identity to Streptomyces sp. HV10. This newly isolated Streptomyces strain produced an extracellular polysaccharide (EPS) composed mainly of glucose and mannose in a ratio of 1:4.1, as was characterized by Fourier transform infrared spectroscopy (FTIR), HPLC and ¹H-NMR. The antioxidant activities of the partially purified MOE6-EPS were determined by measuring the hydroxyl free radical scavenging activity and the scavenging of 2,2-diphenyl-2-picryl-hydrazyl (DPPH) radicals. In addition, the partially purified MOE6-EPS showed high ferrous ion (Fe2+) chelation activity which is another antioxidant activity. Interestingly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays that were colorimetric assays for NAD(P)H-dependent cellular oxidoreductases and a proxy of the number of viable cells, showed that the partially purified MOE6-EPS inhibited the proliferation of the human breast cancer cells (MDA-MB-231). The scratch wound assay showed that MOE6-EPS reduced the migration of mouse breast cancer cells (4T1). This study reports the production of EPS from Streptomyces species with promising antioxidant, metal chelating and mammalian cell inhibitory activities.

Possible correlation between levansucrase production and probiotic activity of Bacillus sp. isolated from honey and honey bee.

Five bacterial isolates from honey and bee gut were selected based on their high levansucrase activity and levan yield which were strongly positively correlated. All isolates showed good tolerance to temperature up to 70 °C, to NaCl up to 3 M and to 0.1% H2O2. They maintained over 59 and 64% survival at pH 9.0 and 2.0 respectively, but showed varying tolerance to 0.1% bile salts and pancreatic enzymes. Most isolates were susceptible to widely used antibiotics, but demonstrated diverse antimicrobial activity. Non hemolytic isolates were identified on the basis of 16S rRNA sequencing as Bacillus subtilis HMNig-2 and B. subtilis MENO2 with 97% homology. They exhibited promising probiotic characteristics and achieved highest levansucrase activity of 94.1 and 81.5 U/mL respectively. Both exhibited highest biofilm formation ability in static microtiter plate assay. Also, they achieved 34 and 26% adhesion respectively to Caco-2cells and had highest free radical scavenging activity of 30.8 and 26.2% respectively. The levans of the two isolates showed good antimicrobial activity against some pathogens and exhibited positive prebiotic effect (prebiotic index >1) with Lactobacillus casei and Lactobacillus reuteri. Results suggest a correlation between levansucrase production, levan yield and pre-probiotic activities of the studied strains.

Cell surface glycan engineering of neural stem cells augments neurotropism and improves recovery in a murine model of multiple sclerosis.

Neural stem cell (NSC)-based therapies offer potential for neural repair in central nervous system (CNS) inflammatory and degenerative disorders. Typically, these conditions present with multifocal CNS lesions making it impractical to inject NSCs locally, thus mandating optimization of vascular delivery of the cells to involved sites. Here, we analyzed NSCs for expression of molecular effectors of cell migration and found that these cells are natively devoid of E-selectin ligands. Using glycosyltransferase-programmed stereosubstitution (GPS), we glycan engineered the cell surface of NSCs ("GPS-NSCs") with resultant enforced expression of the potent E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) and of an E-selectin-binding glycoform of neural cell adhesion molecule ("NCAM-E"). Following intravenous (i.v.) injection, short-term homing studies demonstrated that, compared with buffer-treated (control) NSCs, GPS-NSCs showed greater neurotropism. Administration of GPS-NSC significantly attenuated the clinical course of experimental autoimmune encephalomyelitis (EAE), with markedly decreased inflammation and improved oligodendroglial and axonal integrity, but without evidence of long-term stem cell engraftment. Notably, this effect of NSC is not a universal property of adult stem cells, as administration of GPS-engineered mouse hematopoietic stem/progenitor cells did not improve EAE clinical course. These findings highlight the utility of cell surface glycan engineering to boost stem cell delivery in neuroinflammatory conditions and indicate that, despite the use of a neural tissue-specific progenitor cell population, neural repair in EAE results from endogenous repair and not from direct, NSC-derived cell replacement.

Acute hemolytic anemia as an initial presentation of Wilson disease in children.

BACKGROUND: Wilson disease (WD) is an inherited disorder of copper metabolism. Hemolytic anemia in WD occurs in up to 17% of patients at some point during their illness. AIM: To screen for WD among children presenting with hemolytic anemia. METHODOLOGY: Twenty cases (mean age, 8.8 ± 3.9 y) with Coombs-negative hemolytic anemia, attending the hematology clinic of children hospital, Cairo University, were screened for WD by serum ceruloplasmin level, 24 hours urinary copper before and after D-penicillamine challenge test, and slit-lamp examination for detecting Kayser-Fleischer rings. RESULTS: No case had low ceruloplasmin, whereas bilateral Kayser-Fleischer rings was detected in 5% of our cases. Urinary copper was elevated in 5% before and in 40% after D-penicillamine challenge test. According to the scoring system used, 1 case had definite WD and 7 cases were likely to have WD. These 8 (40%) cases were referred to as group B. Group B had a significantly lower hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, and reticulocytes (P=0.04, 0.001, 0.04, and 0.04, respectively) and a significantly higher urinary copper after penicillamine (P=0.000) when compared with group A (unlikely WD). CONCLUSION: WD is not uncommon in children with hemolytic anemia after exclusion of other common causes.

Ileal prolapse through patent omphalomesenteric duct in a two year-old boy: a case report.

BACKGROUND: Patent omphalomesenteric duct is one of the birth defects included in the spectrum of vitelline duct abnormalities. It is a rare anomaly with estimated prevalence of 0.13-0.2% in the general population. The most common presentation of patent vitelline duct is yellowish or mucoid type umbilical discharge which is usually noted in neonatal age or infancy. The main stay of diagnosis is clinical and outcome is favorable as long as timely surgical correction is offered. Here we present a 2 years old male child who presented with ileal prolapse through patent vitelline duct which is an exceptional mode of presentation of this pathology. CASE PRESENTATION: 2 years old Ethiopian male child who was noticed to have umbilical discharge since early infancy presented with protrusion of pinkish mass per the umbilicus of 4 h duration. He had no signs and symptoms of bowel obstruction. Abdominal examination revealed a prolapsed bowel which was viable via the umbilicus which was about 6 cm long. Otherwise, he had no abdominal tenderness or rigidity. He was explored with a smiley incision just above the umbilicus. The prolapsed bowel was reduced gently to the abdominal cavity. The tract of the Patent vitelline duct was identified and completely resected along with a wedge of ileum at its base. Primary repair of the ileal end where the tract was inserted was done in two layers and abdomen was closed in layers. The child had smooth post op course and was discharged on the 4th post-operative day. CONCLUSION: Prolapse of a bowel through the umbilicus is unusual presentation of a rare anomaly namely patent vitelline duct. This presentation warrants early surgical intervention before bowel ischemia issues. Hence, all clinicians dealing with children should be aware of this rare pathology so that urgent surgical management can be offered.