Mechanism and Molecular Targets of Ejiao Siwu Decoction for Treating Primary Immune Thrombocytopenia Based on High-Performance Liquid Chromatograph, Network Pharmacology, Molecular Docking and Cytokines Validation.

We explored the mechanisms and molecular targets of Ejiao Siwu Decoction (EJSW) for treating primary immune thrombocytopenia (ITP) using network pharmacology and molecular docking. Active compounds of EJSW were identified by high-performance liquid chromatography-diode array detector (HPLC-DAD) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) and their targets were obtained from HERB and SwissTargetPrediction, and ITP targets were obtained from Comparative Toxicogenomics Database (CTD) and GeneCards. STRING and Cytoscape were used for protein-protein interaction (PPI) network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses by WebGestalt yielded a gene-pathway network, Autodock molecular docking was applied to screen targets and active compounds, and cytokines were detected using a cytometric bead array (CBA) human inflammation kit. We identified 14 compounds and 129 targets, and 1,726 ITP targets. RAC-alpha serine/threonine-protein kinase (AKT1), tumour necrosis factor (TNF), interleukin-6 (IL6), caspase-3 (CASP3) and tumour suppressor protein (TP53) were core targets (nodes and edges). Functional annotation identified cofactor binding and coenzyme binding, and 20 significantly enriched pathways. Active compounds of EJSW were successfully docked with ITP targets. Tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) were upregulated in ITP patients, vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor D (VEGF-D) were downregulated, and EJSW treatment reversed these trends. EJSW may regulate key ITP targets based on the in silico analyses, and protect vascular integrity through AGE-RAGE signalling, complement and coagulation cascades, and VEGF signalling by downregulating TNF-α, IL-1ß and other inflammatory factors.

Protocol for quantitative nuclear magnetic resonance for deciphering electrolyte decomposition reactions in anode-free batteries.

In this protocol, we describe the quantification of electrolytes using nuclear magnetic resonance. We detail the steps involved for battery cycling, sample preparation, instrument operation, and data analysis. The protocol can be used to quantify electrolyte decomposition reactions and the apparent electron transfer numbers of different electrolyte components. The protocol is optimized for lithium-based anode-free batteries but can also be applied to other rechargeable batteries. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2022).1.

[Expression of MicroRNAs in Peripheral Blood of Patients with Primary Immune Thrombocytopenia and Its Correlation with the Imbalance of Th1/Th2 Cell].

OBJECTIVE: To investigate the expressions of microRNAs in peripheral blood of patients with primary immune thrombocytopenia(ITP) and its correlation with the imbalance of Th1/Th2 cells. METHODS: Thirty patients with ITP (ITP group) and 15 healthy people (control group) were enrolled．Real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of six miRNAs (miR-107,miR-205-5p,miR-138-5p,miR-326,miR-1827,miR-185-5p) and Th1-specific transcription factor T-bet mRNA and Th2-specific transcription factor GATA-3 mRNA in the peripheral blood of the two groups. Th1 and Th2 cells were detected by flow cytometry. The expressions of Th1-cytokines TNF-α and IFN-γ and Th2-cytokines IL-4 and IL-10 were detected by AimPlex multiple immunoassays for Flow. The expression difference of miRNAs, mRNA, Th1, Th2 cells and cytokines of the two groups were compared, and the correlations of miRNAs to mRNA, Th1, Th2 cells and cytokines were analyzed in ITP group. RESULTS: The expressions of miRNAs（miR-107, miR-205-5p, miR-138-5p, miR-326, miR-1827, miR-185-5p）and Th2-specific transcription factor GATA-3 mRNA of the patients in ITP group were significantly decreased (P<0.05) as compared with those in control, while the expressions of Th1 cells and Th1-specific transcription factor T-bet mRNA and Th1-cytokines TNF-α were significantly increased (P<0.05), also for the ratios of T-bet mRNA/GATA-3 mRNA and Th1/Th2 cells were significantly increased (P<0.05). The relative expressions of miR-107, miR-205-5p, miR-138-5p in ITP patients were negatively correlated with Th2 cells (r=-0.411, r=-0.593, r=-0.403,P<0.05) and the relative expression of miR-1827 was negatively correlated with TNF-α (r=-0.390). CONCLUSION: The relative expressions of the six miRNAs in peripheral blood of patients with ITP are significantly decreased, which result in the increasing ratio of T-bet mRNA/GATA-3 mRNA, then lead to the imbalance of Th1/Th2.

Inhibitory effects and molecular mechanisms of pentagalloyl glucose in combination with 5-FU on aggressive phenotypes of HepG2 cells.

This study examined the inhibition and mechanism of natural product pentagalloyl glucose (PGG) against HepG2 cells and determined the effects of its combination with the clinical chemotherapeutic drug, 5-FU. PGG was found to inhibit the proliferation, migration and invasion of HepG2 cells, induced G1 arrest and apoptosis in both concentration- and time- dependent manners. The combination of PGG and 5-FU had synergistic effects on reversal the aggressive phenotypes of HepG2 cells, increasing the proportion of Bax/Bcl-2, promoting the activation of caspase-9 and caspase-3, and inducing apoptosis. This combination upregulated P27 and cyclin B1, and downregulated cyclin E1, leading to G1 phase arrest. The combination significantly downregulated MDR1 and LRP1, suggesting the potential to reverse the resistance to 5-FU.

A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application.

BACKGROUND: Recently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application. METHODS: Taking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis. RESULTS: We found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number. CONCLUSION: This study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories.

Proteomic Profiling of Serum Exosomes From Patients With Metastatic Gastric Cancer.

Background: Clinical management of metastatic gastric cancer (mGC) remains a major challenge due to a lack of specific biomarkers and effective therapeutic targets. Recently, accumulating evidence has suggested that exosomes play an essential role in cancer metastasis and can be an excellent reservoir of novel biomarkers and candidate therapeutic targets for cancer. Therefore, in this study, we aimed to reveal the proteomic profile of mGC-derived exosomes. Methods: Exosomes were isolated from pooled serum samples of 20 mGC patients and 40 healthy controls (HC) by ultracentrifugation. Next, quantitative proteomic analyses were applied to analyze the protein profiles of the exosomes, and bioinformatic analyses were conducted on the proteomic data. Finally, the expression of exosomal protein candidates was selectively validated in individual subjects by western blot analysis. Results: We isolated exosomes from serum samples. The size of the serum derived exosomes ranged from 30 to 150 nm in diameter. The exosomal markers CD9 and CD81 were observed in the serum exosomes. However, the exosomal negative marker calnexin, an endoplasmic reticulum protein, was not detected in exosomes. Overall, 443 exosomal proteins, including 110 differentially expressed proteins (DEPs) were identified by quantitative proteomics analyses. The bioinformatics analyses indicated that the upregulated proteins were enriched in the process of protein metabolic, whereas the downregulated proteins were largely involved in cell-cell adhesion organization. Surprisingly, 10 highly vital proteins (UBA52, PSMA1, PSMA5, PSMB6, PSMA7, PSMA4, PSMA3, PSMB1, PSMA6, and FGA) were filtered from DEPs, most of which are proteasome subunits. Moreover, the validation data confirmed that PSMA3 and PSMA6 were explicitly enriched in the serum derived exosomes from patients with mGC. Conclusion: The present study provided a comprehensive description of the serum exosome proteome of mGC patients, which could be an excellent resource for further studies of mGC.

KRAS Mutant Allele Fraction in Circulating Cell-Free DNA Correlates With Clinical Stage in Pancreatic Cancer Patients.

Background: The research on circulating tumor DNA (ctDNA) in pancreatic cancer (PC) has emerged recently. Although the detection rate of the KRAS mutation in ctDNA was relatively consistent with that in tumor tissue, whether the KRAS mutant allele fraction (MAF) differed was still not reported. So far, the clinical application of ctDNA detection in PC remains inconclusive. Methods: Plasma samples were collected from 110 PC and 52 pancreatic benign (PB) disease patients. The detection of KRAS mutation in ctDNA was performed using droplet digital PCR and compared with that in matched tumor tissue. We assessed the utility of KRAS MAFs in ctDNA and tissue for pancreatic malignancy assessment. Results: We found that KRAS MAF in ctDNA of PC patients was higher than that of PB patients, and was obviously associated with tumor staging and distant metastasis. However, KRAS MAF in ctDNA was significantly different from that in matched tissue. KRAS MAF in tumor tissue had no significant correlation with the clinical status. In addition, a ROC curve analysis revealed that mutant KRAS ctDNA combined with CA19-9 could increase the sensitivity rate of early-stage PC prediction, compared with CA19-9 test alone. Conclusion: The MAF of KRAS in ctDNA was related to the clinical stage of PC (p = 0.001). Mutant KRAS ctDNA could improve the sensitivity in early diagnosis of PC as a complement to CA19-9. Our study suggested that KRAS mutation in ctDNA could be a valuable circulating biomarker for the malignancy assessment in PC.

Microbial induced solidification and stabilization of municipal solid waste incineration fly ash with high alkalinity and heavy metal toxicity.

This paper presents an experimental study on the applicability of microbial induced carbonate precipitation (MICP) to treat municipal solid waste incineration (MSWI) fly ash with high alkalinity and heavy metal toxicity. The experiments were carried out on fly ashes A and B produced from incineration processes of mechanical grate furnace and circulating fluidized bed, respectively. The results showed that both types of fly ashes contained high CaO content, which could supply sufficient endogenous Ca for MICP treatment. Moreover, S. pasteurii can survive from high alkalinity and heavy metal toxicity of fly ash solution. Further, the unconfined compressive strength (UCS) of MICP treated fly ashes A and B reached 0.385MPa and 0.709 MPa, respectively. The MICP treatment also resulted in a reduction in the leaching toxicity of heavy metals, especially for Cu, Pb and Hg. MICP had a higher solidification and stabilization effect on fly ash B, which has finer particle size and higher Ca content. These findings shone a light on the possibility of using MICP technique as a suitable and efficient tool to treat the MSWI fly ash.

Laboratory method of microbial induced solidification/stabilization for municipal solid waste incineration fly ash.

This paper presents a method to solidify/stabilize the municipal solid waste incineration (MSWI) fly ash by originally employing the microbial induced carbonate precipitation (MICP) technique. In this method, the rich endogenous calcium in the MSWI fly ash was utilized to induce calcite precipitation, which is different from the operation of adding extra calcium source in previous researches. The fly ash sample had a CaO content of 44.5%, and its leaching concentrations of Zn, Cr and Pb exceed the limits of the identification standard for hazardous wastes in China. The optical density at 600 nm (OD600) of the bacterial solution was about 1.0 after the processes of bacterial activation and reproduction. The prepared fly ash sample was well mixed with bacterial solution at an ash-liquid ratio of 1 kg: 0.3 L and cured at a temperature of 20 °C and a humidity of ≥95% for 7 days. After treatment, the heavy metal leachability significantly reduced to meet the standard for pollution control of landfill site, and the unconfined compressive strength increased approximately 40%. The precipitated carbonates were verified by SEM-EDS analysis and quantified by measurement of carbonate content via acid-dissolving method. The results shone a light on the possibility of using MICP technique as a useful and efficient tool to stabilize the MSWI fly ash before being reused or properly stored in landfills. •The MICP method is efficient to reduce the heavy metal leachability and increase the compressive strength of MSWI fly ash.•The endogenous calcium in MSWI fly ash was utilized to induce calcite precipitation.•The heavy metals in MSWI fly ash were well immobilized by the formation of carbonates.

[Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR].

OBJECTIVE: To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR). METHODS: By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique. RESULTS: The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected. CONCLUSION: A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.

[Level of Regulatory B Cells in Patients with Immune Thrombocytopenia and Its Clinical Significance].

OBJECTIVE: To investigate the role of regulatory B cells (Breg) in pathogenesis of immune thrombocytopenia(ITP) and its clinical significance. METHODS: A total of 40 ITP patients and 20 normal controls were enrolled in this study. The content of Breg, Th1, Th2, Th17 and Treg cells were detected by flow cytometry (FCM). The expression level of IL-10,TGF-ß, CD40 and CD40L was detected by AimPlex Flow High Throughput Screening Technology. RESULTS: The of Breg cells in ITP patients was significantly lower than that in normal controls (P<0.05)，the expression levels of IL-10,TGF-ß and CD40L in ITP patients were also significantly lower than those in normal controls (P<0.05). The contents of Th1 cells in ITP patients were significantly higher than that in normal controls (P<0.05), whereas the contents of Th2, Th17 and Treg cells in ITP patients were significantly lower than those in normal controls (P<0.05). CONCLUSION: The Breg cells may play an important role in the pathogenesis of ITP.

Curcumin and allopurinol ameliorate fructose-induced hepatic inflammation in rats via miR-200a-mediated TXNIP/NLRP3 inflammasome inhibition.

Excess fructose consumption causes high prevalence of metabolic syndrome and inflammatory liver diseases. The aim of the current study was to investigate the therapeutic effects and underlying molecular mechanisms of curcumin and allopurinol in high fructose-induced hepatic inflammation. Male Sprague-Dawley rats were supplied with standard rat chow and drinking water containing 10% (w/v) fructose for consecutive 12 weeks. Curcumin (15, 30 and 60 mg/kg) and allopurinol (5 mg/kg) were administered to rats via oral gavage daily from Week 7 to 12. For in vitro experiments, curcumin (2.5 µM) and allopurinol (100 µM) were treated to 5 mM fructose-exposed Buffalo rat liver cell line (BRL-3 A) and human hepatoblastoma cell line (HepG2), respectively. The data from these animal and hepatocyte models showed that curcumin and allopurinol ameliorated fructose-induced metabolic symptom, especially hepatic inflammation in rats. Interestingly, down-regulation of microRNA-200a (miR-200a) was screened out in livers of fructose-fed rats and then validated in fructose-exposed BRL-3 A and HepG2 cells. Fructose-induced miR-200a low-expression was identified as a negative mediator of thioredoxin interacting protein (TXNIP) by direct targeting of 3'UTR-rTXNIP, subsequently activating the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome in BRL-3 A cells. Curcumin, as well as allopurinol, notably up-regulated miR-200a expression, accordingly, down-regulated TXNIP and inhibited NLRP3 inflammasome activation in fructose-fed rat livers and fructose-exposed BRL-3 A and HepG2 cells. Taken together, this study firstly identified miR-200a as a biomarker of fructose-induced hepatic inflammation, and revealed the hepatoprotection of curcumin and allopurinol via up-regulating miR-200a-mediated TXNIP/NLRP3 inflammasome pathway.

[Secretory Imbalance between Pro-inflammatory and Anti-inflammatory Cytokines in the Patients with Immune Thrombocytopenia].

OBJECTIVE: To analyze the imbalance of pro-inflammatory and anti-inflammatory cytokines in the patients of immune thrombocytopenia (ITP). METHODS: Thirty-five patients with ITP were enrolled in ITP group, while 28 healthy persons were included in control group. The expressions of IL-8, IL-17A, IL-22, TNF-α, IFN-γ, IL-4, CD40, CD40L, TGF-ß and IL-10 were detected by flow cytometry with aimPlex multiple immunoassay Flow. RESULTS: The expressions of pro-inflammatory factors IL-8, IL-17A, IL-22, IFN-γ and TNF-α in ITP group all were significantly higher than that in the control group (P<0.05), however the expressions of anti-inflammatory factors IL-4, CD40L, TGF-ß and IL-10 in ITP group all were significantly lower than that in the control group (P<0.05). The expression of CD40 was not significantly different between ITP group and control group (P>0.05). Expressions of TNF-α significantly related with platelet counts in both ITP group and control group (ITP group, r=0.64, P<0.05; control group, r=-0.41, P<0.05). However the expression of CD40, TGF-ß, CD40L, IL-8, IL-17A, IL-22, IL-10, IL-1ß, IFN-γ and IL-4 significantly did not relate with platelet counts in both ITP and control group. CONCLUSIONS: The secretory imbalance between pro-inflammatory and anti-inflammatory cytokines exists in the patients of ITP. The decrease of Plt regulated may be regulated by the abnormal expression of TNF-α.

The roles of ING5 expression in ovarian carcinogenesis and subsequent progression: a target of gene therapy.

Here, we found that ING5 overexpression suppressed cell viability, glucose metabolism, migration, invasion and epithelial-mesenchymal transition, and induced cell arrest, apoptosis, senescence, autophagy and fat accumulation in ovarian cancer cells. ING5-mediated chemoresistance was positively linked to apoptotic resistance and chemoresistance-related gene expression. ING5 overexpression suppressed tumor growth of ovarian cancer by decreasing proliferation, and inducing apoptosis and autophagy. ING5 mRNA level was lower in ovarian cancer than normal ovary, and borderline than benign tumors (p < 0.05), and negatively correlated with vascular invasion, lymphatic invasion and FIGO staging of ovarian cancer (p < 0.05). ING5 protein was less expressed in primary cancer than normal ovary (p < 0.05). There was a negative correlation between ING5 mRNA expression and the overall or progression-free survival time of the cancer patients with Grade 2, Grade 3, and stage I cancer (p < 0.05). Immunohistochemically, ING5 was less expressed in serous and mucinous adenocarcinoma than miscellaneous subtypes, and positively correlated with dedifferentiation and ki-67 expression of ovarian cancer (p < 0.05). These data suggested that down-regulated ING5 expression might be involved in ovarian carcinogenesis possibly by suppressing aggressive phenotypes, including proliferation, tumor growth, migration, invasion, and anti-apoptosis.

The nucleocytoplasmic translocation and up-regulation of ING5 protein in breast cancer: a potential target for gene therapy.

Here, we found that ING5 overexpression resulted in a lower proliferation, reduced glucose metabolism, S arrest, decreased migration and invasion, apoptotic induction, fat accumulation, autophagy, senescence and mesenchymal-epithelial-transition of breast cancer cells. It also suppressed the tumor growth of breast cancer cells by inhibiting proliferation, inducing apoptosis and autophagy. ING5-mediated chemoresistance was positively linked to Akt and NF-κB activation, MRP1 and GST-π overexpression, and FBXW7 hypoexpression. ING5 expression was higher in breast cancer than normal tissue at both mRNA and protein levels. ING5 mRNA expression was positively correlated with relapse- and distant metastasis-free survival rates. Nuclear ING5 expression showed gradual decrease from breast normal tissue, fibroadenoma, adenomatosis, primary to metastatic cancers, while versa for cytoplasmic ING5. Nuclear ING5 expression was negatively correlated with distant metastasis and p53 hypoexpression, while cytoplasmic ING5 expression was positively correlated with tumor size and ER expression. These data suggested that up-regulated expression and nucleocytoplasmic translocation of ING5 protein were observed in breast cancer. The higher expression of nuclear ING5 was inversely linked to worse clinicopathological behaviors of breast cancer by in vivo and vitro reversing aggressive phenotypes. Therefore, it should be employed as a biomarker to indicate the tumorigenesis and aggressiveness of breast cancer, and as a potential target for gene therapy.

Coronary Artery Diameter is Inversely Associated with the Severity of Coronary Lesions in Patients Undergoing Coronary Angiography.

BACKGROUND: The diameters of the coronary arteries have been suggested to be a potential predictor of coronary artery disease (CAD). However, whether the diameters of the coronary arteries are associated with the coronary lesion severity on angiography has not been determined. METHODS: One hundred sixty-seven consecutive adult patients (109 men and 58 women) aged 31-84 years who underwent coronary angiography for suspected or known CAD were enrolled. The known catheter tip diameter was used as the calibration to measure the diameters of coronary arteries, and the severity of coronary lesions was evaluated with the vessel score and Gensini score. RESULTS: In patients with a higher vessel score and Gensini score, the diameters of the left main (LM), left anterior descending (LAD), left circumflex (LCX), and right coronary arteries (RCA) were smaller (all p<0.05) than those in patients with lower scores. Multiple linear regression analysis indicated that the average coronary artery diameter was significantly associated with the Gensini score (ß=-0.444, p<0.00001). Moreover, the diameters of the coronary arteries were potential predictors of CAD, with areas under the receiver operating characteristic curves of 0.268 for average diameter (95% confidence interval [CI]: 0.183-0.353, p<0.00001), 0.356 for the LM diameter (95% CI: 0.266-0.445, p=0.005), 0.214 for the LAD diameter (95% CI: 0.136-0.291, p<0.00001), 0.366 for the LCX diameter (95% CI: 0.271-0.461, p=0.009), and 0.346 for the RCA diameter (95% CI: 0.245-0.447, p=0.003). CONCLUSION: The diameters of coronary arteries are inversely associated with the severity of CAD.

The roles of ING5 in gliomas: a good marker for tumorigenesis and a potential target for gene therapy.

To elucidate the anti-tumor effects and molecular mechanisms of ING5 on glioma cells, we overexpressed it in U87 cells, and examined the phenotypes and their relevant molecules. It was found that ING5 overexpression suppressed proliferation, energy metabolism, migration, invasion, and induced G2/M arrest, apoptosis, dedifferentiation, senescence, mesenchymal- epithelial transition and chemoresistance to cisplatin, MG132, paclitaxel and SAHA in U87 cells. There appeared a lower expression of N-cadherin, Twist, Slug, Zeb1, Zeb2, Snail, Ac-H3, Ac-H4, Cdc2, Cdk4 and XIAP, but a higher expression of Claudin 1, Histones 3 and 4, p21, p53, Bax, ß-catenin, PI3K, Akt, and p-Akt in ING5 transfectants. ING5 overexpression suppressed tumor growth of U87 cells in nude mice by inhibiting proliferation and inducing apoptosis. Down-regulated ING5 expression was closely linked to the tumorigenesis and histogenesis of glioma. These data indicated that ING5 expression might be considered as a good marker for the tumorigenesis and histogenesis of gliomas. It might be employed as a potential target for gene therapy of glioma. PI3K/Akt or ß-catenin/TCF-4 activation might be positively linked to chemotherapeutic resistance, mediated by ING5.

Predictive Effects of Circulating miR-221, miR-130a and miR-155 for Coronary Heart Disease: A Multi-Ethnic Study in China.

BACKGROUND: Differences in microRNA (miRNA) profiles between patients with and without coronary heart disease (CHD)have not been fully determined. The purpose of the study was to evaluate in a multi-ethnic population in China the predictive value of miRNAs previously suggested to have a role in CHD. SUBJECT AND METHOD: 932 participants were included, and plasma samples obtained. A quantitative reverse-transcription PCR(RT-qPCR) assay was conducted to confirm the concentration of plasma miRNAs. Circulating levels of miRNAs were quantified using the 2-Δct method. The severity of coronary atherosclerosis was evaluated via Gensini Scores. RESULT: The circulating levels of the nine proposed miRNAs were not different among the five main ethnicities examined (all p > 0.05). The Spearman correlation analyses indicated that miR-221 and miR-130a were negatively associated with the severity of CHD as indicated by Gensini Scores (r = -0.106, p = 0.001;r = -0.073, p = 0.026). Results of the univariate analysis showed that lower circulating miR-221 (OR, 1.663; 95 % CI, 1.255-2.202, p = <0.001), miR-155 (OR, 1.520; 95 % CI, 1.132-2.042, p = 0.005), and miR-130a (OR, 1.943; 95% CI, 1.410-2.678, p = <0.001) were potential risk factors for CHD. Moreover, miR-130a (OR, 2.405; 95 % CI, 1.691-3.421, p = <0.001) remained independently associated with the risk of CHD after adjusting for potential confounding factors. The analysis of the possible positive/negative associations between miR-221, miR-155 and miR-130awere conducted. A positive association between miR-130a and miR-155 was found (SI = 1.60, SIM = 1.21 and AP = 0.22), and in these groups, the proportion of CHD attributable to the interaction between miR-130a and miR-155 was as high as 22 %. A negative interaction was found between miR-221 and miR-130a (SI = 0.68, SIM = 0.60 and AP = 0.27). CONCLUSION: Plasma levels of miR-221, miR-130a and miR-155 decreased in patients with CHD, and miR-130a may be an independent predictor for CHD.

Cytokeratin 19 promoter directs the expression of Cre recombinase in various epithelia of transgenic mice.

Cytokeratin 19 (K19) is expressed in various differentiated cells, including gastric, intestinal and bronchial epithelial cells, and liver duct cells. Here, we generated a transgenic mouse line, K19-Cre, in which the expression of Cre recombinase was controlled by the promoter of K19. To test the tissue distribution and excision activity of Cre recombinase, K19-Cre transgenic mice were bred with Rosa26 reporter strain and a mouse strain that carries PTEN conditional alleles (PTENLoxp/Loxp). At mRNA level, Cre was strongly expressed in the stomach, lung and intestine, while in stomach, lung, and liver at protein level. The immunoreactivity to Cre was strongly observed the cytoplasm of gastric, bronchial and intestinal epithelial cells. Cre activity was detectable in gastric, bronchial and intestinal epithelial cells, according to LacZ staining. In K19-Cre/PTEN Loxp/Loxp mice, PTEN was abrogated in stomach, intestine, lung, liver and breast, the former two of which were verified by in situ PCR. There appeared breast cancer with PTEN loss. These data suggest that K19 promoter may be a useful tool to study the pathophysiological functions of cytokeratin 19-positive cells, especially gastrointestinal epithelial cells. Cell specificity of neoplasia is not completely attributable to the cell-specific expression of oncogenes and cell-specific loss of tumor suppressor genes.

Manipulating PML SUMOylation via Silencing UBC9 and RNF4 Regulates Cardiac Fibrosis.

The promyelocytic leukemia protein (PML) is essential in the assembly of dynamic subnuclear structures called PML nuclear bodies (PML-NBs), which are involved in regulating diverse cellular functions. However, the possibility of PML being involved in cardiac disease has not been examined. In mice undergoing transverse aortic constriction (TAC) and arsenic trioxide (ATO) injection, transforming growth factor ß1 (TGF-ß1) was upregulated along with dynamic alteration of PML SUMOylation. In cultured neonatal mouse cardiac fibroblasts (NMCFs), ATO, angiotensin II (Ang II), and fetal bovine serum (FBS) significantly triggered PML SUMOylation and the assembly of PML-NBs. Inhibition of SUMOylated PML by silencing UBC9, the unique SUMO E2-conjugating enzyme, reduced the development of cardiac fibrosis and partially improved cardiac function in TAC mice. In contrast, enhancing SUMOylated PML accumulation, by silencing RNF4, a poly-SUMO-specific E3 ubiquitin ligase, accelerated the induction of cardiac fibrosis and promoted cardiac function injury. PML colocalized with Pin1 (a positive regulator for TGF-ß1 mRNA expression in PML-NBs) and increased TGF-ß1 activity. These findings suggest that the UBC9/PML/RNF4 axis plays a critical role as an important SUMO pathway in cardiac fibrosis. Modulating the protein levels of the pathway provides an attractive therapeutic target for the treatment of cardiac fibrosis and heart failure.