Integrin Beta Research Articles

Reduced susceptibility to COVID-19 associated with ABO blood group and pre-existing anti-A and anti-B antibodies

BackgroundSusceptibility to severe acute respiratory syndrome coronavirus 2 shows individual variability in un-vaccinated and previously un-exposed individuals. We investigated the impact of ABO blood group, titers of anti-A and anti-B, other blood group antigens, and the extracellular deposition of ABH antigens as controlled by secretor fucosyltransferase 2 (FUT2) status. Study design and methodsWe studied incidents in three different hospitals between April to September 2020, where un-diagnosed coronavirus disease 2019 (COVID-19) patients were cared for by health care workers without use of personal protection and with close contact while delivering therapy. We recruited 108 exposed staff, of whom 34 were diagnosed with COVID-19. ABO blood type, titer of anti-A and -B, blood group specific alleles, and secretor status were determined. ResultsBlood group O was associated with lower risk of COVID-19 (OR 0.39, 95 %CI (0.16–0.92), p = 0.03) compared to non-O, i.e., blood groups A, B and AB. High titer anti-A immunoglobulin G (IgG) compared to low titer was associated with lower risk of COVID-19 (OR 0.24 95 %CI (0.07–0.78), p = 0.017). High titer of anti-B immunoglobulin M (IgM) compared to no anti-B (IgM) was associated with lower risk of COVID-19 (OR 0.16, 95 %CI (0.039–0.608), p = 0.006) and the same applies to low titer anti-B (IgM) compared to no titer (OR 0.23, 95 %CI (0.07–0.72), p = 0.012).The 33Pro variant in Integrin beta-3, that is part of human platelet antigen 1b (HPA-1b), was associated with lower risk of COVID-19 (OR 0.23, 95 %CI (0.034–0.86), p = 0.028). ConclusionOur data showed that blood group O, anti-A (IgG) titer, anti-B (IgM) titer as well as HPA-1b are associated with lower risk for COVID-19.

Assessment Of Endometrial Integrin Beta 5 In Women With Unexplained Recurrent Pregnancy Loss.

To examine the relationship between endometrial integrin beta 5 level and risk of recurrent pregnancy loss. The descriptive, prospective, observational, case-controlstudy was conducted at the Kafrelsheikh University Hospital, Egypt, from January to May 2022, and comprised women aged up to 35 years with at least 1 live birth delivery beyond 20-week gestation with normal thyroid and prolactin levels. Age-matched normal fertile women were enrolled as controls. All the participants were subjected to detailed history and complete clinical examination. Endometrial integrin beta 5 was assessed using an antibody sandwich enzyme-linked immunosorbent assay. Data was analysed using SPSS 20. Of the 50 subjects, 25(50%) were cases with a mean age of 26.72±2.64 years, and 25(50%) were controls with a mean age of 25.36±2.16 years. The integrin beta 5 level was significantly lower among the cases than the controls (p<0.05). The best cut-off level of serum integrin beta 5 was ≤2.5765 with area under curve 0.886, sensitivity 88%, specificity 76%, positive predictive value 78.6%, negative predictive value 86.4%, and accuracy 82%. Therewas an inverse correlationbetween endometrial integrinbeta 5 andthe risk ofrecurrentpregnancy loss.

Expression and diagnostic significance of integrin beta-2 in synovial fluid of patients with osteoarthritis.

Osteoarthritis (OA) is characterized by synovial cartilage degeneration and is the leading cause of disability and pain worldwide. This study sought to investigate the expression of integrin beta-2 (ITGB2) in synovial fluid of OA patients and its clinical significance. A total of 110 OA patients were enrolled, who were classified into grade I (N = 35), II (N = 42), and III (N = 33) according to the Kellgren-Lawrence classification, with 110 healthy subjects as controls, and their clinical data were compared. ITGB2 level was detected by RT-qPCR. The receiver operating characteristic curve was used to analyze the predictive value of ITGB2 on OA occurrence. The correlation between ITGB2 and bone metabolism indexes procollagen type I N-terminal peptide (PINP), bone glaprotein (BGP), bone alkaline phosphatase (BALP), and β-collagen I telopeptide (β-CTX) was analyzed by the Pearson method. Logistic regression model was performed to analyze the influencing factors of OA. The content of red blood cells, white blood cells, PINP, BGP, and BALP was lowered in OA patients, while β-CTX was elevated. ITGB2 was highly-expressed in OA patients, negatively-correlated with PINP, BGP, and BALP, but positively-correlated with β-CTX. ITGB2 level increased with the elevation of OA grade. The ITGB2 level >1.375 had certain diagnostic values for OA. ITGB2 level is related to OA severity and may be a biomarker for OA classification. ITGB2 was an independent risk factor for OA. High expression of ITGB2 in synovial fluid can assist OA diagnosis and may be a biomarker for OA grade.

A new antibody for the study of human CD157 expression and function

Abstract CD157 is an ectoenzyme that has both cyclic ADP-ribose hydrolase and ADP-ribosyl cyclase activities. In the human hematopoietic system, CD157 is prevalently expressed by cells of the myelomonocytic lineage, and interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signaling. CD157 is implicated in the pathophysiology of several neurological disorders. Although it lacks a cytoplasmatic domain, CD157 is able to transduce intracellular signals by establishing functional and structural crosstalk with β1 (CD29) and β2 (CD18) integrins. CD157 ligation causes integrin– dependent cytoskeletal remodeling and increase in F-actin content. Activation of downstream signaling pathways leads to cell polarization, with CD157 migrating prevalently to the rear of the cells whereas F-actin is localized at the opposite pole. However, the molecular and cellular functions of CD157 still remain poorly understood. Here, we describe the generation of a monoclonal antibody specific for human CD157 (clone W21007F) that can be used in flow cytometry, immunofluorescence and blocking of CD157 mediated adhesion of monocytes to fibronectin. Treatment of the pro-monocytic, myeloid leukemia cell line U937 with clone W21007F induces cell polarization and a marked increase in filamentous actin, with CD157 clustering at the uropod. The majority of F-Actin fibers are located at the opposite end of CD157, and CD157 co-localizes with integrin beta-1 in distinct membrane domains. The reference clone SY/11B5 and isotype control antibody do not show this effect. In conclusion, anti-human CD157 (clone W21007F) is a valuable tool for the study of CD157 function and expression in human cells.

Sex-specific and immune-cell-specific contributions of beta2 integrins in myocardial ischemia-reperfusion injury

Leukocyte recruitment and their mediated inflammatory responses are critical for cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury, which accounts for 9% mortality and 10% morbidity rates in ischemic heart disease patients. Blocking leukocyte recruitment in mouse knockouts (KO) of beta2 integrin (CD18) or blocking beta2 integrin with anitibodies in multiple animals significantly reduced infarct size after myocardial I/R injury. However, the cell-specific contribution of leukocyte beta2 integrin to I/R injury is unknown. In this study, we used the newly established CD18flox/flox (hITGB2 KI) mouse strain to address this knowledge gap. We crossed them to CSF1R-cre (CD115) and MRP8-cre (S100A8) and tested the KO of beta2 integrins in different leukocyte populations. Interestingly, CSF1R-cre CD18flox/flox unexpectedly deleted beta2 integrins in all peripheral blood leukocyte populations, including blood neutrophils, monocytes, CD4 T cells, CD8 T cells, B cells, and NK cells. It also elevated the cell number of these leukocyte populations in peripheral blood. In MRP8-cre CD18flox/flox mice, beta2 integrins were only knocked out in neutrophils but not other peripheral blood leukocytes. And only neutrophil number was elevated in peripheral blood. After 35 minutes of myocardial ischemia and 24 hours of reperfusion, we found both CSF1R-cre CD18flox/flox and MRP8-cre CD18flox/flox mice have significantly reduced infarct size compared to cre- controls. However, if we distinguish the sex in analysis, we only found a significant alleviation in female but not male CSF1R-cre CD18flox/flox mice. In contrast, we observed a significant alleviation only in male but not female MRP8-cre CD18flox/flox mice. These results suggested sex-specific and immune-cell-specific contributions of beta2 integrins in myocardial ischemia-reperfusion injury and provided new insights into beta2 integrin targeting therapies. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454, R41HL156322, and R44HL152710) and a startup fund from UConn Health. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeting fatty acid oxidation via Acyl-CoA binding protein hinders glioblastoma invasion

The diffuse nature of Glioblastoma (GBM) tumors poses a challenge to current therapeutic options. We have previously shown that Acyl-CoA Binding Protein (ACBP, also known as DBI) regulates lipid metabolism in GBM cells, favoring fatty acid oxidation (FAO). Here we show that ACBP downregulation results in wide transcriptional changes affecting invasion-related genes. In vivo experiments using patient-derived xenografts combined with in vitro models demonstrated that ACBP sustains GBM invasion via binding to fatty acyl-CoAs. Blocking FAO mimics ACBPKD-induced immobility, a cellular phenotype that can be rescued by increasing FAO rates. Further investigation into ACBP-downstream pathways served to identify Integrin beta-1, a gene downregulated upon inhibition of either ACBP expression or FAO rates, as a mediator for ACBP’s role in GBM invasion. Altogether, our findings highlight a role for FAO in GBM invasion and reveal ACBP as a therapeutic vulnerability to stall FAO and subsequent cell invasion in GBM tumors.

Biological injection therapy with leukocyte-poor platelet-rich plasma induces cellular alterations, enhancement of lubricin, and inflammatory downregulation in vivo in human knees: A controlled, prospective human clinical trial based on mass spectrometry imaging analysis.

To investigate the in vivo biological effects of leukocyte-poor platelet-rich plasma (LpPRP) treatment in human synovial layer to establish the cellular basis for a prolonged clinical improvement. Synovial tissues (n = 367) were prospectively collected from patients undergoing arthroscopic surgery. Autologous-conditioned plasma, LpPRP, was injected into the knees of 163 patients 1-7 days before surgery to reduce operative trauma and inflammation, and to induce the onset of regeneration. A total of 204 patients did not receive any injection. All samples were analyzed by mass spectrometry imaging. Data analysis was evaluated by clustering, classification, and investigation of predictive peptides. Peptide identification was done by tandem mass spectrometry and database matching. Data analysis revealed two major clusters belonging to LpPRP-treated (LpPRP-1) and untreated (LpPRP-0) patients. Classification analysis showed a discrimination accuracy of 82%-90%. We identified discriminating peptides for CD45 and CD29 receptors (receptor-type tyrosine-protein phosphatase C and integrin beta 1), indicating an enhancement of musculoskeletal stem cells, as well as an enhancement of lubricin, collagen alpha-1-(I) chain, and interleukin-receptor-17-E, dampening the inflammatory reaction in the LpPRP-1 group following LpPRP injection. We could demonstrate for the first time that injection therapy using "autologic-conditioned biologics" may lead to cellular changes in the synovial membrane that might explain the reported prolonged beneficial clinical effects. Here, we show in vivo cellular changes, possibly based on muscular skeletal stem cell alterations, in the synovial layer. The gliding capacities of joints might be improved by enhancing of lubricin, anti-inflammation by activation of interleukin-17 receptor E, and reduction of the inflammatory process by blocking interleukin-17.

Molecular Pathology of Micropapillary Carcinomas: Is Characteristic Morphology Related to Molecular Mechanisms?

Micropapillary carcinoma is an entity defined histologically in many organs. It is associated with lymph node metastasis and poor prognosis. The main mechanism for its histopathologic appearance is reverse polarization. Although the studies on this subject are limited, carcinomas with micropapillary morphology observed in different organs are examined by immunohistochemical and molecular methods. Differences are shown in these tumors compared with conventional carcinomas regarding the rate of somatic mutations, mRNA and miRNA expressions, and protein expression levels. TP53, PIK3CA, TERT, KRAS, EGFR, MYC, FGFR1, BRAF, AKT1, HER2/ERBB2, CCND1, and APC mutations, which genes frequently detected in solid tumors, have also been detected in invasive micropapillary carcinoma (IMPC) in various organs. 6q chromosome loss, DNAH9, FOXO3, SEC. 63, and FMN2 gene mutations associated with cell polarity or cell structure and skeleton have also been detected in IMPCs. Among the proteins that affect cell polarity, RAC1, placoglobin, as well as CLDNs, LIN7A, ZEB1, CLDN1, DLG1, CDH1 (E-cadherin), OCLN, AFDN/AF6, ZEB1, SNAI2, ITGA1 (integrin alpha 1), ITGB1 (integrin beta 1), RHOA, Jagged-1 (JAG1) mRNAs differentially express between IMPC and conventional carcinomas. Prediction of prognosis and targeted therapy may benefit from the understanding of molecular mechanisms of micropapillary morphology. This review describes the molecular pathologic mechanisms underlying the micropapillary changes of cancers in various organs in a cell polarity-related dimension.

Exploring the pharmacological and adverse reaction mechanism of a drug by network pharmacology strategy: Using colchicine to treat Behcet syndrome as an example

Colchicine (COLC) is a natural alkaloid used to treat Behcet syndrome (BS), but its adverse reactions limit its clinical application in treating BS. However, the adverse reaction mechanism of COLC in the treatment of BS remains unclear. Herein, a network pharmacology-based strategy was designed to analyze the pharmacological and adverse reaction mechanism of COLC in treating BS. The biological functions of COLC and BS pathogenesis were analyzed through a series of network construction and analysis. The data above predicted the pharmacological and adverse reaction mechanism of COLC in BS treatment. The pharmacological mechanism of COLC against BS was predicted to control inflammatory responses. Interleukin-8, interleukin-18, integrin alpha-4, integrin beta-2, and tubulin targets are crucial in treating BS. The adverse reactions of COLC in BS treatment were predicted as neurotoxicity and hepatotoxicity. The mechanism of hepatotoxicity may be related to the decrease of cytochrome P450 family 3 subfamily A activity caused by various factors, such as poor hepatic function, the dosage of COLC, and combination with inhibitors. The mechanism of neurotoxicity may be related to the disruption of microtubules in the nervous system by COLC transport across the blood-brain barrier. This study provided basic evidence for the medication safety management of COLC used in treating BS. Moreover, this study demonstrated that it is feasible to analyze the adverse reaction mechanisms of drugs using a network pharmacology strategy, which facilitates systematic drug safety management and evaluation.

Abstract 6234: Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression

Abstract Integrins have long been sought as therapeutic targets in cancer, but there are no FDA approved drugs to target this gene family. Since integrins require the dimerization between an alpha and beta subunit to function, we developed a CRISPR library targeting all 26 alpha and beta subunits to identify which pair is the most important. We found a strong correlation between ITGAV and ITGB5 (Integrin Beta 5), which form a known heterodimer, in solid tumors. CRISPR targeting either ITGAV or ITGB5 led to attenuated cell proliferation with increased cell cycle stalling and apoptosis. As the more essential partner, we designed a CRISPR tiling scan targeting the entire coding region of ITGAV and determine the essential nature of each amino acid. We found a sub-domain within the beta propeller to be important for ITGAV function, the CRISPR identified pocket (CIP). Genetic targeting of this pocket disrupted the ability to detect the heterodimer signal of ITGAV-ITGB5 by a specific antibody. Furthermore, we confirmed that essential amino acids within the CIP responsible for the heterodimer stability by the NanoBRET energy transfer system. Knowing the role of the CIP in heterodimer stability we searched for a small molecule to bind in the CIP to phenocopy our genetic findings. A combined in-silico and in-vitro high throughput drug screen started tested compounds for cell killing and disruption of the ITGAV-ITGB5 heterodimer. “Compound 2” (cpd_2) showed both strong cell killing and dissociation of the heterodimer. Disruption was measured by detection by flow cytometry and through the NanoBRET system. Cpd_2’s binding was confirmed with thermal shift studies with purified ITGAV beta propeller, validating that this compound is behaving and binding as we expect at the CIP. Next, we sought to understand how the ITGAV-ITGB5 heterodimer controls cancer cell survival. From the Dependency Map Public database (DepMap) we found that Rac1 dependency is significantly correlated only with ITGAV and ITGB5. We hypothesized that this meant that ITGAV-ITGB5 regulated Rac1 activity. Loss of Rac1 phenocopies knock out of ITGAV or ITGB5 and loss of either Rac1 or ITGAV led to dysregulation of the actin cytoskeleton, a known downstream feature of Rac1. A constitutively Rac1 (CA_Rac1) rescues the effects of targeting of ITGAV or treatment with cpd_2. Altogether this data suggests that ITGAV-ITGB5 signals to Rac1 and that attenuation of their function can be reversed by an active Rac1 In summary, our study highlights a novel strategy of targeting the integrin heterodimer ITGAV-ITGB5 with a small molecule binding to a novel subdomain, CIP, of ITGAV. The CIP is responsible for the heterodimer stability of ITGAV and ITGB5. Disruption of this heterodimer led to the dysregulation of Rac1 and destabilization of the actin cytoskeleton. This mechanism highlights a novel strategy for targeting integrins as a therapeutic target in cancer. Citation Format: Nicole Mattson, Kazuya Miyashita, Anthony Chan, Lu Yang, Sheela Pangeni Pokharel, Wei Liu, Ming li, Qiao Liu, Xiaobao Xu, Priyanka Singh, Leisi Zhang, Mingye Feng, Chun-Wei Chen. Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6234.

The Role of Integrin Beta 3 Polymorphisms in Coronary Artery Disease: A Systematic Review and Meta-analysis

Objectives: The literature on the role of integrin beta 3 (ITGB3) exonic variants in coronary artery disease (CAD) and lipid outcomes is scarce. However, the findings remained uncertain and still not clear. Therefore, the current study aims to determine the association of rs5918 polymorphism with coronary artery disease. Methodology: All the eligible literature published in the English language from February 3, 2005, up to December 19, 2021, were searched by using different electronic databases and extracted all the required information from the available literature. The statistical analysis was performed through the MetaGenyo program, and pooled odds ratios (ORs) were calculated to determine the association between rs5918 and CAD. Results: The final analysis includes four studies, and the overall rs5918 risk allele in all the tested genetic models as follow: allelic model: OR 0.80 CI 0.41-1.58; homozygote model: OR 1.66, 95% CI 0.20-2.16; recessive model: OR 0.71 CI 0.44-1.14; dominant model: OR 0.81 CI 0.22-3.03. In addition, the lipid outcomes, including lipoproteins, cholesterol, and triglycerides were associated with increased disease risk. The shapes of the funnel plots suggest no publication bias in our study. Conclusion: In conclusion, our final pooled analysis revealed a non-significant role of this exonic polymorphism in coronary artery disease that may exert its effect by modulating various lipid parameters. However, more studies are required with a larger cohort size that may give us conclusive results in the future.

MP08-01 IMMUNOSUPPRESSIVE MECHANISMS OF INTEGRIN BETA8 FOR IMMUNE RESPONSE INDUCED BY BCG THERAPY

You have accessJournal of UrologyCME1 Apr 2023MP08-01 IMMUNOSUPPRESSIVE MECHANISMS OF INTEGRIN BETA8 FOR IMMUNE RESPONSE INDUCED BY BCG THERAPY Seiji Hoshi, Hiroki Natsuya, Yuki Harigane, Kei Yaginuma, Syunta Makabe, Yuki Yoshida, Satoru Meguro, Akifumi Onagi, Kanako Matsuoka, Tomoyuki Koguchi, Junya Hata, Yuichi Sato, Hidenori Akaihata, Masao Kataoka, Soichiro Ogawa, Motohide Uemura, and Yoshiyuki Kojima Seiji HoshiSeiji Hoshi More articles by this author , Hiroki NatsuyaHiroki Natsuya More articles by this author , Yuki HariganeYuki Harigane More articles by this author , Kei YaginumaKei Yaginuma More articles by this author , Syunta MakabeSyunta Makabe More articles by this author , Yuki YoshidaYuki Yoshida More articles by this author , Satoru MeguroSatoru Meguro More articles by this author , Akifumi OnagiAkifumi Onagi More articles by this author , Kanako MatsuokaKanako Matsuoka More articles by this author , Tomoyuki KoguchiTomoyuki Koguchi More articles by this author , Junya HataJunya Hata More articles by this author , Yuichi SatoYuichi Sato More articles by this author , Hidenori AkaihataHidenori Akaihata More articles by this author , Masao KataokaMasao Kataoka More articles by this author , Soichiro OgawaSoichiro Ogawa More articles by this author , Motohide UemuraMotohide Uemura More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003223.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In non-muscle invasive bladder cancer (NMIBC), immunosuppressive factors responsible for post-BCG therapy recurrence remains unclear. In this study, we performed comprehensive genetic analysis and functional analysis in untreated NMIBC to explore key players of immunosuppression associated with recurrence after BCG therapy. METHODS: 13 patients (73.5±8.9 years old) with untreated NMIBC who were treated over 6 months BCG and could be followed up for 5 years were included. They were divided into two groups; recurrence (n=6) and non-recurrence groups (n=7). Genes that are upregulated in bladder cancer tissues compared to normal bladder tissues and associated with recurrence and immunosuppression were extracted from the microarray of untreated bladder cancer tissue. In bladder cancer cells (RT4 and UMUC5), the expression of the genes was evaluated by qPCR and Western blot (WB). Cell proliferation (WST assay), adhesion (CytoSelectTM), invasion (scratch assay) and secretion of immunosuppressive cytokine TGF-β1 (ELISA and immunostaining) were also assessed after knockdown of the genes to evaluate the function of the gene in bladder cancer cells. RESULTS: Microarray analysis revealed integrin beta 8 (ITGB8) was significantly upregulated in the recurrence group (p<0.0001). The microarray results were validated with ITGB8 immunostaining of the same patient's tissue and showed similar results to the microarray. qPCR and WB results showed that ITGB8 was highly expressed in UMUC5 compared to RT4. In UMUC5, ITGB8 knockdown significantly decreased cell proliferation, adhesion, invasion (p<0.01) and TGF-β1 secretion (p<0.01). It also significantly reduced staining for TGF-β1 and TGF-β1 LAP-D, which indicates TGF-β1 activation (p<0.01). CONCLUSIONS: In untreated NMIBC, ITGB8 may contribute to recurrence after BCG by suppressing tumor immunity through the activation of TGF-β1. Thus, inhibition of ITGB8 was considered to prevent the recurrence of NMIBC via inducing an immune response by BCG therapy. Source of Funding: This study was supported by a Grant-in-Aid for Young Investigators from the Japan Society for Promotion of Science (JSPS KAKENHI 19K18590, S. Hoshi) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e93 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Seiji Hoshi More articles by this author Hiroki Natsuya More articles by this author Yuki Harigane More articles by this author Kei Yaginuma More articles by this author Syunta Makabe More articles by this author Yuki Yoshida More articles by this author Satoru Meguro More articles by this author Akifumi Onagi More articles by this author Kanako Matsuoka More articles by this author Tomoyuki Koguchi More articles by this author Junya Hata More articles by this author Yuichi Sato More articles by this author Hidenori Akaihata More articles by this author Masao Kataoka More articles by this author Soichiro Ogawa More articles by this author Motohide Uemura More articles by this author Yoshiyuki Kojima More articles by this author Expand All Advertisement PDF downloadLoading ...

Single-cell analyses reveal the dynamic functions of Itgb2+ microglia subclusters at different stages of cerebral ischemia-reperfusion injury in transient middle cerebral occlusion mice model.

The underlying pathophysiological mechanisms of cerebral ischemia reperfusion injury (CIRI) is intricate, and current studies suggest that neuron, astrocyte, microglia, endothelial cell, and pericyte all have different phenotypic changes of specific cell types after ischemic stroke. And microglia account for the largest proportion after CIRI. Previous transcriptomic studies of ischemic stroke have typically focused on the 24 hours after CIRI, obscuring the dynamics of cellular subclusters throughout the disease process. Therefore, traditional methods for identifying cell types and their subclusters may not be sufficient to fully unveil the complexity of single-cell transcriptional profile dynamics caused by an ischemic stroke. In this study, to explore the dynamic transcriptional profile of single cells after CIRI, we used single-cell State Transition Across-samples of RNA-seq data (scSTAR), a new bioinformatics method, to analyze the single-cell transcriptional profile of day 1, 3, and 7 of transient middle cerebral artery occlusion (tMCAO) mice. Combining our bulk RNA sequences and proteomics data, we found the importance of the integrin beta 2 (Itgb2) gene in post-modeling. And microglia of Itgb2+ and Itgb2- were clustered by the scSTAR method. Finally, the functions of the subpopulations were defined by Matescape, and three different time points after tMCAO were found to exhibit specific functions. Our analysis revealed a dynamic transcriptional profile of single cells in microglia after tMCAO and explored the important role of Itgb2 contributed to microglia by combined transcriptomics and proteomics analysis after modeling. Our further analysis revealed that the Itgb2+ microglia subcluster was mainly involved in energy metabolism, cell cycle, angiogenesis, neuronal myelin formation, and repair at 1, 3, and 7 days after tMCAO, respectively. Our results suggested that Itgb2+ microglia act as a time-specific multifunctional immunomodulatory subcluster during CIRI, and the underlying mechanisms remain to be further investigated.

Multiplex protein analysis and ensemble machine learning methods of fine needle aspirates from prostate cancer patients reveal potential diagnostic signatures associated with tumour grade.

Improved molecular diagnosis is needed in prostate cancer (PC). Fine needle aspiration (FNA) is a minimally invasive biopsy technique, less traumatic compared to core needle biopsy, and could be useful for diagnosis of PC. Molecular biomarkers (BMs) in FNA-samples can be assessed for prediction, eg of immunotherapy efficacy before treatment as well as at treatment decision time points during disease progression. In the present pilot study, the expression levels of 151 BM proteins were analysed by proximity extension assay in FNA-samples from 16 patients, including benign prostate lesions (n= 3) and cancers (n= 13). An ensemble data analysis strategy was applied using several machine learning models. Twelve potentially predictive BM proteins correlating with International Society of Urological Pathology grade groups were identified, among them vimentin, tissue factor pathway inhibitor 2, and integrin beta-5. The validity of the results was supported by network analysis that showed functional associations between most of the identified putative BMs. We also showed that multiple immune checkpoint targets can be assessed (eg PD-L1, CD137, and Galectin-9), which may support the selection of immunotherapy in advanced PC. Results are promising but need further validation in a larger cohort. Our pilot study represents a "proof of concept" and shows that multiplex profiling of potential diagnostic and predictive BM proteins is feasible on tumour material obtained by FNA sampling of prostate cancer. Moreover, our results demonstrate that an ensemble data analysis strategy may facilitate the identification of BM signatures in pilot studies when the patient cohort is limited.

Characterization and Proteomic Analysis of Plasma EVs Recovered from Healthy and Diseased Dogs with Canine Leishmaniosis.

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Claudin-4: A New Molecular Target for Epithelial Cancer Therapy.

Claudin-4 (CLDN4) is a key component of tight junctions (TJs) in epithelial cells. CLDN4 is overexpressed in many epithelial malignancies and correlates with cancer progression. Changes in CLDN4 expression have been associated with epigenetic factors (such as hypomethylation of promoter DNA), inflammation associated with infection and cytokines, and growth factor signaling. CLDN4 helps to maintain the tumor microenvironment by forming TJs and acts as a barrier to the entry of anticancer drugs into tumors. Decreased expression of CLDN4 is a potential marker of epithelial-mesenchymal transition (EMT), and decreased epithelial differentiation due to reduced CLDN4 activity is involved in EMT induction. Non-TJ CLDN4 also activates integrin beta 1 and YAP to promote proliferation, EMT, and stemness. These roles in cancer have led to investigations of molecular therapies targeting CLDN4 using anti-CLDN4 extracellular domain antibodies, gene knockdown, clostridium perfringens enterotoxin (CPE), and C-terminus domain of CPE (C-CPE), which have demonstrated the experimental efficacy of this approach. CLDN4 is strongly involved in promoting malignant phenotypes in many epithelial cancers and is regarded as a promising molecular therapeutic target.

Glycation-mediated tissue-level remodeling of brain meningeal membrane by aging.

Collagen is a prominent target of nonenzymatic glycation, which is a hallmark of aging and causes functional alteration of the matrix. Here, we uncover glycation-mediated structural and functional changes in the collagen-enriched meningeal membrane of the human and mouse brain. Using an in vitro culture platform mimicking the meningeal membrane composed of fibrillar collagen, we showed that the accumulation of advanced glycation end products (AGEs) in the collagen membrane is responsible for glycation-mediated matrix remodeling. These changes influence fibroblast-matrix interactions, inducing cell-mediated ECM remodeling. The adherence of meningeal fibroblasts to the glycated collagen membrane was mediated by the discoidin domain-containing receptor 2 (DDR2), whereas integrin-mediated adhesion was inhibited. A-kinase anchoring protein 12 (AKAP12)-positive meningeal fibroblasts in the meningeal membrane of aged mice exhibited substantially increased expression of DDR2 and depletion of integrin beta-1 (ITGB1). In the glycated collagen membrane, meningeal fibroblasts increased the expression of matrix metalloproteinase 14 (MMP14) and less tissue inhibitor of metalloproteinase-1 (TIMP1). In contrast, the cells exhibited decreased expression of type I collagen (COL1A1). These results suggest that glycation modification by meningeal fibroblasts is intimately linked to aging-related structural and functional alterations in the meningeal membrane.

Structural N-glycoproteomics characterization of cell-surface N-glycosylation of MCF-7/ADR cancer stem cells

Breast cancer is responsible for the highest mortality all over the world. Cancer stem cells (CSCs) along with epithelial mesenchymal transition (EMT) are identified as a driver of cancer which are responsible for cancer metastasis and drug resistance. Several signaling pathways are associated with drug resistance. Additionally, glycosyltransferases regulate different types of glycosylation which are involved in drug resistance. To the end, it is urgent to figure out the knowledge on cell-surface altered N-glycosylation and putative markers. Here, differential cell-surface intact N-glycopeptides in adriamycin (ADR)-resistant michigan breast cancer foundation-7 stem cells (MCF-7/ADR CSCs) relative to ADR-sensitive MCF-7 CSCs were analyzed with site- and structure-specific quantitative N-glycoproteomics. The intact N-glycopeptides and differentially expressed intact N-glycopeptides (DEGPs) were determined and quantified via intact N-glycopeptide search engine GPSeeker. Totally, 4777 intact N-glycopeptides were identified and N-glycan sequence structures among 2764 IDs were distinguished from their isomers by structure-diagnostic fragment ions. Among 1717 quantified intact N-glycopeptides, 104 DEGPs were determined (fold change ≥ 1.5 and p value < 0.05). Annotation of protein–protein interaction and biological processes among others of DEGPs were finally carried out; down-regulated intact N-glycopeptide with bisecting GlcNAc from p38-interacting protein and up-regulated intact N-glycopeptide with β1,6-branching N-glycan from integrin beta-5 were found.

Systemic proinflammatory−profibrotic response in aortic stenosis patients with diabetes and its relationship with myocardial remodeling and clinical outcome

BackgroundPrevious studies have mainly focused more on how diabetes affects the valve than the myocardium in aortic stenosis (AS). In the pressure-overloaded heart, myocardial fibrosis is an important driver of the progression from compensated hypertrophy to heart failure. Using comprehensive noninvasive imaging and plasma proteomics, we investigated whether and how diabetes aggravates the remodeling of the myocardium and its relation with prognosis in AS patients.MethodsSevere AS patients were enrolled in two prospective cohorts for imaging and biomarker analysis. The imaging cohort (n = 253) underwent echocardiography and cardiac magnetic resonance, and the biomarker cohort (n = 100) blood sampling with multiplex proximity extension assay for 92 proteomic biomarkers. The composite outcome of hospitalization for heart failure admissions and death was assessed in the imaging cohort.ResultsDiabetic patients were older (70.4 ± 6.8 versus 66.7 ± 10.1 years) with more advanced ventricular diastolic dysfunction and increased replacement and diffuse interstitial fibrosis (late gadolinium enhancement % 0.3 [0.0–1.6] versus 0.0 [0.0–0.5], p = 0.009; extracellular volume fraction % 27.9 [25.7–30.1] versus 26.7 [24.9–28.5], p = 0.025) in the imaging cohort. Plasma proteomics analysis of the biomarker cohort revealed that 9 proteins (E-selectin, interleukin-1 receptor type 1, interleukin-1 receptor type 2, galectin-4, intercellular adhesion molecule 2, integrin beta-2, galectin-3, growth differentiation factor 15, and cathepsin D) were significantly elevated and that pathways related to inflammatory response and extracellular matrix components were enriched in diabetic AS patients. During follow-up (median 6.3 years), there were 53 unexpected heart failure admissions or death in the imaging cohort. Diabetes was a significant predictor of heart failure and death, independent of clinical covariates and aortic valve replacement (HR 1.88, 95% CI 1.06−3.31, p = 0.030).ConclusionsPlasma proteomic analyses indicate that diabetes potentiates the systemic proinflammatory−profibrotic milieu in AS patients. These systemic biological changes underlie the increase of myocardial fibrosis, diastolic dysfunction, and worse clinical outcomes in severe AS patients with concomitant diabetes.

Genetic screening of PLA1/PLA2 polymorphous marker of integrin beta 3 (ITGB3) GP IIIA gene in adolescent girls with puberty menorrhagia.

Puberty menorrhagia is one of the urgent problems of modern reproductive medicine. The study aimed to investigate the relationship between polymorphism of the GP IIIa (PLA1/PLA2) gene and improve the diagnosis of puberty menorrhagia in girls with thyroid gland pathology. Ninety-seven girls at puberty age were divided into three groups: group 1 (main) - girls with puberty menorrhagia and thyroid gland pathology (30 individuals), group 2 (comparison) - 40 girls with puberty menorrhagia, group 3 (control) - 27 practically healthy girls. Polymorphism of the GP IIIa (PLA1/PLA2) gene was studied by isolating genomic DNA from peripheral blood leukocytes, followed by amplification with a polymerase chain reaction. Results showed that mutation in the 17th chromosome of q21.32 of the GP IIIa gene occurred in 8.6% of cases among adolescents with menorrhagia, in contrast to the control group, where it was not observed at all. The A1A1-genotype occurred by 11.7% (X2=4.01, p=0.041) more often in adolescents with menorrhagia than in girls with concomitant thyroid gland pathology and by 15.0% (X2=4.54, p=0.033) more often than in the control group. It was also found that the presence of the A1A2-genotype unreliably reduced the chances of uterine bleeding in adolescent girls by 1.45 times (OR=2.12) and was a protective factor in the puberty menorrhagia occurrence (OR=0.47). It may be concluded that the identification of a hereditary factor of the reproductive system diseases of adolescent girls fundamentally changes the point of view on the tactics of disease management and subsequent therapy.