Roflumilast Reduced the IL-18-Induced Inflammatory Response in Fibroblast-Like Synoviocytes (FLS).

Pro-inflammatory cytokines, such as the IL-18-induced inflammatory response and associated damage in fibroblast-like synoviocytes (FLS), play an important role in the pathogenesis of rheumatoid arthritis (RA). Roflumilast, an inhibitor of phosphodiesterase-4 (PDE-4), has been licensed for the treatment of chronic obstructive pulmonary disease (COPD). However, it is unknown whether roflumilast possesses a protective effect against the IL-18-induced inflammatory response in FLS. We found that roflumilast attenuated IL-18-induced oxidative stress by reducing the production of reactive oxygen species and malondialdehyde (MDA) in MH7A fibroblast-like synoviocytes (FLS). Additionally, roflumilast prevented IL-18-induced expressions and secretions of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. Importantly, we found that roflumilast inhibited IL-18-induced expressions of chemokines such as CCL5, CXCL9, and CXCL10. Further, roflumilast inhibited the expression of extracellular matrix degradative enzymes, such as matrix metalloproteinase-3 (MMP-3) and MMP-13. Mechanistically, we found that roflumilast suppressed the activation of the transcriptional factor AP-1 and NF-κB. Our results suggest that roflumilast might be a potential therapeutic agent for the treatment of RA.

miR-155-5p regulates mesenchymal stem cell osteogenesis and proliferation by targeting GSK3B in steroid-associated osteonecrosis.

microRNAs (miRNAs) have recently been recognized as playing an important role in bone-associated diseases. This study investigated whether the reduced miR-155-5p in steroid-associated osteonecrosis of the femoral head (ONFH) attenuated osteogenic differentiation and cell proliferation by targeting GSK3B. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 10) and patients with new femoral neck fracture (n = 10) and mesenchymal stem cells (MSCs) were isolated. The expression profile, the biological function of miR-155-5p, and the interaction between miR-155-5p and GSK3B were investigated by cell viability measurement, western blot, real-time polymerase chain reaction, luciferase reporter assay, and Alizarin Red S (ARS) staining of MSCs. The MSCs that were obtained from the femoral neck fracture group and from the steroid-associated ONFH group were transfected with or without miR-155-5p. We found that, in ONFH samples, the level of mature miR-155-5p was significantly lower than that of control samples. By inhibiting GSK3B, miR-155-5p promoted the nuclear translocation of ß-catenin, increased the expression of osteogenesis-related genes, and facilitated the proliferation and differentiation of MSCs. Restoring the expression of GSK3B in MSCs partially reversed the effect of miR-155-5p. These findings suggest that reduced miR-155-5p in steroid-associated ONFH attenuates osteogenic differentiation and cell proliferation by increased levels of GSK3B and inhibition of Wnt signaling.

Develop a circular RNA-related regulatory network associated with prognosis of gastric cancer.

BACKGROUND: In gastric cancer (GC), circular RNAs (circRNAs) mainly play an important role in miRNA sponge, which not only indicate long-term survival and prognosis but also increase resistance to the apoptosis. The purpose of the study is to explore new circRNAs and their underlying mechanisms in GC. METHOD: Through rigorous retrieval strategies, we used the sva package to analyze and identify differentially expressed circRNAs (DECs) from three Gene Expression Omnibus microarray datasets (GSE83521, GSE89143, and GSE78092). Online website CSCD and CircInteractome were used to reveal the binding sites between miRNAs and DECs. The possible target miRNAs of the DECs identified based on miRNAs, and Cytoscape was used to create a regulatory network of circRNA-miRNA-mRNA and identified the hub genes which were further validated using The Cancer Genome Atlas database and Human Protein Atlas. RESULTS: Twenty-eight DECs were obtained using the sva package. A regulatory network of circRNA-miRNA-mRNA (competing endogenous RNA) containing 15 circRNAs, 24 miRNAs, and 158 genes was identified. A protein-protein interaction network based on the 158 genes was established, and further determined that 10 hub genes (SKA1, ANLN, CHEK1, SKA3, TOP2A, BIRC5, RRM2, NCAPG2, FANCI, and RAD51) were associated with some cancer-related pathways based on the functional enrichment analysis. Finally, six hub genes (BIRC5, TOP2A, FANCI, NCAPG2, RAD51, and RRM2) were proven to influence the overall survival of GC. CONCLUSION: Our study established a circRNA-miRNA-mRNA regulatory network and defined six circRNA-related hub genes in GC, which could serve as potential therapeutic targets or prognostic biomarker for GC treatment.

A cone-beam computed tomography study of the root and canal morphology of maxillary premolars / 口腔疾病防治

Objective @# To study root morphology, the incidence of three root canals and the root canal anatomy of maxillary premolars.@*Methods@#The cone-beam computed tomography (CBCT) data of 779 maxillary first premolars and 728 maxillary second premolars were collected from 412 patients in Zhuhai Stomatological Hospital. The root and canal morphology, incidence of three canals, bilateral symmetry and location of root canal bifurcation were analyzed. @*Results @#The incidence of three canals in the maxillary first premolars was 1.8% and that in the maxillary second premolars was 0.3%. The incidence of three canals in the maxillary first premolars was significantly higher than that in the maxillary second premolars (c2=8.304, P=0.004). The symmetrical ratio of the three-canal maxillary first premolar was 27.3%. There was no symmetrical three-canal maxillary second premolar. The anatomical morphology of the maxillary premolar can be single root, double root or trident root. Its internal root canal system is complex and diverse. There are seven kinds of Vertucci morphology: the first maxillary premolar is mainly Vertucci IV type, and the second maxillary premolar is mainly Vertucci I type. Most of the root canal bifurcations of the three-canal maxillary premolars were observed in the midthird or the cervical third of the root. All three-canal maxillary premolars had three independent apical foramens. @*Conclusion @#The root canal morphology of maxillary premolars is complex and changeable. CBCT plays an important role in the discovery of variation and extra root canals.

MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis.

BACKGROUND: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. OBJECTIVE: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. METHOD: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. RESULTS: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased ß-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. CONCLUSION: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

Targeting high Aurora kinases expression as an innovative therapy for hepatocellular carcinoma.

The Aurora kinases A and B control tumorigenesis by inhibiting apoptosis and promoting proliferation and metastasis, however, it remains unknown whether Aurora A and B overexpressed concomitantly and its clinical significance in hepatocellular carcinoma (HCC). Here, we obsearved Aurora A and B tended to overexpress parallelly on protein level (r = 0.8679, P < 0.0001) and their co-overexpression (Aurora AHBH), associated with the worst prognosis, was an independent predictor for the survival. Importantly, with the lower IC50 and stronger anti-tumor effect than selective inhibitors, SNS-314, the pan-inhibitor of Aurora kinases, which induced YAP (Yes-associated protein) reduction and resulted in P21 accumulation, significantly promoted the polyploidy (> 4N) formation and apoptosis in HCC. High YAP expression (YAPH) was associated with Aurora AHBH, and appeared to be an independent predictor for survival, but P21 not. Moreover, silencing YAP also induced P21 accumulation, and knockdown P21, which enhanced YAP accumulation and weakened the SNS-314-induced YAP reduction, impaired SNS-314-induced apoptosis. Therefore, P21 enhanced the apoptotic effect of SNS-314 in HCC. Taken together, our findings indicated Aurora kinases/YAP/P21 was an oncogenic signaling axis in HCC, and revealed targeting Aurora AHBH induced apoptosis by YAP suppression. Our results also provided a solid evidence for SNS-314 as a potential targeted therapy, and a proof-of-concept evidence for a possible combined therapy of SNS-314 plus Hippo pathway inhibitors on HCC.

Cap-assisted hemoclip application with forward-viewing endoscope for hemorrhage induced by endoscopic sphincterotomy: a prospective case series study.

BACKGROUND: Endoscopic sphincterotomy (ES) is a therapeutic technique developed as an advanced application of endoscopic retrograde cholangiopancreatography (ERCP). An important adverse event associated with this procedure is hemorrhage, which may sometimes be uncontrollable. We sought to examine whether cap-assisted hemoclip application is effective in controlling ES-induced hemorrhage. METHODS: In this prospective study, we investigated the outcomes in 10 patients who had uncontrolled ES-induced hemorrhage and were treated by cap-assisted application of hemoclip with a forward-viewing endoscope. RESULTS: Nine of the 10 investigated patients were successfully treated using the cap-assisted hemoclip technique with forward-viewing endoscope, yielding a success rate of 90 %. The patient with hemorrhage non-responsive to hemoclipping required catheter embolization of the bleeding artery after its identification by digital subtraction angiography. One of the 10 patients developed mild pancreatitis after the procedure, but was successfully managed conservatively. CONCLUSIONS: Cap-assisted hemoclip application with a forward-viewing endoscope appears to be an effective therapeutic modality for achieving hemostasis in cases of ES-induced hemorrhage, without the occurrence of any severe adverse events; we believe that this method should be considered as an option in the management of ES-induced hemorrhage.

Abnormal intracellular calcium homeostasis associated with vulnerability in the nerve cells from heroin-dependent rat.

The cellular mechanisms by which opiate addiction develops with repetitive use remain largely unresolved. Intercellular calcium homeostasis is one of the most critical elements to determine neuroadaptive changes and neuronal fate. Heroin, one of the most addictive opiates, may induce neurotoxicity potentially inducing brain impairment, especially for those chronic users who get an overdose. Here we examined changes in intracellular calcium concentration ([Ca2+]i) after repeated exposure to heroin using cultured cerebral cortical neurons. Dynamic changes in [Ca2+]i indicated by fluo-3-AM were monitored using confocal laser scan microscopy, followed by cytotoxicity assessments. It showed that the cells dissociated from heroin-dependent rats had a smaller depolarization-induced [Ca2+]i responses, and a higher elevation in [Ca2+]i when challenged with a high concentration of heroin (500 µM). The restoration ability to remove calcium after washout of these stimulants was impaired. Calcium channel blocker verapamil inhibited the heroin-induced [Ca2+]i elevations as well as the heroin-induced cell damage. The relative [Ca2+]i of the nerve cells closely correlated with the number of damaged cells induced by heroin. These results demonstrate that nerve cells from heroin-dependent rats manifest abnormal [Ca2+]i homeostasis, as well as vulnerability to heroin overdose, suggesting involvement of [Ca2+]i regulation mechanisms in heroin addiction and neurotoxicity.

High-throughput discovery of chloroplast and mitochondrial DNA polymorphisms in Brassicaceae species by ORG-EcoTILLING.

BACKGROUND: Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used. METHODOLOGY/PRINCIPAL FINDINGS: To address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results. CONCLUSIONS/SIGNIFICANCE: These results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.

[Influence of visual attention in visual evoked potential examination].

OBJECTIVE: To explore the influence of visual attention in visual evoked potential (VEP) examination. METHODS: The pattern visual evoked potential (PVEP) and flash visual evoked potential (FVEP) were used to examine 110 normal subjects whose visual acuity or corrected visual acuity is in the range of 4.8-5.2, and age between 20 to 30 years old. In PVEP inspection, subjects were asked to watch with three ways, to stare at the central screen (at the same time count silently the number of alternating of black and white squares to improve the subjects' attention), to stare at the central screen at the same time thinking other things, to look sideways (the left eye stared at the left bottom corner of the screen or the right eye stared at the right one). In FVEP inspection, subjects were checked with eyes open and closed respectively. The latency and amplitude of P100 wave were recorded. The data were statistically analyzed by SPSS 13.0 software. RESULTS In PVEP inspection, the latency and amplitude of P100 wave were statistically different among three watching ways. In FVEP inspection, latency and amplitude of P100 wave were statistically different between eyes open and closed group. CONCLUSION: Although the visual evoked potential test is an objective electrophysiological visual function test, it is susceptible to the influence of various subjective factors such as visual attention. It should arise the attention of forensic experts.