Bioinformatics-Based Identification of CircRNA-MicroRNA-mRNA Network for Calcific Aortic Valve Disease.

Background: Calcific aortic valve disease (CAVD) is the most common native valve disease. Valvular interstitial cell (VIC) osteogenic differentiation and valvular endothelial cell (VEC) dysfunction are key steps in CAVD progression. Circular RNA (circRNAs) is involved in regulating osteogenic differentiation with mesenchymal cells and is associated with multiple disease progression, but the function of circRNAs in CAVD remains unknown. Here, we aimed to investigate the effect and potential significance of circRNA-miRNA-mRNA networks in CAVD. Methods: Two mRNA datasets, one miRNA dataset, and one circRNA dataset of CAVD downloaded from GEO were used to identify DE-circRNAs, DE-miRNAs, and DE-mRNAs. Based on the online website prediction function, the common mRNAs (FmRNAs) for constructing circRNA-miRNA-mRNA networks were identified. GO and KEGG enrichment analyses were performed on FmRNAs. In addition, hub genes were identified by PPI networks. Based on the expression of each data set, the circRNA-miRNA-hub gene network was constructed by Cytoscape (version 3.6.1). Results: 32 DE-circRNAs, 206 DE-miRNAs, and 2170 DE-mRNAs were identified. Fifty-nine FmRNAs were obtained by intersection. The KEGG pathway analysis of FmRNAs was enriched in pathways in cancer, JAK-STAT signaling pathway, cell cycle, and MAPK signaling pathway. Meanwhile, transcription, nucleolus, and protein homodimerization activity were significantly enriched in GO analysis. Eight hub genes were identified based on the PPI network. Three possible regulatory networks in CAVD disease were obtained based on the biological functions of circRNAs including: hsa_circ_0026817-hsa-miR-211-5p-CACNA1C, hsa_circ_0007215-hsa-miR-1252-5p-MECP2, and hsa_circ_0007215-hsa-miR-1343-3p- RBL1. Conclusion: The present bionformatics analysis suggests the functional effect for the circRNA-miRNA-mRNA network in CAVD pathogenesis and provides new targets for therapeutics.

Multi-omics analyses of serum metabolome, gut microbiome and brain function reveal dysregulated microbiota-gut-brain axis in bipolar depression.

The intricate processes of microbiota-gut-brain communication in modulating human cognition and emotion, especially in the context of mood disorders, have remained elusive. Here we performed faecal metagenomic, serum metabolomics and neuroimaging studies on a cohort of 109 unmedicated patients with depressed bipolar disorder (BD) patients and 40 healthy controls (HCs) to characterise the microbial-gut-brain axis in BD. Across over 12,000 measured metabolic features, we observed a large discrepancy (73.54%) in the serum metabolome between BD patients and HCs, spotting differentially abundant microbial-derived neuroactive metabolites including multiple B-vitamins, kynurenic acid, gamma-aminobutyric acid and short-chain fatty acids. These metabolites could be linked to the abundance of gut microbiota presented with corresponding biosynthetic potentials, including Akkermansia muciniphila, Citrobacter spp. (Citrobacter freundii and Citrobacter werkmanii), Phascolarctobacterium spp., Yersinia spp. (Yersinia frederiksenii and Yersinia aleksiciae), Enterobacter spp. (Enterobacter cloacae and Enterobacter kobei) and Flavobacterium spp. Based on functional neuroimaging, BD-related neuroactive microbes and metabolites were discovered as potential markers associated with BD-typical features of functional connectivity of brain networks, hinting at aberrant cognitive function, emotion regulation, and interoception. Our study combines gut microbiota and neuroactive metabolites with brain functional connectivity, thereby revealing potential signalling pathways from the microbiota to the gut and the brain, which may have a role in the pathophysiology of BD.

Integrated bioinformatics analysis of microarray data from non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), with its high mortality rate, lack of early diagnostic markers and prevention of distant metastases are the main challenges in treatment. To identify potential miRNAs and key genes in NSCLC to find new biomarkers and target gene therapies. The GSE102286, GSE56036, GSE25508, GSE53882, GSE29248 and GSE101929 datasets were obtained from the Gene Expression Omnibus (GEO) database and screened for differentially co-expressed miRNAs (DE-miRNAs) and lncRNAs (DElncRs) by GEO2R and R software package. Pathway enrichment analysis of DE-miRNAs-target genes was performed by String and Funrich database to construct protein-protein interaction (PPI) and competing endogenous RNA (ceRNA) network and visualized with Cytoscape software. Nineteen co-expressed DE-miRNAs were screened from five datasets. The 7683 predicted up- and down-regulated DE-miRNAs-target genes were significantly concentrated in cancer-related pathways. The top 10 hub nodes in the PPI were identified as hub genes, such as MYC, EGFR, HSP90AA1 and TP53, MYC, and ACTB. By constructing miRNA-hub gene networks, hsa-miR-21, hsa-miR-141, hsa-miR-200b and hsa-miR-30a, hsa-miR-30d, hsa-miR-145 may regulate most hub genes and hsa-miR-141, hsa-miR-200, hsa-miR-145 had higher levels in the miRNA and ceRNA regulatory networks, respectively. In conclusion, the identification of hsa-miR-21, hsa-miR-141, hsa-miR-200b hsa-miR-30a, hsa-miR-30d and hsa-miR-145 provides a new theoretical basis for understanding the development of NSCLC.

Integrated Network Analysis to Determine CNN1, MYL9, TAGLN, and SORBS1 as Potential Key Genes Associated with Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is challenging to treat. It is necessary to screen for related biological markers to accurately predict the prognosis and recurrence of prostate cancer. METHODS: Three data sets, GSE28204, GSE30521, and GSE69223, from the Gene Expression Omnibus (GEO) database were integrated into this study. After the identification of differentially expressed genes (DEGs) between PCa and normal prostate tissues, network analyses including protein-protein interaction (PPI) network, and weighted gene co-expression network analysis (WGCNA) were used to select hub genes. Gene Ontology (GO) term analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to annotate the functions of DEGs and hub modules of the networks. Survival analysis was performed to validate the correlation between the key genes and PCa relapse. RESULTS: In total, 867 DEGs were identified, including 201 upregulated and 666 downregulated genes. Three hub modules of the PPI network and one hub module of the weighted gene co-expression network were determined. Moreover, four key genes (CNN1, MYL9, TAGLN, and SORBS1) were significantly associated with PCa relapse (p < 0.05). CONCLUSIONS: CNN1, MYL9, TAGLN, and SORBS1 may be potential biomarkers for PCa development.

Integrated microarray for identifying the hub mRNAs and constructed miRNA-mRNA network in coronary in-stent restenosis.

As a major complication after percutaneous coronary intervention (PCI) in patients who suffer from coronary artery disease, in-stent restenosis (ISR) poses a significant challenge for clinical management. A miRNA-mRNA regulatory network of ISR can be constructed to better reveal the occurrence of ISR. The relevant data set from the Gene Expression Omnibus (GEO) database was downloaded, and 284 differentially expressed miRNAs (DE-miRNAs) and 849 differentially expressed mRNAs (DE-mRNAs) were identified. As predicted by online tools, 65 final functional genes (FmRNAs) were overlapping DE-mRNAs and DE-miRNAs target genes. In the biological process (BP) terms of gene ontology (GO) functional analysis, the FmRNAs were mainly enriched in the cellular response to peptide, epithelial cell proliferation, and response to peptide hormone. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the FmRNAs were mainly enriched in breast cancer, endocrine resistance, and Cushing syndrome. Jun proto-oncogene, activator protein-1 (AP-1) transcription factor subunit (JUN), insulin-like growth factor 1 receptor (IGF1R), member RAS oncogene family (RAB14), specificity protein 1 (SP1), protein tyrosine phosphatase nonreceptor type 1 (PTPN1), DDB1 and CUL4 associated factor 10 (DCAF10), retinoblastoma-binding protein 5 (RBBP5), and eukaryotic initiation factor 4A-I (EIF4A1) were hub genes in the protein-protein interaction network (PPI network). The miRNA-mRNA network containing DE-miRNAs and hub genes was built. Hsa-miR-139-5p-JUN, hsa-miR-324-5p-SP1 axis pairs were found in the miRNA-mRNA network, which could promote ISR development. The aforementioned results indicate that the miRNA-mRNA network constructed in ISR has a regulatory role in the development of ISR and may provide new approaches for clinical treatment and experimental development.

SYT-SSX1 enhances the invasiveness and maintains stem-like cell properties in synovial sarcoma via induction of TGF-ß1/Smad signaling.

BACKGROUND: Synovial sarcoma (SS) is a type of soft tissue sarcoma (STS) of undetermined tissue origin, which is characterized by the recurrent pathognomonic chromosomal translocation t (X;18)(p11.2; q11.2). Studies have shown that SS is a malignant tumor originating from cancer stem cells or pluripotent mesenchymal stem cells and may be related to fusion genes. In addition, some studies have indicated that the induction of epithelial-mesenchymal transition (EMT) via the TGF-ß1/Smad signaling pathway leads to SS metastasis. METHODS: We analyzed the effects of SYT-SSX1 on the stemness of SS cells via TGF-ß1/Smad signaling in vitro. The SYT-SSX1 fusion gene high expression cell was constructed by lentiviral stable transfer technology. SYT-SSX1 and SW982 cells were cultured and tested for sphere-forming ability. The transwell migration assay and flow cytometry were used to assess the migration ability of the sphere cells as well as the expression of CSC-related markers. We treated SYT-SSX1 cells with rhTGF-ß1 (a recombinant agent of the TGF-ß1 signaling pathway) and SB431542 and observed morphological changes. A CCK-8 experiment and a western blot (WB) experiment were conducted to detect the expression of TGF-ß1 signaling pathway- and EMT-related proteins after treatment. The SYT-SSX1 cells were then cultured and their ability to form spheres was tested. Flow cytometry, WB, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of CSC surface markers on SYT-SSX1 sphere cells. RESULTS: It was found that SYT-SSX1 has stronger sphere-forming ability, migration ability, and higher expression of CSC-related molecules than SW982 cells. Through treating SYT-SSX1 and SW982 cells with rhTGF-ß1 and SB431542, we found that TGF-ß1 enhanced the proliferation of cells, induced EMT, and that TGF-ß1 enhanced the characteristics of tumor stem cells. CONCLUSIONS: Our results suggest that SYT-SSX1 enhances invasiveness and maintains stemness in SS cells via TGF-ß1/Smad signaling. These findings reveal an effective way to potentially improve the prognosis of patients with SS by eliminating the characteristics of cancer stem cells (CSCs) during treatment.

Predictive values of baseline matrix metalloproteinase 9 levels in peripheral blood on 3-month outcomes of high-risk patients with minor stroke or transient ischemic attack.

BACKGROUND AND PURPOSE: To explore the relationship between baseline levels of matrix metalloproteinase 9 (MMP9) in peripheral blood and the outcomes in patients with acute minor stroke and transient ischemic attack (TIA). METHODS: We assessed data from patients with acute minor ischemic stroke or TIA who were included in the CHANCE (Clopidogrel in High-Risk Patients with Acute Nondisabling Cerebrovascular Events) trial. Baseline level of MMP9 in peripheral blood is classified into five quintiles. We assessed the relationship between the baseline MMP9 and outcomes of stroke recurrence, composite vascular events, and poor functional outcomes within 90 days after stroke onset. RESULTS: Of the 3014 patients included, 295 (9.79%) had recurrent stroke, 289 (9.59%) had recurrent ischemic stroke, 297 (9.85%) had combined vascular events, and 199 (6.64%) had poor functional outcomes within 90 days. We used MMP9 concentrations near hazard ratio (HR) = 1 (Q3) in restricted cubic splines as the reference. The result showed that, compared to patients in the Q3 group, patients in the highest quintile (Q5 group) had an increased risk of poor functional outcomes at 90 days after adjusting the risk factors and confounders (p = 0.030), which may be associated with an increased risk of combined vascular events (p = 0.052). Using Cox regression models or logistic regression models with restricted cubic spline, we also observed that higher MMP9 ratios were associated with an increased risk of stroke recurrence, combined events, and poor functional outcomes at a range of concentrations. CONCLUSIONS: For patients with acute minor stroke or TIA, higher baseline MMP9 level was associated with an increased risk of poor functional outcomes, which might be related to stroke recurrence and combined vascular events.

Long non-coding RNA MIR31HG as a prognostic predictor for malignant cancers: A meta- and bioinformatics analysis.

BACKGROUND: The possible regulatory mechanism of MIR31HG in human cancers remains unclear, and reported results of the prognostic significance of MIR31HG expression are inconsistent. METHODS: The meta-analysis and related bioinformatics analysis were conducted to evaluate the role of MIR31HG in tumor progression. RESULTS: The result showed that high MIR31HG expression was not related to prognosis. However, in the stratified analysis, we found that the overexpression of MIR31HG resulted in worse OS, advanced TNM stage, and tumor differentiation in respiratory system cancers. Moreover, our results also found that MIR31HG overexpression was related to shorter OS in cervical cancer patients and head and neck tumors. In contrast, the MIR31HG was lower in digestive system tumors which contributed to shorter overall survival, advanced TNM stage, and distant metastasis. Furthermore, the bioinformatics analysis showed that MIR31HG was highly expressed in normal urinary bladder, small intestine, esophagus, stomach, and duodenum and low in colon, lung, and ovary. The results obtained from FireBrowse indicated that MIR31HG was highly expressed in LUSC, CESC, HNSC, and LUAD and low in STAD and BLCA. Gene Ontology analysis showed that the co-expressed genes of MIR31HG were most enriched in the biological processes of peptide metabolism and KEGG pathways were most enriched in Ras, Rap1, and PI3K-Akt signaling pathway. CONCLUSION: MIR31HG may serve as a potential biomarker in human cancers.

IFITM1, CD10, SMA, and h-caldesmon as a helpful combination in differential diagnosis between endometrial stromal tumor and cellular leiomyoma.

BACKGROUND: The differential diagnosis of endometrial stromal tumor (EST) and uterine cellular leiomyoma (CL) remains a challenge in clinical practice, especially low grade endometrial stromal sarcoma (ESS) and CL, suggesting the need for novel immunomarkers panels for differential diagnosis. Interferon-induced transmembrane protein 1 (IFITM1) is a novel immunomarker for endometrial stromal cells, h-caldesmon is an immunomarker for smooth muscle cells and has a higher specificity than smooth muscle actin (SMA). So this study aimed to evaluate whether IFITM1, cluster of differentiation 10(CD10), SMA, and h-caldesmon are useful biomarker combinations for the differential diagnosis of EST and CL. METHODS: Tissue microarrays were used to detect IFITM1, CD10, SMA, and h-caldesmon immunohistochemical staining in 30 EST and 33 CL cases. RESULTS: The expressions of IFITM1 and CD10 were high in EST (86.7 and 63.3%, respectively) but low in CL (18.2 and 21.2%), whereas those of h-caldesmon and SMA were high in CL (87.9 and 100%) and low in EST (6.9 and 40%). In diagnosing EST, IFITM1 shows better sensitivity and specificity (86.7 and 81.8%, respectively) than CD10 (63.3 and 78.8%). The specificity of h-caldesmon in diagnosing CL was significantly higher (93.1%) than that of SMA (60%). When all four antibodies were combined for the differential diagnosis, the area-under-the-curve (AUC) predictive value was 0.995. The best combination for diagnosing EST was IFITM1 (+) or CD10 (+) and h-caldesmon (-) (sensitivity 86.7%, specificity 93.9%). CONCLUSION: The best combination for diagnosing CL were h-caldesmon (+) and SMA (+) (sensitivity 87.9%, specificity 100%). IFITM1, CD10, SMA, and h-caldesmon are a good combination for the differential diagnosis of EST and CL.

Prognostic Significance of Matrix Metalloproteinase 14 in Patients with Cancer: a Systematic Review and Meta-Analysis.

BACKGROUND: There is increasing evidence that matrix metalloproteinase 14 (MMP-14) is involved in tumor progression and prognosis. MMP-14 exhibits different expression in patients with various cancers, suggesting that it may be considered as a potential prognostic biomarker for cancer. METHODS: Therefore, this meta-analysis was performed to elucidate the prognostic value and association of MMP-14 over-expression in several types of cancers. Eligible studies based on eligibility criteria from various online databases were searched. The pooled hazard ratios (HRs) with 95% confidence intervals (CIs) for overall survival (OS) were analyzed to determine the prognostic value of MMP-14 using STATA software 12.0. RESULTS: We identified sixteen applicable studies in this meta-analysis comprising 2,766 samples. Over-expression MMP-14 was significantly correlated with a poor overall survival (OS) outcome in multiple cancers (HR: 2.22; 95% CI: 1.72 - 2.87). Moreover, high levels of MMP-14 were markedly associated with tumor progression and metastasis (HR: 1.83; 95% CI: 1.36 - 2.46). MMP-14 expression was also associated with histological differentiation (OR: 0.37; 95% CI: 0.18 - 0.77). CONCLUSIONS: MMP-14 over-expression suggested aggressive biological behaviors and implied that MMP-14 may be a useful prognostic biomarker in human cancers.

Prognostic impact of tumor-associated macrophage infiltration in esophageal cancer: a meta-analysis.

Aim: To provide clarity surrounding the association between tumor-associated macrophages (TAMs) and esophageal cancer prognosis. Materials & methods: Several databases were searched. The meta-analysis was conducted by using software Stata 12.0 and Revman. Results: Sixteen studies were included in this analysis (2292 samples). CD68+ TAM density was not associated with overall survival (OS; hazard ratio [HR]: 0.88, 95% CI: 0.67-1.15; p = 0.33) and disease-free survival (HR: 1.25, 95% CI: 0.66-2.35; p = 0.49). M2-like TAMs were associated with poor overall survival (HR: 1.47, 95% CI: 1.10-1.98; p = 0.01), Tumor, Node, Metastasis staging and vessel metastasis. Conclusion: CD68+ TAM density is not associated with esophageal cancer progression, while CD163+ M2-like TAMs is a potential risk factor.

miR-30 Family Reduction Maintains Self-Renewal and Promotes Tumorigenesis in NSCLC-Initiating Cells by Targeting Oncogene TM4SF1.

Increasing evidence indicates that tumor-initiating cells (TICs) are responsible for the occurrence, development, recurrence, and development of the drug resistance of cancer. MicroRNA (miRNA) plays a significant functional role by directly regulating targets of TIC-triggered non-small-cell lung cancer (NSCLC), but little is known about the function of the miR-30 family in TICs. In this study, we found the miR-30 family to be downregulated during the spheroid formation of NSCLC cells, and patients with lower miR-30a/c expression had shorter overall survival (OS) and progression-free survival (PFS). Moreover, transmembrane 4 super family member 1 (TM4SF1) was confirmed to be a direct target of miR-30a/c. Concomitant low expression of miR-30a/c and high expression of TM4SF1 correlated with a shorter median OS and PFS in NSCLC patients. miR-30a/c significantly inhibited stem-like characteristics in vitro and in vivo via suppression of its target gene TM4SF1, and then it inhibited the activity of the mTOR/AKT-signaling pathway. Thus, our data provide the first evidence that TM4SF1 is a direct target of miR-30a/c and miR-30a/c inhibits the stemness and proliferation of NSCLC cells by targeting TM4SF1, suggesting that miR-30a/c and TM4SF1 may be useful as tumor biomarkers for the diagnosis and treatment of NSCLC patients.

KinITC-One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists.

Affinity data, such as dissociation constants (KD ) or inhibitory concentrations (IC50 ), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein-ligand complexes and their half-life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure-kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re-analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off-rate constant koff ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant kon ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and koff could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on kon .

Reduced hsa-miR-124-3p levels are associated with the poor survival of patients with hepatocellular carcinoma.

Hsa-MicroRNA-124a-3p (hsa-miR-124-3p) is involved in tumor progression in certain malignant tumors. However, its function and clinical implication in hepatocellular carcinoma (HCC) have not yet been illustrated. In this study, we explored the expression and prognostic value of hsa-miR-124-3p in patients with HCC. Hsa-miR-124-3p expression in HCC was analyzed in silico, which was subsequently confirmed by quantitative PCR in 155 HCC biopsy samples. Overall survival (OS) and disease-free survival in HCC patients was evaluated by Kaplan-Meier survival analysis, and univariate and multivariate Cox proportional hazard models were used. The in silico results demonstrated that hsa-miR-124-3p was reduced in cell lines and tissues of HCC, and hsa-miR-124-3p expression was lower in HCC tumor samples than in normal liver tissues. Moreover, a decrease in hsa-miR-124-3p expression was closely correlated with tumor diameter (≥ 5 cm) and number of lesions (multiple). Lower hsa-miR-124-3p expression was shown to be correlated with a shorter OS and poor prognosis in HCC. Our findings demonstrate that hsa-miR-124-3p might be a potential target for the diagnosis and prognosis of HCC.

Laser capture microdissection for detecting the expression of epithelial-mesenchymal transition-related genes in epithelial and spindle cells of paraffin-embedded formalin-fixed biphasic synovial sarcoma.

Synovial sarcoma (SS) is a mesenchymal malignant neoplasm showing characteristics of epithelial-mesenchymal biphasic differentiation. SS is of uncertain cellular origin; however, studies have suggested that SS originates from a somatic stem cell population. In this study, we aim to determine whether differential morphological features of the epithelial-mesenchymal transition (EMT) contributed to the tumourigenesis of SS invasion and metastasis. Twelve paraffin-embedded formalin-fixed tissue (FFPE) SS tissue specimens were obtained, and laser capture microdissection (LCM) with the ArcturusXT system and small chip method (SCM) were used to isolate and purify spindle and epithelial cells from SS specimens. The TRIzol method was used to extract RNA, and the mRNA levels of EMT-related genes in epithelial and spindle cells of SS specimens were measured using real-time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results show that collection of about 2 × 104 cells from FFPE samples using LCM was sufficient for qRT-PCR, with an efficiency of 75%. Compared with LCM, 72.2% (13 of 18) RNA samples were successfully extracted using SCM to isolate cells from FFPE SS tissues. In the 16 samples (11 spindle cell samples and 5 epithelial cell samples), Snail mRNA was significantly upregulated in spindle cell areas compared with that in epithelial cell areas (P = .001). Expression levels of the epithelial marker E-cadherin and the mesenchymal marker N-cadherin were not significantly different between epithelial and spindle cell areas. In spindle cells of recurrent SS samples, the mRNA levels of E-cadherin, N-cadherin, Snail, and Slug were higher in primary SS samples than in recurrent samples. Taken together, our results indicated that in SS samples, Snail mRNA was upregulated in spindle cell areas compared with that in epithelial cell areas and that the expression of EMT-related genes was increased in primary SS. LCM could be used to isolate and purify RNA from FFPE samples.

[Involvement of interaction between TRPC1 and Orai1 in calcium sensing receptor-mediated calcium influx and nitric oxide generation in human umbilical vein endothelial cells].

The present study was to investigate the role of the interaction between canonical transient receptor potential channel 1 (TRPC1) and calcium release-activated calcium modulator 1 (Orai1) in extracellular Ca2+-sensing receptor (CaR)-induced extracellular Ca2+ influx and nitric oxide (NO) production. Human umbilical vein endothelial cells (HUVECs) were incubated with CaR agonist Spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC)] alone or in combination with the following reagents: CaR negative allosteric modulator Calhex231 plus ROC analogue TPA (activating ROC and blocking SOC), Ro31-8220 (PKC inhibitor that activates SOC and blocks ROC) or Go6967 (PKCs and PKCµ inhibitor that activates SOC and blocks ROC). The protein expressions and co-localization of TRPC1 and Orai1 were determined using immunofluorescent staining. The interaction between TRPC1 and Orai1 was examined by co-immunoprecipitation. We silenced the expressions of their genes in the HUVECs by transfection of constructed TRPC1 and Orai1 shRNA plasmids. Intracellular Ca2+ concentration ([Ca2+]i) was detected using Ca2+ indicator Fura-2/AM, and NO production was determined by DAF-FM staining. The results showed that TRPC1 and Orai1 protein expressions were co-located on the cell membrane of the HUVECs. Compared with Spermine+Ca2+ group, Calhex231+ TPA+Spermine+Ca2+, Ro31-8220+Spermine+Ca2+ and Go6976+Spermine+Ca2+ groups exhibited down-regulated protein expressions of TRPC1 and Orai1 in cytoplasm and decreased co-localization on the cell membrane. Co-immunoprecipitation results showed that the interaction between TRPC1 and Orai1 was reduced by Calhex231 plus TPA, Ro31-8220 or Go6976 addition in the Spermine-stimulated HUVECs. Double knockdown of Trpc1 and Orai1 genes significantly decreased [Ca2+]i level and NO production in all of the Spermine+Ca2+, Calhex231+TPA+Spermine+Ca2+, Ro31-8220+Spermine+Ca2+ and Go6976+Spermine+Ca2+ groups. These results suggest that TRPC1/Orai1 may form a complex that mediates Ca2+ influx and No production via SOC and ROC activation.

Encapsulating an Immunosuppressant Enhances Tolerance Induction by Siglec-Engaging Tolerogenic Liposomes.

Unwanted antibody responses significantly impact human health, and current options for treating deleterious antibody responses largely rely on broad immunosuppressants that can compromise overall immunity. A desirable alternative is to induce antigen-specific immune tolerance. We have shown that co-presentation of antigen and ligands of B cell sialic acid-binding immunoglobulin-like lectins (Siglecs) on a liposomal nanoparticle induces antigen-specific tolerance. Although Siglec-engaging tolerance-inducing antigenic liposomes (STALs) induce robust B cell tolerance in naïve mice, the full potential of STALs requires long-term tolerance induction and suppression of an ongoing immune response. We hypothesized that STALs encapsulated with rapamycin (RAPA), an immunomodulator, could improve the efficacy of STALs and potentially enable their use in the context of immunological memory. Here, we showed that formulation of STALs with RAPA produced enhanced tolerance induction in naïve mice compared to STALs without RAPA but had minimal impact on inducing tolerance in previously sensitized mice. These findings indicate that the addition of immunomodulators to STALs could be beneficial in tolerance induction and support future development of STALs for the treatment of allergy and autoimmune diseases.

Target-directed Dynamic Combinatorial Chemistry: A Study on Potentials and Pitfalls as Exemplified on a Bacterial Target.

Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established.

The expression profile and clinicopathological significance of Notch1 in patients with colorectal cancer: a meta-analysis.

AIM: This meta-analysis was conducted to evaluate the clinicopathological significance of Notch1 expression in patients with colorectal cancer (CRC). METHODS: Available articles were searched from diverse databases, and the meta-analysis was done by using Stata 12.0 software. RESULTS: Thirteen studies were included in this analysis (3401 samples). The Notch1 expression in CRC tissues was significantly higher than that in normal tissues statistically (OR: 15.46; 95% CI: 8.11-29.45; p = 0.003), and were associated with lymph node metastasis, tumor stage, depth of infiltration and histological differentiation. DISCUSSION:  There is a close relationship between higher Notch1 expression in CRC. Notch1 may be involved in tumor progression, invasion and metastasis with CRC. CONCLUSION: Notch1 overexpression in CRC suggested aggressive biological behaviors and thus implying that Notch1 may be a useful biomarker in CRCs.

The clinicopathological parameters significance of CD133 and Nestin in epithelial ovarian cancer: a meta-analysis.

This meta-analysis was conducted to evaluate the association of CD133 and Nestin with epithelial ovarian cancer. Databases (PubMed, EMBASE, Web of Science, China National Knowledge Infrastructure, Wanfang) were searched for relevant studies updated in August 2017. CD133 and Nestin expression were estimated by immunohistochemistry. Statistical analysis was performed by RevMan. A total of 18 studies were included in this meta-analysis. High expression of both CD133 and Nestin was associated with late International Federation of Gynecology and Obstetrics stage (p < 0.00001), larger size of residual cancer (p < 0.05). CD133 overexpression was also associated with higher histological grade (p = 0.0006) and lymph node metastases (p < 0.00001). Nestin overexpression was associated with a higher rate of treatment resistance (p = 0.0007). Positive expression of CD133 and Nestin may be associated with aggressive biological behaviors in epithelial ovarian cancer.