Regulation Of Cell Adhesion Research Articles

Antitumor activities of anti‑CD44 monoclonal antibodies in mouse xenograft models of esophageal cancer.

CD44 is a type I transmembrane glycoprotein associated with poor prognosis in various solid tumors. Since CD44 plays a critical role in tumor development by regulating cell adhesion, survival, proliferation and stemness, it has been considered a target for tumor therapy. Anti‑CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody‑drug conjugates and chimeric antigen receptor‑T cell therapy. Anti-pan‑CD44 mAbs, C44Mab‑5 and C44Mab‑46, which recognize both CD44 standard (CD44s) and variant isoforms were previously developed. The present study generated a mouse IgG2a version of the anti‑pan‑CD44 mAbs (5‑mG2a and C44Mab‑46‑mG2a) to evaluate the antitumor activities against CD44‑positive cells. Both 5‑mG2a and C44Mab‑46‑mG2a recognized CD44s‑overexpressed CHO‑K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5‑mG2a and C44Mab‑46‑mG2a could activate effector cells in the presence of CHO/CD44s cells and exhibited complement-dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5‑mG2a and C44Mab‑46‑mG2a significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control mouse IgG2a. These results indicate that 5‑mG2a and C44Mab‑46‑mG2a could exert antitumor activities against CD44‑positive cancers and be a promising therapeutic regimen for tumors.

Expression of cardiomyopathy-related genes in mononuclear blood cells predicts all-cause mortality in patients presenting with acute versus chronic coronary syndrome requiring revascularisation

Abstract Objective Despite advances in the revascularisation techniques their net clinical benefit differs substantially between patients presenting with myocardial infarction (ACS) and chronic coronary syndrome (CCS) due to obstructive coronary atherosclerosis. Considering that leukocytes represents a key pathophysiological component of myocardial ischaemia, exploration of mononuclear blood cell (PBMC) gene expression profile in CCS and ACS setting could reveal clinically relevant transcriptomic signals. The aim of the present study was to identify differences in PBMC transcriptome between ACS and CCS patients which are linked to mortality after revascularisation. Methods and Results We analysed PBMC transcriptomes from719 ACS and 486 CCS patients creating discovery cohort. The results of transcriptomic analysis were verified in replication cohort (ACS: N=682 for CCS: N=489) generated based on propensity score matching. Median time from revascularisation to blood collection (IQR) was 15.68 (-4.52, 23.23) hrs. Patients with ACS showed consistent differential expression of 8173 genes (4544 upregulated, 3629 downregulated) when compared with CCS patients. The subsequent pathway analysis also showed significant enrichment of several canonical pathways involved in major cardiovascular pathologies i.e. atherosclerosis (p = 1.22-06, enrichment = 3.39), vascular endothelial growth factor production (p = 1.16-06, enrichment = 16.73), positive regulation of vascular development (p = 2.78-06, enrichment = 5.30) and heterotopic cell adhesion including gene desmocollin 2 (DSC2, p = 3.64-04, enrichment = 6.30). In der multivariate survival analysis with differentially expressed genes, increased expression of DCS2, lamin A/C (LMNA), and UGGT2, and decreased expression of SNRNP70 predicted poor survival over the median follow-up 11.35 yrs. Consistently, controlling the false discovery rate at ≤ 5% these genes enriched pathways involved in cardiomyopathies such as arrhythmogenic right-ventricular cardiomyopathy (p = 4.19x10-4, enrichment = 58.9) and collagen disease (p = 2.29x10-3, enrichment = 23.8). Conclusion We found altered PBMC expression of multiple genes in patients requiring revascularisation for ACS versus CCS. Several differentially expressed genes included in cardiomyopathy pathways predicted long-term all-cause mortality in these patients.

Limosilactobacillus reuteri HY7503 and Its Cellular Proteins Alleviate Endothelial Dysfunction by Increasing Nitric Oxide Production and Regulating Cell Adhesion Molecule Levels.

Endothelial dysfunction, which is marked by a reduction in nitric oxide (NO) production or an imbalance in relaxing and contracting factor levels, exacerbates atherosclerosis by promoting the production of cell adhesion molecules and cytokines. This study aimed to investigate the effects of Limosilactobacillus reuteri HY7503, a novel probiotic isolated from raw milk, on endothelial dysfunction. Five lactic acid bacterial strains were screened for their antioxidant, anti-inflammatory, and endothelium-protective properties; L. reuteri HY7503 had the most potent effect. In a mouse model of angiotensin II-induced endothelial dysfunction, L. reuteri HY7503 reduced vascular thickening (19.78%), increased serum NO levels (226.70%), upregulated endothelial NO synthase (eNOS) expression in the aortic tissue, and decreased levels of cell adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and serum cytokines (tumor necrosis factor-alpha [TNF-α] and interleukin-6 [IL-6]). In TNF-α-treated human umbilical vein endothelial cells (HUVECs), L. reuteri HY7503 enhanced NO production and reduced cell adhesion molecule levels. In HUVECs, surface-layer proteins (SLPs) were more effective than extracellular vesicles (exosomes) in increasing NO production and decreasing cell adhesion molecule levels. These findings suggested that L. reuteri HY7503 may serve as a functional probiotic that alleviates endothelial dysfunction.

More than a mutagenic Aflatoxin B1 precursor: The multiple cellular targets of Versicolorin A revealed by global gene expression analysis

Versicolorin A (VerA), a precursor of the potent carcinogen Aflatoxin B1 (AFB1), is an emerging mycotoxin. Recent research has highlighted the mutagenic and genotoxic properties of VerA, yet several facets of its pronounced toxicity remain unexplored. In the present study, we investigated early (6 h) transcriptomic changes induced by VerA in differentiated intestinal cells in non-cytotoxic conditions (1 and 3 μM) and compared its effects to those of AFB1 at 1 μM. Our findings indicated that VerA led to substantial alterations in global gene expression profiles, while AFB1 did not exhibit the same effects. As expected, both toxins caused alterations in gene expression associated with well-known aspects of their toxicity, including mutagenicity, genotoxicity, oxidative stress, and apoptosis. However, we also observed novel features of VerA toxicity, including the ability to cause mitochondrial dysfunction and to trigger a type-1 interferon response, at least partially mediated by cGAS-STING. VerA also induced changes in the expression of genes involved in the regulation of cell shape and adhesion, transcription/translation as well as genes associated with tumor biology. Our results provide new evidence of the high toxicity of VerA and underscore the importance of further assessing the risks associated with its presence in food.

Focal Adhesion Kinase (FAK) and c-Src Dependent Signal Transduction in Cell Adhesion.

This review predominantly acquaints the role of focal adhesion kinase (FAK) and cellular-Src (c-Src) in cell adhesion. Cell adhesion is a crucial phenomenon that causes the cells to interact with the extracellular matrix (ECM) or with each other. There are different proteins involved in cell adhesion including cell adhesion molecules (CAMs)/receptors that are present on the cell surface and various cytoplasmic proteins. FAK and c-Src are two proteins in the cytoplasm, which serve as regulators of different proteins involved in cell adhesion. They activate talin, vinculin and paxillin in turn connect the integrins with the cytoskeleton and in this way strengthen the integrin interaction with ECM. FAK-Src signalling also modulates cell-cell adhesion by regulating actin interactions. Being a key modulator of cell adhesion, FAK and c-Src signalling are linked with different pathological conditions like cancer, cardiovascular diseases, and embryonic developmental disorders. Thus, comprehensive research into FAK-Src signalling is of great importance in the exploration of different signalling targets for therapeutic interpretations. Different inhibitors and antibodies against various cell adhesion proteins, such as FAK, c-Src, and integrins, have already been used in preclinical and clinical trials to treat a variety of diseases, including cancer and chronic inflammatory conditions. Furthermore, this review presents different challenges to FAK-Src and cell adhesion signalling targeted drug development, which include, cytotoxicity and cell resistance to the drug. Finally, this review remarks that FAK and c-Src are important regulators of cell adhesion and are linked to various pathologies, nevertheless, more comprehensive research on these proteins would be a significant step forward in the development of effective therapies for the diseases associated with them.

Impact of zirconia-based oxide on endothelial cell dynamics and extracellular matrix remodeling

IntroductionZirconia (ZrO2) is highly regarded in dental restoration due to its aesthetic compatibility and mechanical properties that align with biological tissues. This study explores the effects of stabilized ZrO2 on endothelial cell function and extracellular matrix (ECM) remodeling, processes critical to successful osseointegration in dental implants. MethodologyHuman Umbilical Vein Endothelial Cells (HUVECs) were cultured in ZrO2 -enriched medium under both static and shear stress conditions. Newly implemented techniques, including detailed zirconia surface characterization using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD), were used to verify material properties. Gene and protein expression related to cell adhesion, proliferation, and ECM remodeling were assessed through RT-qPCR and Western blotting. Zymography was used to evaluate the activity of matrix metalloproteinases (MMP2 and MMP9) involved in ECM remodeling. ResultsCharacterization data confirmed the stability and structural properties of ZrO2, revealing a tetragonal crystalline structure and rough surface morphology conducive to cell adhesion. ZrO2 exposure led to the downregulation of Src, a key regulator of cell adhesion, while upregulating cell cycle regulators p15, CDK2, and CDK4, indicating enhanced cell proliferation. Under shear stress, ZrO2 modulated TGF-β and MAPK signaling, affecting cell proliferation and angiogenesis. MMP2 and MMP9 activity increased in static conditions but decreased under shear stress, suggesting ZrO2 dynamic role in ECM remodeling. ConclusionThis study shows that stabilized zirconia (ZrO2) modulates endothelial cell dynamics and ECM remodeling, key for osseointegration. ZrO2 downregulated Src expression and upregulated cell cycle regulators, enhancing endothelial proliferation. It also affected TGF-β and MAPK pathways, influencing angiogenesis, and differentially modulated MMP2 and MMP9 activity depending on mechanical conditions. These findings highlight ZrO2 has potential ability to enhance vascular and tissue integration in dental applications.

Family-Wide Photoproximity Profiling of Integrin Protein Social Networks in Cancer.

Integrin family transmembrane receptors mediate dynamic interactions between cells and their extracellular microenvironment. The heterogeneous interaction partners of integrins directly regulate cell adhesion, motility, proliferation, and intracellular signaling. Despite the recognized importance of protein-protein interactions and the formation of signaling hubs around integrins, the ability to detect and quantify these dynamic binding partners with high spatial and temporal resolution remains challenging. Here, we developed an integrin-family-directed quantitative photoproximity protein interaction (PhotoPPI) profiling method to detect and quantify native integrin-centered protein social networks on live cells and tissues without the need for genetic manipulation, antibodies, or non-physiologic cell culture conditions. We drafted quantitative maps of integrin-centered protein social networks, highlighting conserved and unique binding partners between different cell types and cellular microenvironments. Comparison of integrin social networks in cancer cell lines of diverse tissue of origin and disease state identified specific AND-gate binding partners involved cell migration, microenvironmental interactions and proliferation that serve as markers of tumor cell metastatic state. Finally, we identified unique combinations - or barcodes - of integrin-proximal proteins on the surface of pre- and post-metastatic triple negative breast cancer (TNBC) cells whose expression strongly correlate with both positive and negative disease progression and outcomes in TNBC patients. Taken together, these data provide the first family-wide high-resolution maps of native protein interactors on live cells and identify dynamic integrin-centered social networks as potential AND-gate markers of cell identity, microenvironmental context and disease state.

A Computational Approach in the Systematic Search of the Interaction Partners of Alternatively Spliced TREM2 Isoforms.

Alzheimer's disease is the most common form of dementia, characterized by the pathological accumulation of amyloid-beta (Aβ) plaques and tau neurofibrillary tangles. Triggering receptor expressed on myeloid cells 2 (TREM2) is increasingly recognized as playing a central role in Aβ clearance and microglia activation in AD. The TREM2 gene transcriptional product is alternatively spliced to produce three different protein isoforms. The canonical TREM2 isoform binds to DAP12 to activate downstream pathways. However, little is known about the function or interaction partners of the alternative TREM2 isoforms. The present study utilized a computational approach in a systematic search for new interaction partners of the TREM2 isoforms by integrating several state-of-the-art structural bioinformatics tools from initial large-scale screening to one-on-one corroborative modeling and eventual all-atom visualization. CD9, a cell surface glycoprotein involved in cell-cell adhesion and migration, was identified as a new interaction partner for two TREM2 isoforms, and CALM, a calcium-binding protein involved in calcium signaling, was identified as an interaction partner for a third TREM2 isoform, highlighting the potential role of cell adhesion and calcium regulation in AD.

Evaluation of diagnostic value and Mendelian randomization study of appendicitis hub genes obtained by WGCNA analysis.

The timely and precise diagnosis of appendicitis was deemed essential. This study sought to examine the diagnostic significance of hub genes linked to appendicitis and to delve deeper into the pathophysiology of the condition. Differential gene expression analysis revealed distinct genes in the appendicitis group compared to other abdominal pain group, while weighted gene co-expression network analysis identified appendicitis-associated modules. Further analysis of common genes was conducted using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis. The diagnostic efficiency of hub genes was explored through the use of nomograms and receiver operator characteristic curves. Additionally, immunoinfiltration analysis was performed to investigate the immune cell infiltration in both groups. The causal relationship between hub genes and appendicitis, as well as gut microbiota and appendicitis, was ultimately examined through Mendelian randomization. By conducting differential expression analysis and weighted gene co-expression network analysis, a total of 757 common genes were identified. Subsequent Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses revealed that these common genes were primarily associated with positive regulation of cell adhesion, focal adhesion, protein serine kinase activity, and amyotrophic lateral sclerosis. Utilizing Cytoscape software, the top 10 genes with the highest degree of interaction were identified as RPS3A, RPSA, RPL5, RPL37A, RPS27L, FLT3LG, ARL6IP1, RPL32, MRPL3, and GSPT1. Evaluation using nomograms and receiver operator characteristic curves demonstrated the diagnostic value of these hub genes. Ultimately, a causal relationship between hub genes and appendicitis was not identified in our study. Nevertheless, our findings indicate that appendicitis is correlated with 9 gut microbiota. This study identified 5 hub genes, specifically HSP90AA1, RPL5, MYC, CD44, and RPS3A, which exhibit diagnostic significance of appendicitis. Furthermore, the elucidation of these hub genes aids in enhancing our comprehension of the molecular pathways implicated in the development of appendicitis.

Anti-CD44 Variant 10 Monoclonal Antibody Exerts Antitumor Activity in Mouse Xenograft Models of Oral Squamous Cell Carcinomas.

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3-10-overexpressed CHO-K1 (CHO/CD44v3-10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3-10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3-10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3-10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.

A conserved protein tyrosine phosphatase, PTPN-22, functions in diverse developmental processes in C. elegans.

Protein tyrosine phosphatases non-receptor type (PTPNs) have been studied extensively in the context of the adaptive immune system; however, their roles beyond immunoregulation are less well explored. Here we identify novel functions for the conserved C. elegans phosphatase PTPN-22, establishing its role in nematode molting, cell adhesion, and cytoskeletal regulation. Through a non-biased genetic screen, we found that loss of PTPN-22 phosphatase activity suppressed molting defects caused by loss-of-function mutations in the conserved NIMA-related kinases NEKL-2 (human NEK8/NEK9) and NEKL-3 (human NEK6/NEK7), which act at the interface of membrane trafficking and actin regulation. To better understand the functions of PTPN-22, we carried out proximity labeling studies to identify candidate interactors of PTPN-22 during development. Through this approach we identified the CDC42 guanine-nucleotide exchange factor DNBP-1 (human DNMBP) as an in vivo partner of PTPN-22. Consistent with this interaction, loss of DNBP-1 also suppressed nekl-associated molting defects. Genetic analysis, co-localization studies, and proximity labeling revealed roles for PTPN-22 in several epidermal adhesion complexes, including C. elegans hemidesmosomes, suggesting that PTPN-22 plays a broad role in maintaining the structural integrity of tissues. Localization and proximity labeling also implicated PTPN-22 in functions connected to nucleocytoplasmic transport and mRNA regulation, particularly within the germline, as nearly one-third of proteins identified by PTPN-22 proximity labeling are known P granule components. Collectively, these studies highlight the utility of combined genetic and proteomic approaches for identifying novel gene functions.

Biomolecule‐responsive polymers and their bio‐applications

AbstractPrecise recognition and specific interactions between biomolecules are key prerequisites for ensuring the performance of all actives within living organisms. The convergence of biomolecular recognition systems into synthetic materials could endow the materials with high specificity and biological sensitivity; this, in turn, enables precise drug release, monitoring or detection of important biomolecules, and cell manipulation through targeted capture or release of specific biomolecules. Meanwhile, from the perspective of materials science, the application of conventional polymers in practical biological systems poses several challenges, such as low responsiveness and sensitivity, inadequate targetability, insufficient anti‐interference capacities, and unsatisfactory biocompatibility. These problems could be partly attributed to the polymers' weak discrimination abilities toward target biomolecules in the presence of interfering substances with high abundance. In particular, the proposition of “precision medicine” project raises higher demands for the design of biomaterials in terms of their precision and targetability. Therefore, there is an urgent demand for the development of new‐generation biomaterials with precise recognition and sensitive responsiveness comparable to biomacromolecules. This promotes a new research direction of biomolecule‐responsive polymers and their diverse applications. This review focuses on the origin and construction of biomolecule‐responsive polymers, as well as their attractive applications in drug delivery systems, bio‐detection, bio‐sensing, separation, and enrichment, as well as regulating cell adhesion.

The effects of BMP2 and the mechanisms involved in the invasion and angiogenesis of IDH1 mutant glioma cells

PurposeThis study investigated the effect of an isocitrate dehydrogenase 1 (IDH1) mutation (mutIDH1) on the invasion and angiogenesis of human glioma cells.MethodsDoxycycline was used to induce the expression of mutIDH1 in glioma cells. Transwell and wound healing assays were conducted to assess glioma cell migration and invasion. Western blotting and cell immunofluorescence were used to measure the expression levels of various proteins. The influence of bone morphogenetic protein 2 (BMP2) on invasion, angiogenesis-related factors, BMP2-related receptor expression, and changes in Smad signaling pathway-related proteins were evaluated after treatment with BMP2. Differential gene expression and reference transcription analysis were performed.ResultsSuccessful infection with recombinant lentivirus expressing mutIDH1 was demonstrated. The IDH1 mutation promoted glioma cell migration and invasion while positively regulating the expression of vascularization-related factors and BMP2-related receptors. BMP2 exhibited a positive regulatory effect on the migration, invasion, and angiogenesis of mutIDH1-glioma cells, possibly mediated by BMP2-induced alterations in Smad signaling pathway-related factors.After BMP2 treatment, the differential genes of MutIDH1-glioma cells are closely related to the regulation of cell migration and cell adhesion, especially the regulation of Smad-related proteins. KEGG analysis confirmed that it was related to BMP signaling pathway and TGF-β signaling pathway and cell adhesion. Enrichment analysis of gene ontology and genome encyclopedia further confirmed the correlation of these pathways.ConclusionMutation of isocitrate dehydrogenase 1 promotes the migration, invasion, and angiogenesis of glioma cells, through its effects on the BMP2-driven Smad signaling pathway. In addition, BMP2 altered the transcriptional patterns of mutIDH1 glioma cells, enriching different gene loci in pathways associated with invasion, migration, and angiogenesis.

Time and space modulation of substrate curvature to regulate cell mechanical identity

Recently, a variety of microenvironmental biophysical stimuli have been proved to play a crucial role in regulating cell functions. Among them, morpho-physical cues, like curvature, are emerging as key regulators of cellular behavior. Changes in substrate curvature have been shown to impact the arrangement of Focal Adhesions (FAs), influencing the direction and intensity of cytoskeleton generated forces and resulting in an overall alteration of cell mechanical identity. In their native environment, cells encounter varying degrees of substrate curvature, and in specific organs, they are exposed to dynamic changes of curvature due to periodic tissue deformation. However, the mechanism by which cells perceive substrate curvature remains poorly understood. To this aim, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. Employing a combined experimental and simulative approach, human adipose-derived stem cells were exposed to controlled curvature intensity and frequency. During this exposure, measurements were taken on FAs extension and orientation, cytoskeleton organization and cellular/nuclear alignment. The data clearly indicated a significant influence of the substrate curvature on cell adhesion processes. These findings contribute to a better understanding of the mechanisms through which cells perceive and respond to substrate curvature signals. Statement of significanceThis work is our contribution to the comprehension of substrate curvature's function as a crucial regulator of cell adhesion at the scale of focal adhesions and cell mechanical identity. In recent years, a large body of knowledge is continuously growing providing comprehension of the role of various microenvironmental biophysical stimuli in regulating cell functions. Nevertheless, little is known about the role of substrate curvature, in particular, when cells are exposed to this stimulus in a dynamic manner. To address the role of substrate curvature on cellular behavior, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. The experiment data made it abundantly evident that the substrate curvature had a major impact on the mechanisms involved in cell adhesion.

The Novel-m0230-3p miRNA Modulates the CSF1/CSF1R/Ras Pathway to Regulate the Cell Tight Junctions and Blood-Testis Barrier in Yak.

The yak (Bos grunniens) is a valuable livestock animal endemic to the Qinghai-Tibet Plateau in China with low reproductive rates. Cryptorchidism is one of the primary causes of infertility in male yaks. Compared with normal testes, the tight junctions (TJs) of Sertoli cells (SCs) and the integrity of the blood-testis barrier (BTB) in cryptorchidism are both disrupted. MicroRNAs are hairpin-derived RNAs of about 19-25 nucleotides in length and are involved in a variety of biological processes. Numerous studies have shown the involvement of microRNAs in the reproductive physiology of yak. In this study, we executed RNA sequencing (RNA-seq) to describe the expression profiles of mRNAs and microRNAs in yaks with normal testes and cryptorchidism to identify differentially expressed genes. GO and KEGG analyses were used to identify the biological processes and signaling pathways which the target genes of the differentially expressed microRNAs primarily engaged. It was found that novel-m0230-3p is an important miRNA that significantly differentiates between cryptorchidism and normal testes, and it is down-regulated in cryptorchidism with p < 0.05. Novel-m0230-3p and its target gene CSF1 both significantly contribute to the regulation of cell adhesion and tight junctions. The binding sites of novel-m0230-3p with CSF1 were validated by a dual luciferase reporter system. Then, mimics and inhibitors of novel-m0230-3p were transfected in vitro into SCs, respectively. A further analysis using qRT-PCR, immunofluorescence (IF), and Western blotting confirmed that the expression of cell adhesion and tight-junction-related proteins Occludin and ZO-1 both showed changes. Specifically, both the mRNA and protein expression levels of Occludin and ZO-1 in SCs decreased after transfection with the novel-m0230-3p mimics, while they increased after transfection with the inhibitors, with p < 0.05. These were achieved via the CSF1/CSF1R/Ras signaling pathway. In summary, our findings indicate a negative miRNA-mRNA regulatory network involving the CSF1/CSF1R/Ras signaling pathway in yak SCs. These results provide new insights into the molecular mechanisms of CSF1 and suggest that novel-m0230-3p and its target protein CSF1 could be used as potential therapeutic targets for yak cryptorchidism.

Abstract We040: Molecular Characterization of Human Highly Proliferative Cells Isolated from Coronary Artery Bypass Graft Surgery Patients Identifies IGFBP3, SOST, and ISLR as Downstream Targets of BMP9/ALK1 Signaling

Stem/progenitor cells are being investigated as a potential cell source to reverse cardiac remodeling and restore cardiac function, but the molecular mechanisms required for improved repair are not well understood. Circulating BMP9 is negatively associated with heart disease risk factors. BMP9/ALK1 signaling is a critical pathway involved in several mechanisms of vascular repair such as angiogenesis and inflammation. We recently identified ALK1 expression in human highly proliferative cells (hHiPCs) with progenitor capabilities isolated from patients undergoing coronary artery bypass graft (CABG) surgery as measured by flow cytometry. We have determined that circulating BMP9 in patients is negatively associated with number of hHiPC (p&lt;0.001) and positively associated with endothelial cell (EC) number (p=0.008) suggesting that BMP9 may play a role in hHiPC differentiation into EC. Further, we found upregulation of novel BMP9 regulated proteins, IGFBP3, SOST, and ISLR, in hHiPC upon BMP9 treatment using LC-MS/MS analysis of the secretome (p&lt;0.01, Fold-change (FC)&gt;2.0) and RT-qPCR (p&lt;0.001, FC&gt;2.0). Conditioned media collected from BMP9 treated hHiPCs or ECs improved tube formation in the tube formation assay. IGFBP3, SOST, and ISLR upregulation by BMP9 is inhibited upon knockdown and pharmacological inhibition of ALK1 as measured by proteomics (p&lt;0.01) and RT-qPCR analysis (p&lt;0.05). ISLR is a novel marker of mesenchymal stem cells found to have an important role in maintaining “stemness” and colony formation. hHiPCs treated with novel BMP9 target, ISLR, were analyzed using LC-MS/MS and STRING. We found ISLR treatment enriched for differential regulation of cell adhesion, cell migration, and angiogenesis proteins such as CXCL12, EMILIN1, and ANXA5 (p&lt;0.05). Together, these data lay the foundation for investigating BMP9/ALK1 autocrine signaling in potential regulation of progenitor cell maintenance and angiogenesis in heart disease.

Abstract We122: Suppression of defender against cell death 1 (Dad1) induces cardiomyocyte death by regulating cell adhesion proteins.

Introduction: Suppression of cardiomyocyte loss is considered a promising strategy for the prevention of the onset of heart failure (HF). Previously, defender against cell death 1 ( Dad1 ) was identified as a cytoprotective gene. Dad1 forms a complex with staurosporine and temperature sensitive 3A (Stt3A), a catalytic subunit of oligosaccharyltransferase (OST) complex which is responsible for N-glycosylation. However, the function of Dad1 in cardiomyocytes remains unknown. Hypothesis: The disruption of cell-extracellular matrix interactions results in cell death, known as anoikis. Because various cell adhesion proteins are N-glycosylated, we hypothesized that Dad1 suppressed anoikis by regulating N-glycosylation of cell adhesion proteins in cardiomyocytes. Methods: Neonatal rat cardiomyocytes were cultured and the expression of Dad1 or Stt3A was knocked down using siRNA. The protein expression was assessed with immunoblot analysis, and cell viability was evaluated using a CellTiter-Blue assay kit. Results: The suppression of Dad1 using siRNAs reduced cardiomyocyte viability ( p &lt;0.01, n=4). Interestingly, suppression of Dad1 impaired the N-glycosylation of integrin β1 and decreased the expression of mature integrin β1 ( p &lt;0.01, n=3). These changes inactivated integrin-mediated signal transduction through dephosphorylation of focal adhesion kinase ( p &lt;0.01, n=5). In addition, enhanced cell-extracellular matrix interactions by using adhesamine rescued the cardiomyocyte death by Dad1 knockdown ( p &lt;0.01, n=4). Next, we pursued the relationship between Dad1 and Stt3A. Dad1 knockdown decreased the expression of Stt3A ( p &lt;0.01, n=6). Suppression of Stt3A induced cardiomyocyte death accompanied by the changes of cell adhesion proteins ( p &lt;0.01, n=4). Finally, double knockdown of Dad1 and Stt3A did not induce a further reduction in cell viability compared with the knockdown of only Dad1 ( p &lt;0.05, n=4), suggesting that cardiomyocyte death by Dad1 knockdown was dependent on Stt3A. Conclusion: Dad1 is required for the stabilization of Stt3A and suppresses cardiomyocyte anoikis by regulating N-glycosylation of cell adhesion proteins in an OST-dependent manner. Dad1-mediated cardiomyocyte survival could be a therapeutic target against HF.

Shikonin Suppresses Cell Tumorigenesis in Gastric Cancer Associated with the Inhibition of c-Myc and Yap-1.

The study aimed to study the potential roles and mechanisms of shikonin in gastric cancer by network pharmacology and biological experiments. The key genes and targets of shikonin in gastric cancer were predicted by network pharmacology and molecular docking study. The effect of shikonin on the proliferation, migration, and invasion of gastric cancer cells was detected by the CCK8 method, and wound healing and transwell assays. The expression levels of c-Myc and Yap-1 were detected via western blotting in gastric cancer cells after shikonin intervention. The results of network pharmacology revealed the key target genes of shikonin on gastric cancer cells to be c-Myc, Yap-1, AKT1, etc. GO and KEGG analysis showed regulation of cell migration, proliferation, adhesion, and other biological processes, including the PI3K-Akt signaling pathway, HIF-1 signaling pathway, necroptosis, and other cancer pathways. Molecular docking showed shikonin to be most closely combined with protooncogenes c-Myc and Yap-1. In vitro experiments showed that the proliferation rate, migration, and invasion ability of the gastric cancer cell group decreased significantly after shikonin intervention for 24h. The expression levels of c-Myc and Yap-1 in gastric cancer cells were found to be significantly decreased after shikonin intervention. This study showed protooncogenes c-Myc and Yap-1 to be the core target genes of shikonin on gastric cancer cells. Shikonin may suppress gastric cancer cells by inhibiting the protooncogenes c-Myc and Yap-1. This suggests that shikonin may be a good candidate for the treatment of gastric cancer.

Nasal microRNA signatures for disease severity in infants with respiratory syncytial virus bronchiolitis: a multicentre prospective study

BackgroundRespiratory syncytial virus (RSV) bronchiolitis contributes to a large morbidity and mortality burden globally. While emerging evidence suggests that airway microRNA (miRNA) is involved in the pathobiology of RSV infection,...

Transcriptome Profiling of Mouse Embryonic Fibroblast Spontaneous Immortalization: A Comparative Analysis.

Cell immortalization, a hallmark of cancer development, is a process that cells can undergo on their path to carcinogenesis. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) have been used for decades; however, changes in the global transcriptome during this process have been poorly described. In our research, we characterized the poly-A RNA transcriptome changes after spontaneous immortalization. To this end, differentially expressed genes (DEGs) were screened using DESeq2 and characterized by gene ontology enrichment analysis and protein-protein interaction (PPI) network analysis to identify the potential hub genes. In our study, we identified changes in the expression of genes involved in proliferation regulation, cell adhesion, immune response and transcriptional regulation in immortalized MEFs. In addition, we performed a comparative analysis with previously reported MEF immortalization data, where we propose a predicted gene regulatory network model in immortalized MEFs based on the altered expression of Mapk11, Cdh1, Chl1, Zic1, Hoxd10 and the novel hub genes Il6 and Itgb2.