circZNF532 promotes endothelial-to-mesenchymal transition in diabetic retinopathy by recruiting TAF15 to stabilize PIK3CD.

Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.

Up-regulation of miR-192-5p inhibits the ELAVL1/PI3Kδ axis and attenuates microvascular endothelial cell proliferation, migration and angiogenesis in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a common complication of diabetes mellitus that poses a threat to adults. MicroRNAs (miRNAs) play a key role in DR progression. However, the role and mechanism of miR-192-5p in DR remain unclear. We aimed to investigate the effect of miR-192-5p on cell proliferation, migration and angiogenesis in DR. METHODS: Expression of miR-192-5p, ELAV-like RNA binding protein 1 (ELAVL1) and phosphoinositide 3-kinase delta (PI3Kδ) in human retinal fibrovascular membrane (FVM) samples and human retinal microvascular endothelial cells (HRMECs) was assessed using RT-qPCR. ELAVL1 and PI3Kδ protein levels were evaluated by Western blot. RIP and dual luciferase reporter assays were performed to confirm the miR-192-5p/ELAVL1/PI3Kδ regulatory networks. Cell proliferation, migration and angiogenesis were assessed by CCK8, transwell and tube formation assays. RESULTS: MiR-192-5p was decreased in FVM samples from DR patients and high glucose (HG)-treated HRMECs. Functionally, overexpressed miR-192-5p inhibited cell proliferation, migration and angiogenesis in HG-treated HRMECs. Mechanically, miR-192-5p directly targeted ELAVL1 and decreased its expression. We further verified that ELAVL1 bound to PI3Kδ and maintained PI3Kδ mRNA stability. Rescue analysis demonstrated that the suppressive effects of HG-treated HRMECs caused by miR-192-5p up-regulation were overturned by overexpressed ELAVL1 or PI3Kδ. CONCLUSION: MiR-192-5p attenuates DR progression by targeting ELAVL1 and reducing PI3Kδ expression, suggesting a biomarker for the treatment of DR.

USP14 Regulates ATF2/PIK3CD Axis to Promote Microvascular Endothelial Cell Proliferation, Migration, and Angiogenesis in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness in diabetic patients. However, the pathogenesis of DR is complex, and no firm conclusions have been drawn so far. It has become a hot spot in ophthalmology research to deeply study the mechanism of DR pathological changes and find effective treatment options. Human retinal microvascular endothelial cells (HRMECs) were induced by high glucose (HG) to construct DR cell model. CCK-8 assay was used to detect the viability of HRMECs. Transwell assay was used to detect the migration ability of HRMECs. Tube formation assay was used to identify the tube formation ability of HRMECs. The expressions of USP14, ATF2 and PIK3CD were detected by Western blot analysis and qRT-PCR assay. Immunoprecipitation (IP) was used to ascertain the relationship of USP14 and ATF2. To explore the regulatory relationship between ATF2 and PIK3CD by dual-luciferase reporter gene assay and Chromatin immunoprecipitation (ChIP) assay. High glucose treatment promoted the proliferation, migration, and tube formation of HRMEC, and the expressions of USP14, ATF2 and PIK3CD were significantly up-regulated. USP14 or ATF2 knockdown inhibited HG-induced HRMECs proliferation, migration, and tube formation. USP14 regulated the expression of ATF2, and ATF2 promoted PIK3CD expression. PIK3CD overexpression attenuated the inhibitory effectiveness of USP14 knockdown on proliferation, migration and tube formation of DR cell model. Here, we revealed that USP14 regulated the ATF2/PIK3CD axis to promote proliferation, migration, and tube formation in HG-induced HRMECs.

LncRNA SNHG1 targets miR-340-5p/PIK3CA axis to regulate microvascular endothelial cell proliferation, migration, and angiogenesis in DR.

Diabetic retinopathy (DR) is a serious long-term complication of diabetes. However, the current treatment of DR is still challenging. We aimed to investigate the role of lncRNA SNHG1/miR-340-5p/PIK3CA in DR and the mechanisms involved. Blood samples from clinical DR patients and healthy subjects were obtained. HRMECs were induced by high glucose for 24 h to establish the DR model. The vector for interfering or overexpressing lncRNA SNHG1, miR-340-5p, and PIK3CA was constructed. LncRNA SNHG1, miR-340-5p, and PIK3CA expressions were detected by qRT-PCR or Western blot. Cell proliferation and migration were detected by CCK-8 and Transwell assays. Blood vessel formation was detected by angiogenesis assay. Dual-luciferase reporter assay tested the interaction of lncRNA SNHG1 with miR-340-5p and miR-340-5p with PIK3CA. RIP measured the binding of miR-340-5p to PIK3CA. In the blood of DR patients and the DR model, lncRNA SNHG1 was increased and miR-340-5p expression was down-regulated. In the DR model, PIK3CA expression was elevated. Downregulation of lncRNA SNHG1 inhibited HRMECs proliferation, migration, and angiogenesis. LncRNA SNHG1 interacted with miR-340-5p, and up-regulation of miR-340-5p inhibited HRMECs proliferation, migration and angiogenesis. The inhibition of cell proliferation, migration, and angiogenesis of HRMECs caused by down-regulation of lncRNA SNHG1 was reversed by knockdown of miR-340-5p. miR-340-5p targeted PIK3CA, and downregulation of PIK3CA inhibited HRMECs proliferation, migration, and angiogenesis. The inhibition of HRMECs proliferation, migration and angiogenesis caused by down-regulation of lncRNA SNHG1 could be reversed by overexpression of PIK3CA. LncRNA SNHG1 targeted miR-340-5p/PIK3CA axis to regulate microvascular endothelial cell proliferation, migration, and angiogenesis in DR.

[Application of Wechat public platform in teaching reform of prosthodontics].

PURPOSE: To explore the law of development and existing problems in prosthodontics teaching in the new era, through flipped classroom teaching based on WeChat public platform. METHODS: WeChat public number was applied and WeChat groups were set up among the students of grade 2013, and 93 students were divided into 20 WeChat groups, with 4 to 5 students in each group. The main platform on reform of prosthodontics teaching was WeChat app, supplemented by platforms of Lediaocha and Youkaoshi. Teachers published courseware and learning resources with WeChat public number and WeChat groups. Two-dimensional codes on questionnaires and tests which were generated by Lediaocha and Youkaoshi could be published with WeChat and the final learning effectiveness was compared using SPSS 19.0 software package for t test. RESULTS: The results showed that 59.1% students admitted WeChat teaching effective, only 5.4% students believed ineffective. Based upon partial chapters of prosthodontics, the score of classroom test (91.35±4.45) was significantly higher than that of pre-class test(90.14±5.03, P＜0.05). CONCLUSIONS: The reform of flipped classroom based on WeChat platform in prosthodontics have some advantages, such as more flexibility in teaching form and time arrangement, promoting students' learner autonomy, and increasing students' motivation and effects of learning prosthodontics.

Toward Eliminating Discontinuous Yielding Behavior of the EA4T Steel.

Cold-rolled EA4T steel was heat-treated by inter-critical holding at 755 °C for 90, 120, 180, and 240 s, respectively, and then quenching in water. The tensile testing results of the EA4T specimens show an evident transition from the discontinuous yielding to the continuous yielding of the steel specimens by prolonging the holding time. A novel relationship between the discontinuous yielding behavior of tensile-deformed steel specimens and the carbide size was proposed based on experimental results and Cottrell's theory. The model may provide a new clue for avoiding discontinuous yielding and improving mechanical properties of metals with static strain aging behaviors.

[Influence of electroacupuncture of "Zhongwan" (CV12) and "Zusanli" (ST36) in different combinations of stimulating strength and sequence on intragastric pressure in normal rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zhongwan" (CV12) and "Zusanli" (ST36) in different combinations of stimulating parameters on intragastric pressure (IGP) in normal rats so as to explore their best combinations for promoting gastrointestinal mobility. METHODS: A total of 90 male SD rats were randomly divided into 6 EA groups：CV12-1 mA＋ST36-1 mA, CV12-1 mA＋ST36-2 mA, CV12-1 mA＋ST36-4 mA, ST36-1 mA＋CV12-1 mA, ST36-2 mA＋CV12-1 mA, and ST36-4 mA＋CV12-1 mA which the first acupoint was stimulated first, followed by the second in each group (n＝15 rats/group). Before (1 min), and 0-30 s, 30-60 s, 60 -90 s, and 90-120 s during EA stimulation of the left ST36 or CV12 first or later, the IGP was measured via an inserted intragastric balloon, a connected pressure transducer and an amplifier. Changes of the IGP were analyzed using 2×3×4 factorial design. RESULTS: 1) During 0-30 s, EA-CV12 showed an obvious inhibitory effect on IGP(P<0.05) while EA-ST36 showed a mild exciting effect (P>0.05). 2) Compared with the IGP level of 0-30 s, the IGP levels of 30-120 s were significantly decreased in all the groups (P<0.01). 3) In the CV12-1 mA/ST 36-1 mA groups, only the IGP level of 0-30 s was affected by the EA-stimulating order (P<0.05). In the CV12-1 mA/ST36-2 mA groups, both the IGP levels during 0-30 s and 90-120 s were obviously affected by EA-stimulating sequence. In the CV12-1 mA/ST36-4 mA groups, the IGP level during 0-120 s was affected by EA-stimulating order. 4) Only in the condition of EA-CV12 stimulating first and EA-ST36 second and at 4 mA, the reduction effect of IGP of EA-CV12 was antagonized. There are marked interaction effects between the EA strength and acupoint EA stimulating sequence, and between the time course and acupoint EA stimulating sequence (P<0.01), but no significant interaction effects were found between the time course and stimulating strength, and among the EA stimulating strength, time course and acupoint EA stimulating sequence (P>0.05). CONCLUSION: Simultaneous EA stimulation of ST36 and CV12 has an antagonistic effect on IGP in normal rats, which is affected by the stimulating sequence, stimulating strength and time course.

Preparation and Characterization of Surface Photocatalytic Activity with NiO/TiO2 Nanocomposite Structure.

This study achieved a nanocomposite structure of nickel oxide (NiO)/titanium dioxide (TiO2) heterojunction on a TiO2 film surface. The photocatalytic activity of this structure evaluated by decomposing methylene blue (MB) solution was strongly correlated to the conductive behavior of the NiO film. A p-type NiO film of high concentration in contact with the native n-type TiO2 film, which resulted in a strong inner electrical field to effectively separate the photogenerated electron-hole pairs, exhibited a much better photocatalytic activity than the controlled TiO2 film. In addition, the photocatalytic activity of the NiO/TiO2 nanocomposite structure was enhanced as the thickness of the p-NiO film decreased, which was beneficial for the migration of the photogenerated carriers to the structural surface.

Effects of medium components and culture conditions on mycelial biomass and the production of bioactive ingredients in submerged culture of Xylaria nigripes (Ascomycetes), a Chinese medicinal fungus.

The optimal culture conditions were investigated to maximize the production of mycelial biomass and bioactive ingredients in submerged cultivation of Xylaria nigripes, a Chinese medicinal fungus. The one-factor-at-a-time method was used to explore the effects of medium components, including carbon, nitrogen, mineral sources, and initial pH of the medium and environmental factors, such as culture temperature and rotation speed, on mycelial growth and production of bioactive ingredients. The results indicated that the optimal culture temperature and rotation speed were 25°C and 100 rpm in a medium with 20 g fructose, 6 g yeast extract, and 2 g magnesiun sulfate heptahydrate as carbon, nitrogen, and mineral sources, respectively, in 1 L distilled water with an initial medium pH of 5.5. With optimal medium components and conditions of cultivation, the maximal production of mycelial biomass was 6.64 ± 0.88 g/L, with maximal production of bioactive ingredients such as extracellular polysaccharides (2.36 ± 0.18 mg/mL), intracellular polysaccharides (2.38 ± 0.07 mg/g), adenosine (43.27 ± 2.37 mg/g), total polyphenols (36.57 ± 1.36 mg/g), and triterpenoids (31.29 ± 1.17 mg/g) in a shake flask culture. These results suggest that different bioactive ingredients including intracellular polysaccharides, adenosine, total polyphenols and triterpenoids in mycelia and extracellular polysaccharides in broth can be obtained from one simple medium for submerged cultivation of X. nigripes.

[Attachment and spreading of fibroblasts on titanium coatings by different micro-arc oxidation time].

PURPOSE: To investigate the attachment and spreading of fibroblasts on titanium coatings by different micro-arc oxidation(MAO) time. METHODS: Micro-arc oxidation film was formed on the titanium surface by using micro-arc oxidation. The film morphology and the cell shape on the samples were observed by scanning electron microscopy (SEM) and the surface roughness of film with different time was analyzed by surface coarseness profiling instrument. SPSS11.5 software package was used to analyze the data. RESULTS: The experiment result showed, the coatings were rough and porous, the longer oxidation time, the larger diameter of porous but the less porous numbers. As the treatment time increased, the surface roughness of micro-arc oxidation specimens increased (P<0.05). Compared with titanium, fibroblasts on titanium MAO coatings had more abundant spreading and adhesion. At 24 h, 48 h and 72 h, the cell number on coatings adhesion increased, but with different oxidation time, the cell shape, number and adhesion weren't obviously changed. CONCLUSIONS: The results indicate that microarc oxidation increases the surface roughness, have a good influence on cell adhesion and spreading.

[Effects of the micro-arc oxidized film on pure titanium surface on attachment and growth of BMSCs].

PURPOSE: To evaluate the biocompatibility of oxide film formed by micro-arc oxidation(MAO) on titanium surface by culturing bone marrow stromal cells(BMSCs) on its surface and observing the BMSCs's biological behavior of early adhesion, growth and alkaline phosphatase activity. METHODS: According to the surface treatments, the samples were classified into 4 groups: MAO group, abrator group, abrator and NaOH-treated group, and untreated titanium group. Attachment, growth and alkaline phosphatase (ALP) activity of BMSCs cultured on the titanium surface of each group were assessed at different time. All data were statistically analyzed with one-way ANOVA using SPSS11.5 software package. RESULTS: From a morphological point of view, cell adherence in the MAO group was the best. Cell count and ALP activity in the MAO group were highest(P<0.01). CONCLUSIONS: MAO film on pure titanium surface may increase BMSCs adhesion, growth and ALP activity and titanium surface treated with MAO exhibits better biocompatibility.

Induction of apoptosis in human lung carcinoma A549 epithelial cells with an ethanol extract of Tremella mesenterica.

Tremella mesenterica (TM) is a common food and folk medicine widely used in several Asian countries as a tonic for the lungs. In the present study, we compared the effects of extracellular polysaccharides (EPS), intracellular polysaccharides (IPS), and ethanol extract (EE) of Tremella mesenterica on the induction of apoptosis into human lung carcinoma A549 epithelial cells. The EE, but not the EPS or the IPS, almost completely inhibited the growth of A549 cells. The results of Annexin V-FITC/PI staining and flow cytometric analysis indicated that the percentage of Annexin V(+)/PI(-) cells in EE-treated cells increased to 32.8%. The results of further investigation showed a disruption of mitochondrial transmembrane potential (DeltaPsi(m)), the production of reactive oxygen species (ROS), and the activation of caspase-3 protein in EE-treated cells. These findings suggest that EE can decrease cell viability and induce apoptosis in A549 cell lines by activating a mitochondrial pathway.

[Corrosion behavior of micro-arc oxidation film on titanium in simulated body fluid].

OBJECTIVE: To investigate the variation of the corrosion resistance of micro-arc oxidation film on titanium by electrochemical methods in simulated body fluid. METHODS: Micro-arc oxidation film was formed on the titanium surface using micro-arc oxidation. The morphology was observed with scanning electron microscopy (SEM) and the phase composition was analyzed using X-ray diffraction (XRD). Polarization curves and electrochemical impedance spectroscopy (EIS) in simulated body fluid were examined with electrochemical methods. RESULTS: On the titanium surface with micro-arc oxidation, the film consisted of many volcanic micropores. The film formed was a titanium dioxide (TiO(2)) with peaks for both anatase and rutile phases. In addition, hydroxylapatite was also observed. The self-corrosion potential and self-corrosion current density of titanium with micro-arc oxidation film were -0.255 V and 0.80 microA/cm(2) respectively, while those of untreated titanium were -0.358 V and 0.55 microA/cm(2). Electrochemical impedance spectroscopy confirmed the model of equivalent circuits reasonable. CONCLUSIONS: The results of electrochemical examinations indicate that micro-arc oxidation film increases the corrosion resistance of titanium.

Role of calcineurin in Porphyromonas gingivalis-induced myocardial cell hypertrophy and apoptosis.

BACKGROUND AND OBJECTIVE: Periodontal pathogen Porphyromonas gingivalis (P. gingivalis) increased cardiomyocyte hypertrophy and apoptosis whereas Actinobaeillus actinomycetemcomitans and Prevotella intermedia had no effects. The purpose of this study is to clarify the role of calcineurin signaling pathway in P. gingivalis-induced H9c2 myocardial cell hypertrophy and apoptosis. METHODS: DNA fragmentation, nuclear condensation, cellular morphology, calcineurin protein, Bcl2-associated death promoter (Bad) and nuclear factor of activated T cell (NFAT)-3 protein products in cultured H9c2 myocardial cell were measured by agarose gel electrophoresis, DAPI, immunofluorescence, and Western blotting following P. gingivalis and/or pre-administration of CsA (calcineurin inhibitors cyclosporin A). RESULTS: P. gingivalis not only increased calcineurin protein, NFAT-3 protein products and cellular hypertrophy, but also increased DNA fragmentation, nuclear condensation and Bad protein products in H9c2 cells. The increased cellular sizes, DNA fragmentation, nuclear condensation, and Bad of H9c2 cells treated with P. gingivalis were all significantly reduced after pre-administration of CsA. CONCLUSION: Our findings suggest that the activity of calcineurin signal pathway may be initiated by P. gingivalis and further lead to cell hypertrophy and death in culture H9c2 myocardial cells.

Influence of serum from liver-damaged rats on differentiation tendency of bone marrow-derived stem cells.

AIM: Recent studies in both rodents and humans indicated that bone marrow (BM)-derived stem cells were able to home to the liver after they were damaged and demonstrated plasticity in becoming hepatocytes. However, the question remains as to how these stem cells are activated and led to the liver and where the signals initiating the mechanisms of activation and differentiation of stem cells originate. The aim of this study was to investigate the influence of serum from liver-damaged rats on differentiation tendency of bone marrow-derived stem cells. METHODS: Serum samples were collected from rats treated with a 2-acetylaminofluorene (2-AAF) /carbon tetrachloride (CCl(4)) program for varying time points and then used as stimulators of cultured BM stem cells. Expression of M(2)- and L-type isozymes of rat pyruvate kinase, albumin as well as integrin-beta1 were then examined by reverse transcription polymerase chain reaction (RT-PCR) to estimate the differentiation state of BM stem cells. RESULTS: Expression of M(2)-type isozyme of pyruvate kinase (M(2)-PK), a marker of immature hepatocytes, was detected in each group stimulated with experimental serum, but not in controls including mature hepatocytes, BM stem cells without serum stimulation, and BM stem cells stimulated with normal control serum. As a marker expressed in the development of liver, the expression signal of integrin-beta1 was also detectable in each group stimulated with experimental serum. However, expression of L-type isozyme of pyruvate kinase (L-PK) and albumin, marker molecules of mature hepatocytes, was not detected in groups stimulated with experimental serum. CONCLUSION: Under the influence of serum from rats with liver failure, BM stem cells begin to differentiate along a direction to hepatocyte lineage and to possess some features of immature hepatocytes.

A selective tropism of transfused oval cells for liver.

AIM: To explore the biological behaviors of hepatic oval cells after transfused into the circulation of experimental animals. METHODS: Oval cells from male SD rat were transfused into the circulation of a female rat which were treated by a 2-AAF/CCl(4) program, through caudal vein. Sex-determining gene sry which located on Y chromosome was examined by PCR and in situ hybridization technique in liver, kidney and spleen of the experimental animals, respectively. RESULTS: The results of the cell-transplant experiment showed that the sry gene was detectable only in the liver but not in spleen and kidney of the experimental rats, and no signals could be detected in the control animals. It can be also morphologically proved that some exogenous cells had migrated into the parenchyma of the liver and settled there. CONCLUSION: The result means that there are exogenous cells located in the liver of the experimental animal and the localization is specific to the liver. This indicates that some "signal molecules" must exist in the circulation of the rats treated by 2-AAF/CCl(4). These "signal molecules" might play an important role in specific localization and differentiation of transfused oval cells.