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Two kinds of vacancy defects, rhombic and square nanoholes, were produced in graphene/hexagonal BN (g/h-BN) superlattice nanoribbons by ion irradiation. The effects of the type (rhombus and square), area, number density and position of the nanoholes, and the period length of the nanoribbons on their mechanical properties were investigated by molecular dynamics simulation. The results show that the failure strength and strain of the nanoribbons are more sensitive to vacancy defects than is the Young's modulus. The mechanical properties are insensitive to the period length of the nanoribbons and those with rhombus nanoholes have inferior mechanical properties to those with square nanoholes under the same conditions. The mechanical properties of the nanoribbons are largely affected by the positions of the nanoholes, such that they should be in the graphene and not in boron nitride as much as possible. By increasing the number density of the nanoholes, the toughness of the nanoribbons is increased.

In this study, we investigate the coupling of an internal field (defect field-sp3 bonds and nanopores) and an external field (strain and temperature). Simultaneously, we provide a design idea of hybrid materials. The mechanical properties of hybrid materials under the condition of internal and external field coupling were studied. When nanopores and sp3 bonds are considered simultaneously, we found that internal (sp3 bonds and defects) and external field (temperature and strain fields) have a negative chain reaction on the mechanical properties of BN-graphene-BN/BN vertically-stacked nanostructures, and the negative chain reaction will gradually increase with the change in parameters (such as the increase in temperature). The sp3 bonds can be regarded as a special defect, which will increase the initial strain of the system. In addition, the mechanical properties of the nanostructure, containing square nanopores in the boron nitride region are most sensitive to temperature change, relative to the nanopore in the other two regions. Atoms (around square nanopores) are more likely to overcome the binding energy and lose stability from the inherent equilibrium position, relative to that of circular nanopores.

Perivascular adipose tissue (PVAT) homeostasis plays an important role in maintaining vascular function, and PVAT dysfunction may induce several pathophysiological situations. In this study, we investigated the effect and mechanism of the local angiotensin II (Ang II) on PVAT. High-throughput comparative proteomic analysis, based on TMT labeling combined with LC-MS/MS, were performed on an in vivo Ang II infusion mice model to obtain a comprehensive view of the protein ensembles associated with thoracic PVAT (tPVAT) dysfunction induced by Ang II. In total, 5037 proteins were confidently identified, of which 4984 proteins were quantified. Compared with the saline group, 145 proteins were upregulated and 146 proteins were downregulated during Ang II-induced tPVAT pathogenesis. Bioinformatics analyses revealed that the most enriched GO terms were annotated as gene silencing, monosaccharide binding, and extracellular matrix. In addition, some novel proteins, potentially associated with Ang II infusion, were identified, such as acyl-CoA carboxylase α, very long-chain acyl-CoA synthetase (ACSVL), uncoupling protein 1 (UCP1), perilipin, RAS protein-specific guanine nucleotide-releasing factor 2 (RasGRF2), and hypoxia inducible factor 1α (HIF-1α). Ang II could directly participate in the regulation of lipid metabolism, transportation, and adipocyte differentiation by affecting UCP1 and perilipin. Importantly, the key KEGG pathways were involved in fatty acid biosynthesis, FABP3-PPARα/γ, RasGRF2-ERK-HIF-1α, RasGRF2-PKC-HIF-1α, and STAT3-HIF-1α axis. The present study provided the most comprehensive proteome profile of mice tPVAT and some novel insights into Ang II-mediated tPVAT dysfunction and will be helpful for understanding the possible relationship between local RAS activation and PVAT dysfunction.

RhoGTPase is involved in PDGF-BB-mediated VSMC phenotypic modulation. RhoGDIs are key factors in regulating RhoGTPase activation. In the present study, we investigated the regulatory effect of RhoGDI1 on the activation of RhoGTPase in VSMC transformation and neointima formation. Western blot and co-immunoprecipitation assays showed that the PDGF receptor inhibition by crenolanib promoted RhoGDI1 polyubiquitination and degradation. Inhibition of RhoGDI1 degradation via MG132 reversed the decrease in VSMC phenotypic transformation. In addition, RhoGDI1 knockdown significantly inhibited VSMC phenotypic transformation and neointima formation in vitro and in vivo. These results suggest that PDGF-BB promotes RhoGDI1 stability via the PDGF receptor and induces the VSMC synthetic phenotype. The co-immunoprecipitation assay showed that PDGF-BB enhanced the interaction of RhoGDI1 with Cdc42 and promoted the activation of Cdc42, these enhancements were blocked by crenolanib and RhoGDI1 knockdown. Moreover, RhoGDI1 knockdown and crenolanib pretreatment prevented the localization of Cdc42 to the plasma membrane (PM) to activate and improve the accumulation of Cdc42 on endoplasmic reticulum (ER). Furthermore, Cdc42 inhibition or suppression significantly reduced VSMC phenotypic transformation and neointima formation in vitro and in vivo. This study revealed the novel mechanism by which RhoGDI1 stability promotes the RhoGDI1-Cdc42 interaction and Cdc42 activation, thereby affecting VSMC phenotypic transformation and neointima formation.

Background and aims The physiological role of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) in vascular remodeling remains unknown. We investigated the function of RhoGDI in angiotensin II (Ang II)-induced vascular remodeling in cultured human aortic vascular smooth muscle cells (HA-VSMCs) and in an Ang II-infusion vascular remodeling mouse model. Methods For in vitro assays of HA-VSMCs, proliferation was assessed by BrdU and EdU assays and immunofluorescence analysis of ki-67 expression. RhoGDI1 and RhoGDI2 function and expression were assessed by RNAi, Western blotting and real-time RT-PCR. RhoGDI ubiquitination and SUMOylation levels were evaluated by co-immunoprecipitation and Western blotting. The functions of proteosomal-mediated degradation, ubiquitination, SUMOylation and Ang II receptors were assessed using specific inhibitors. To evaluate the in vivo effects of Ang II and RhoGDI, H & E staining, Masson's trichrome staining, and immunostaining were employed. Results Ang II treatment of HA-VSMCs for 6 or 48 h promoted RhoGDI1 and RhoGDI2 protein degradation and reduced cell proliferation, which was reversed by proteosome inhibition. In contrast, treatment with Ang II for 12 or 24 h induced dose-dependent cell proliferation without affecting RhoGDI expression. RNA interference of either RhoGDI1 or RhoGDI2 blocked proliferation induced by 12 or 24 h treatment of Ang II. Moreover, Ang II-dependent degradation at 6 and 48 h correlated with RhoGDI ubiquitination and inversely correlated with RhoGDI SUMOylation and cell proliferation. Treatment with specific inhibitors suggests that ubiquitin and SUMO competitively bind to RhoGDI1 and RhoGDI2 to reciprocally regulate RhoGDI stability and HA-VSMC proliferation. Furthermore, inhibition of the Ang II receptor 1 (AT1 receptor), but not the Ang II receptor 2, blocked Ang II-dependent RhoGDI stabilization and proliferation at 12 and 24 h. In mice, Ang II infusion increased the intima-media thickness, collagen and myofiber production and VSMC proliferation, and these effects were shown to be dependent on RhoGDI1, RhoGDI2 and AT1 receptor. Ang II infusion exerted no significant effect on RhoGDI1 and RhoGDI2 protein levels, which were decreased after AT1 receptor inhibition. Conclusions Together, the results of this study reveal a novel mechanism by which Ang II regulates RhoGDI stability by SUMOylation and ubiquitination via AT1 receptor activation and thus affects VSMC proliferation and vascular remodeling.

Graphene and graphene oxide (GO) usually have grain boundaries (GBs) in the process of synthesis and preparation. Here, we &ldquo, attach&rdquo, GBs into GO, a new molecular configuration i.e., polycrystalline graphene oxide (PGO) is proposed. This paper aims to provide an insight into the stability and mechanical properties of PGO by using the molecular dynamics method. For this purpose, the &ldquo, bottom-up&rdquo, multi-structure-spatial design performance of PGO and the physical mechanism associated with the spatial structure in mixed dimensions (combination of sp2 and sp3) were studied. Also, the effect of defect coupling (GBs and functional groups) on the mechanical properties was revealed. Our results demonstrate that the existence of the GBs reduces the mechanical properties of PGO and show an &ldquo, induction&rdquo, role during the tensile fracture process. The presence of functional groups converts in-plane sp2 carbon atoms into out-of-plane sp3 hybrid carbons, causing uneven stress distribution. Moreover, the mechanical characteristics of PGO are very sensitive to the oxygen content of functional groups, which decrease with the increase of oxygen content. The weakening degree of epoxy groups is slightly greater than that of hydroxyl groups. Finally, we find that the mechanical properties of PGO will fall to the lowest values due to the defect coupling amplification mechanism when the functional groups are distributed at GBs.

Background: In this study, we investigated the mechanism of Rho GTPases signaling on Ang II-mediated cell migration and dedifferentiation in human aortic vascular smooth muscle cells (HA-VSMCs) and an Ang II-infusion mouse model. Methods: Cells were pretreated with different inhibitors or Ang II. Cell migration was detected by Wound healing and Transwell assay. Mice were treated with Ad-RhoA-shRNA virus or Irbesartan or fasudil and then infused with Ang II. Results: Ang II treatment induced HA-VSMCs migration in a dose- and time-dependent manner and reduced the expression of VSMC contractile proteins. These effects were significantly suppressed by the inhibition of Ang II type 1 receptor (AT1 receptor), RhoA, and Rho-associated kinase (ROCK). Furthermore, Ang II treatment promoted the activation of RhoA and ROCK, which was reduced by AT1 receptor inhibition. Meanwhile, Ang II treatment induced F-actin polymerization, which was inhibited after ROCK inhibition. In mice, Ang II infusion increased VSMC migration into the neointima and reduced VSMC differentiation proteins levels, and these effects were shown to be dependent on AT1 receptor and RhoA/ROCK pathway. Conclusion: This study reveals a novel mechanism by which Ang II regulates RhoA/ROCK signaling and actin polymerization via AT1 receptor and then affects VSMC dedifferentiation.

We analyzed the role of the RhoA/ROCK pathway in regulating endothelial dysfunction triggered by LPS and the protective effects of TSG (2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucoside). Human umbilical vein endothelial cells (HUVECs) were treated with LPS at different concentrations or at different time points. Cells were also pretreated with 30 μM ROCK inhibitor Y27632 for 30 min or different concentrations of TSG for 24 h and then were incubated with 100 μg/ml LPS for another 24 h. The results showed that LPS treatment significantly reduced endothelial cell viability, increased LDH release, and promoted cell necrosis in a dose- and time-dependent manner, which was dramatically inhibited by TSG pretreatment. Furthermore, LPS induction significantly enhanced the expression of RhoA, ROCK1, and ROCK2 and the activation of ROCK; these effects were reduced by TSG pretreatment. The suppression of either RhoA or ROCK significantly improved LPS-induced endothelial cell viability, and reduced cell necrosis and LDH release. In addition, LPS treatment promoted F-actin skeleton rearrangement and contraction ring formation around the plasma membrane, which was greatly inhibited by the suppression of the RhoA/ROCK pathway or TSG pretreatment. In conclusion, TSG may inhibit F-actin cytoskeletal remodeling by blocking RhoA/ROCK signaling and thus reduce LPS-induced endothelial cell toxicity.

Sirtuin 3 (SIRT3) is a deacetylase involved in the development of many inflammation-related diseases including liver fibrosis. Withaferin A (WFA) is a bioactive constituent derived from the Withania somnifera plant, which has extensive pharmacological activities; however, little is known about the regulatory role of SIRT3 in the WFA-induced antifibrogenic effect. The current study is aimed at investigating the role of SIRT3 in WFA-induced antioxidant effects in liver fibrosis. Our study verified that WFA attenuated platelet-derived growth factor BB- (PDGF-BB-) induced liver fibrosis and promoted PDGF-BB-induced SIRT3 activity and expression in JS1 cells. SIRT3 silencing attenuated the antifibrogenic and antioxidant effects of WFA in activated JS1 cells. Moreover, WFA inhibited carbon tetrachloride- (CCl4-) induced liver injury, collagen deposition, and fibrosis; increased the SIRT3 expression; and suppressed the CCl4-induced oxidative stress in fibrotic livers of C57/BL6 mice. Furthermore, the antifibrogenic and antioxidant effects of WFA could be available in CCl4-induced WT (129S1/SvImJ) mice but were unavailable in CCl4-induced SIRT3 knockout (KO) mice. Our study suggested that WFA inhibited liver fibrosis through the inhibition of oxidative stress in a SIRT3-dependent manner. WFA could be a potential compound for the treatment of liver fibrosis.

Background/Aims: This study used Rho-associated protein kinase (ROCK) isoform-selective suppression or a ROCK inhibitor to analyze the roles of ROCK1 and ROCK2 in regulating endothelial dysfunction triggered by oxidized low-density lipoprotein (oxLDL). Methods: ROCK1 or ROCK2 expression in human umbilical vein endothelial cells (HUVECs) was suppressed by small interfering RNA (siRNA). HUVECs were pretreated with 30 μM Y27632 (pan ROCK inhibitor) for 30 min before exposure to 200 μg/mL oxLDL for an additional 24 h. Cell viability was determined by the MTT assay, and cell apoptosis was evaluated by the TUNEL assay. Protein expression and phosphorylation were assessed by Western blot analysis. The morphology of total and phosphorylated vimentin (p-vimentin) and the co-localization of vimentin with vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were detected by the immunofluorescence assay. The adhesion of promonocytic U937 cells to HUVECs was observed by light microscopy. Results: ROCK2 suppression or Y27632 treatment, rather than ROCK1 deletion, effectively reduced endothelial cell apoptosis and preserved cell survival. ROCK2 suppression exhibited improved vimentin and p-vimentin cytoskeleton stability and decreased vimentin cleavage by attenuating caspase-3 activity. In addition, increased p-vimentin expression induced by oxLDL was significantly inhibited by ROCK2 deletion or Y27632 treatment. In contrast, ROCK1 suppression showed no obvious effects on the vimentin cytoskeleton, but significantly regulated the expression of adhesion molecules. Endothelial ICAM-1 or VCAM-1 expression induced by oxLDL was obviously inhibited by ROCK1 suppression or Y27632 treatment. Moreover, the expression of ICAM-1 induced by oxLDL could also be reduced by ROCK2 suppression. Furthermore, ROCK2 deficiency or Y27632 treatment inhibited the redistribution of adhesion molecules and their co-localization with vimentin caused by oxLDL. These effects resulted in the significant inhibition of monocyte-endothelial adhesion induced by oxLDL. Conclusion: The results of this study support the novel concept that ROCK1 is involved in oxLDL-induced cell adhesion by regulating adhesion molecule expression, whereas ROCK2 is required for both endothelial apoptosis and adhesion by regulating both the vimentin cytoskeleton and adhesion molecules. Consequently, ROCK1 and ROCK2 have distinct roles in the regulation of oxLDL-mediated endothelial dysfunction.

The RhoA/ROCK signaling pathway regulates cell morphology, adhesion, proliferation, and migration. In this study, we investigated the regulatory role of RhoA/ROCK signaling on PDGF-BB-mediated smooth muscle phenotypic modulation and vascular remodeling and clarified the molecular mechanisms behind these effects. PDGF-BB treatment induced the activation of RhoA, ROCK, PDGF-Rβ, and the expression of PDGF-Rβ in HA-VSMCs (human aortic vascular smooth muscle cells). PDGF-Rβ inhibition and RhoA suppression blocked PDGF-BB-induced RhoA activation and ROCK induction. In addition, PDGF-BB-mediated cell proliferation and migration were suppressed by PDGF-Rβ inhibition, RhoA suppression, and ROCK inhibition, suggesting that PDGF-BB promotes phenotypic modulation of HA-VSMCs by activating the RhoA/ROCK pathway via the PDGF receptor. Moreover, suppressing both ROCK1 and ROCK2 blocked cell cycle progression from G0/G1 to S phase by decreasing the transcription and protein expression of cyclin D1, CDK2, and CDK4 via JNK/c-Jun pathway, thus reducing cell proliferation in PDGF-BB-treated HA-VSMCs. ROCK1 deletion, rather than ROCK2 suppression, significantly inhibited PDGF-BB-induced migration by reducing the expression of vimentin and preventing the remodeling of vimentin and phospho-vimentin. Furthermore, ROCK1 deletion suppressed vimentin by inhibiting the phosphorylation of Smad2/3 and the nuclear translocation of Smad4. These findings suggested that ROCK1 and ROCK2 might play different roles in PDGF-BB-mediated cell proliferation and migration in HA-VSMCs. In addition, PDGF-BB and its receptor participated in neointima formation and vascular remodeling by promoting cell cycle protein expression via the JNK pathway and enhancing vimentin expression in a rat balloon injury model; effects that were inhibited by treatment with fasudil. Together, the results of this study reveal a novel mechanism through which RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.

The geometric imperfections (circular and square nanohole) were created in different regions of graphene/h-BN heterostructure. Defective heterostructures were used as a reinforcing material for copper (defective G/h-BN@Cu). The mechanical properties of defective G/h-BN@Cu were studied, and the effects of defect size and position on mechanical properties were studied by molecular dynamics. The results showed that mechanical properties of the defective G/h-BN@Cu nanostructure decrease with the increase of the diameter of the nanohole. The effects of nanohole on the stress and strain of the G/h-BN@Cu nanostructures are much higher than Young's modulus. The three mechanical values of G/h-BN@Cu nanostructures in the presence of nanoholes change considerably and do not follow the general trends. When the nanohole is created at h-BN region, a greater reduction of mechanical properties is at this location, relative to other positions. The adverse effect of circular defects on mechanical properties of G/h-BN@Cu nanostructures is lower than that of square defects.

Celastrol, a pentacyclic tritepene extracted from Tripterygium Wilfordi plant, showing potent liver protection effects on several liver‐related diseases. However, the anti‐inflammatory potential of celastrol in liver fibrosis and the detailed mechanisms remain uncovered. This study was to investigate the anti‐inflammatory effect of celastrol in liver fibrosis and to further reveal mechanisms of celastrol‐induced anti‐inflammatory effects with a focus on AMPK‐SIRT3 signalling. Celastrol showed potent ameliorative effects on liver fibrosis both in activated hepatic stellate cells (HSCs) and in fibrotic liver. Celastrol remarkably suppressed inflammation in vivo and inhibited the secretion of inflammatory factors in vitro. Interestingly, celastrol increased SIRT3 promoter activity and SIRT3 expression both in fibrotic liver and in activated HSCs. Furthermore, SIRT3 silencing evidently ameliorated the anti‐inflammatory potential of celastrol. Besides, we found that celastrol could increase the AMPK phosphorylation. Further investigation showed that SIRT3 siRNA decreased SIRT3 expression but had no obvious effect on phosphorylation of AMPK. In addition, inhibition of AMPK by employing compound C (an AMPK inhibitor) or AMPK1α siRNA significantly suppressed SIRT3 expression, suggesting that AMPK was an up‐stream protein of SIRT3 in liver fibrosis. We further found that depletion of AMPK significantly attenuated the inhibitory effect of celastrol on inflammation. Collectively, celastrol attenuated liver fibrosis mainly through inhibition of inflammation by activating AMPK‐SIRT3 signalling, which makes celastrol be a potential candidate compound in treating or protecting against liver fibrosis.

One of the miraculous functions of graphene is to use its defects to alter the material properties of graphene composites and, thereby, expand the application of graphene in other fields. In this paper, various defects have been created in graphene by using ion irradiation. Defective graphene is sandwiched between two copper layers. A numerical model of Graphene/Copper layered composites after irradiation damage was established by the molecular dynamics method. The effects of ion irradiation and temperature coupling on defective graphene/copper composites were studied. The results show that there are a lot of empty defects in graphene after irradiation injury, which will produce more incomplete bonding. Although the bonds between carbon atoms can be weakened, defective graphene still enhances the mechanical properties of pure copper. At the same time, the location and arrangement of defects have a great influence on the mechanical stability of graphene/copper composites, and the arrangement of empty defects has different effects on deformation behavior and the stress transfer mechanism. It can be concluded that the defects formed by radiation have an effect on the physical properties of two-dimensional materials. Therefore, irradiation technology can be used to artificially control the formation of defects, and then make appropriate adjustments to their properties. This can not only optimize the radiation resistance and mechanical properties of nuclear materials, but also expand the application of graphene in electronic devices and other fields.

In this paper, a BN-Gr-BN planar heterojunction is established, and the BN-Gr-BN planar heterojunction is used as reinforcing materials for copper-based materials. The coupling effects of defects and temperature on mechanical properties of the graphene-h-BN/copper vertically-stacked heterostructures were investigated. The results show the BN-Gr-BN heterojunction layer exerts a strengthening influence on the Young’ modulus as well as the load transfer ability, thereby enhancing the stability capacity and the plasticity of composite system under axial tension. the introduce of BN-Gr-BN heterojunction layer gives rise to geometrical imperfections at interface, which in turn causes a reduction in the ultimate tensile strength and the associated facture strain. The positive effects of BN-Gr-BN heterojunction layer overpower their negative effects on the mechanical behavior of composite system. The adverse and strengthening effects induced by the BN-Gr-BN heterojunction layer are also strongly dependent of temperature. The present results can serve as a guide for diversified structure design and defect control.

Background and purpose Simvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor with multiple targets and effects. It protects neurons in the brain, but its protective effects on photoreceptors are unclear. In this study, we evaluated the neuroprotective effect of simvastatin on photoreceptors exposed to stress induced by all-trans-retinal (atRAL). Experimental approach AlamarBlue and LDH assays were used to evaluate the viability and metabolic activity of Y79 cells (a retinoblastoma cell line) exposed to atRAL-induced stress with or without simvastatin pretreatment. Changes in cellular ROS were evaluated using flow cytometry and mitochondrial stress markers JC-1 and HSP60. Changes in levels of two photoreceptor-specific markers, cone-rod homeobox protein (CRX) and interphotoreceptor retinoid binding protein (IRBP), were evaluated with western blot. The results were validated in ex vivo human retinal explants and a mouse model of photoreceptor degeneration. Key results Simvastatin improved mitochondrial function, alleviated oxidative stress and up-regulated the photoreceptor-specific markers IRBP and its upstream regulator CRX in Y79 cells and ex vivo human retinal explants under atRAL-induced stress. Simvastatin attenuated photoreceptor degeneration in association with up-regulation of IRBP and CRX expression after knockdown of IRBP in a murine model. Conclusion and implications Our findings suggest that simvastatin has a novel role in protecting photoreceptors from atRAL-induced stress. Simvastatin treatment resulted in up-regulation of IRBP and its upstream transcription factor CRX in Y79 cells, ex vivo human retinal explants, and murine retinas in vivo. Further studies of simvastatin to treat photoreceptor degeneration are warranted.

The Retinal Pigment Epithelium (RPE) is a monolayer of cells located above the choroid. It mediates human visual cycle and nourishes photoreceptors. Hypoxia-induced oxidative stress to RPE is a vital cause of retinal degeneration such as the Age-related Macular Degeneration. Most of these retinal diseases are irreversible with no efficient treatment, therefore protecting RPE cells from hypoxia stress is an important way to prevent or slow down the progression of retinal degeneration. Betulinic acid (BA) and betulin (BE) are pentacyclic triterpenoids with anti-oxidative property, but little is known about their effect on RPE cells. We investigated the protective effect of BA, BE and their derivatives against cobalt chloride-induced hypoxia stress in RPE cells. Human ARPE-19 cells were exposed to BA, BE and their eighteen derivatives (named as H3 H20) that we customized through replacing moieties at C3 and C28 positions. We found that cobalt chloride reduced cell viability, increased Reactive Oxygen Species (ROS) production as well as induced apoptosis and necrosis in ARPE-19 cells. Interestingly, the pretreatment of 3-O-acetyl-glycyl- 28-O-glycyl-betulinic acid effectively protected cells from acute hypoxia stress induced by cobalt chloride. Our immunoblotting results suggested that this derivative attenuated the cobalt chloride-induced activation of Akt, Erk and JNK pathways. All findings were further validated in human primary RPE cells. In summary, this BA derivate has protective effect against the acute hypoxic stress in human RPE cells and may be developed into a candidate agent effective in the prevention of prevalent retinal diseases.

Inducible nitric oxide synthase (iNOS) critically contributes to the development of endotoxin-mediated inflammation. It can be induced by cytokines or endotoxins via distinct signaling pathways. Lipopolysaccharide (LPS) triggers iNOS expression through activation of the inhibitor of κB-α (IκB-α)-nuclear factor κB (NF-κB) cascade, whereas interferon-γ (IFN-γ) acts primarily through Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1). Methylene blue (MB), an agent used clinically to treat numerous ailments, has been shown to reduce NO accumulation through suppression of iNOS activity. But it remains unclear whether MB affects iNOS induction. This knowledge gap is addressed in the present study using cultured cells and endotoxemic mice. With mouse macrophages, MB treatment prevented the LPS- and/or IFN-γ-stimulated iNOS protein expression. Real-time PCR experiments showed that iNOS mRNA transcription was robustly blocked by MB treatment. The inhibitory effect of MB on iNOS expression was confirmed in vivo in endotoxemic mice. Further analysis showed that MB had no significant effect on IκB-α degradation and NF-κB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-κB or STAT1 was also not affected by MB treatment. But MB treatment markedly reduced the binding of NF-κB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that MB reduced NF-κB and STAT1 bindings to iNOS promoter inside the cell. These studies show that MB attenuates transcriptional factor binding amid iNOS mRNA transcription, providing further insight into the molecular mechanism of MB in disease therapy. J. Cell. Biochem. 116: 1730–1740, 2015. © 2015 Wiley Periodicals, Inc.

Dihydromyricetin (DMY) displays a range of biological properties. However, whether DMY attenuates cardiomyocyte hypertrophy is unknown.To investigate whether DMY had potential therapeutic value to protect against angiotensin II (Ang II)-induced cardiomyocyte hypertrophy.Neonatal rat cardiomyocytes were pretreated with DMY (0-320 μM) followed with Ang II (100 nM) stimulation for 24 h, and then degree of hypertrophy was evaluated by cell surface analysis. Levels of reactive oxygen species (ROS) were measured with 2',7'-dichlorfluorescein-diacetate (DCFH-DA) fluorescent staining. Antioxidative activity was evaluated by malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, and total antioxidant capacity (T-AOC). Cyclic guanosine monophosphate (cGMP) was determined with a radioimmunoassay.Pre-incubation with DMY (20, 40, 80, and 160 μM) for 8 h, 12 h, 24 h, or 48 h decreased cell surface areas. It down-regulated mRNA expression of atrial natriuretic factor (1.95- to 1.24-fold) and β-myosin heavy chains (3.51- to 2.32-fold), reduced levels of MDA as well as increased SOD activity and T-AOC. Expression of SOD and thioredoxin were enhanced by DMY, whereas p22(phox) and phosphorylation of mitogen-activated protein kinases were inhibited. Content of cGMP (0.54- to 0.80-fold) and phosphorylation of endothelial nitric oxide synthase at serine 1177 (0.70- to 1.05-fold) were augmented by DMY. Moreover, attenuating effect of DMY on hypertrophy was abolished when NO production was inhibited by l-NAME.Attenuating effects of DMY on Ang II-induced cardiomyocyte hypertrophy related to antioxidative activity in a NO-dependent manner.

In order to investigate the effects of RhoA/ROCK signaling in macrophage differentiation, we used 100 ng/mL PMA to induce macrophage differentiation from U937 cells in vitro. The observation of cell morphology and the expression of CD68 and SR-A were performed to confirm the differentiation induced by PMA. Western blot analysis showed that the expression of ROCK1 and ROCK2 and the phosphorylation of MYPT1 were significantly increased after PMA treatment. Pulldown assay showed that the activation of RhoA was obviously enhanced when U937 cells were treated with PMA. In order to further demonstrate whether RhoA/ROCK signaling could mediate the macrophage differentiation induced by PMA, we successfully suppressed the expression of RhoA, ROCK1 and ROCK2 by performing siRNA technology in U937 cells, respectively. The macrophage differentiation and the expression of CD68 and SR-A were significantly inhibited by the suppression of RhoA, ROCK1 or ROCK2 in PMA-induced U937 cells, indicating that the macrophage differentiation induced by PMA is associated with RhoA/ROCK signaling pathway. In addition, we pretreated U937 cells with Y27632 (ROCK inhibitor, 20 μM) for 30 min and then observed the macrophage differentiation induced by PMA. The result illustrated that Y27632 pretreatment obviously inhibited PMA-induced differentiation and the expression of CD68 and SR-A. In conclusion, the activation of RhoA/ROCK signaling is responsible for the macrophage differentiation induced by PMA.

2,3,4',5-tetrahydroxystilbene 2-O-β-D-glucoside (TSG), a resveratrol analog with glucoside, is purified from a traditional Chinese herbal medicine polygonum multiflorum. It has been extensively studied in last decade and known to exert strong anti-inflammatory, anti-oxidative, anti-apoptotic, and free radical scavenging activities, and therefore has been listed as a potential agent for disease therapies. Recent studies extend well-beyond effects of TSG on the injury of neurons, cardiomyocytes and endothelial cells, and report important functions of TSG in a lot of pathophysiological conditions. For example, TSG has been shown to prevent the production of pro-inflammatory cytokines in microglia and macrophages in vitro, and ameliorate pro-inflammatory responses in animal models with neurodegeneration, atherosclerosis, and rat paw or ear oedema. TSG can prevent the proliferation of vascular smooth cells, gastrointestinal dysfunctions, platelet aggregation, osteoblastic injury, diabetic nephropathy and melanogenesis. TSG is also indicated to facilitate long-term potentiation and learning and memory in both normal and pathological conditions. These effects to some extent enrich the understanding about the role of TSG in disease prevention and therapy. However, to date, we still have no outlined knowledges about the pharmacological effects of TSG, though the role of TSG in aging and Alzheimer's disease has been reviewed in recent years. Here, we summarize the current pharmacological developments of TSG as well as its possible mechanisms in disease prevention and therapy, aiming to push the understanding about the protective role of TSG as well as its preclinical assessment of novel applications.

Background: Endothelial cell dysfunction triggered by oxidized low-density lipoprotein (oxLDL) is the main event occurring during the development of atherosclerosis. 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component of the rhizome extract from Polygonum multiflorum, exhibits significant anti-atherosclerotic activity. However, the protective effects of TSG against oxLDL-induced endothelial dysfunction have not been clarified. We investigated the cytoprotective effects of TSG in human umbilical vein endothelial cells (HUVECs) and explored underlying mechanisms. Methods and Results: TSG pretreatment markedly attenuated oxLDL-mediated loss of cell viability, release of lactate dehydrogenase (LDH), cell apoptosis, and monocyte adhesion. OxLDL increased vimentin mRNA and protein levels, vimentin cleavage, caspase-3 activation, adhesion molecules levels and their colocalization with vimentin in HUVECs. These alterations were attenuated by pretreatment with TSG. Meanwhile, TSG inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by oxLDL. Using shRNA, oxLDL-induced cell apoptosis and monocyte adhesion were significantly inhibited by vimentin suppression in HUVECs. Conclusions: These results suggest that TSG protects HUVECs against oxLDL-induced endothelial dysfunction through inhibiting vimentin expression and cleavage, and the expression of adhesion molecules and their colocalization with vimentin. The interruption of TGFβ/Smad pathway and caspase-3 activation appears to be responsible for the downregulation of TSG on vimentin expression and fragmentation, respectively.

The mechanical properties of graphene/hexagonal boron nitride (G/h-BN) heterobilayer nanosheets coupled by interlayer sp3 bonds and defects have been investigated by using the molecular dynamics (MD) simulation. For this purpose, interlayer sp3 bonds and two kinds of geometric imperfection (including rhombus and square nanopore) are formed in the G/h-BN heterobilayer nanosheets. The effects of adding the nanopore diameter, interlayer sp3 bonds fraction, and the influence of different position of geometric imperfection in heterobilayer nanostructure on mechanical properties have been investigated. The result shows that when sp3 bonds and nanopore coexist, the mechanical properties of defective graphene layer are higher than that of defective hexagonal boron nitride (h-BN) layer. Meanwhile, the negative effect of rhomboid nanohole on mechanical properties of heterostructure is weaker than that of square nanohole. It is observed that that interlayer sp3 bonds have greatest influence on the fracture strain of G/h-BN heterobilayer nanostructure, followed by fracture stress, and finally Young’s modulus. Another important point is that interlayer sp3 bonds can be regarded as a special defect, and its value seems to outweigh the intrinsic defects.

The enzyme φC31 integrase from Streptomyces phage has been documented as functional in mammalian cells and, therefore, has the potential to be a powerful gene manipulation tool. However, the activity of this enzyme is cell-type dependent. The more active mutant forms of φC31 integrase are required. Therefore, a rapid and effective method should be developed to detect the intracellular activity of φC31 integrase. We devised in this study an integrase-inversion cassette that contains the enhanced green fluorescent protein (EGFP) gene and the reverse complementary DsRed gene, which are flanked by attB and reverse complementary attP. This cassette can be inverted by φC31 integrase, thereby altering the fluorescent protein expression. Thus, φC31 integrase activity can be qualitatively or quantitatively evaluated based on the detected fluorescence. Furthermore, this cassette-based method was applied to several cell types, demonstrating that it is an efficient and reliable tool for measuring φC31 integrase activity in mammalian cells.

In this work, we have developed a novel choline biosensor on the basis of immobilization of choline oxidase (ChOx) by the attractive materials layered double hydroxides (LDHs). Amperometric detection of choline was evaluated by holding the modified electrode at 0.5 V (vs. SCE). Due to the special properties of LDHs ([Zn3-Al-Cl]), such as chemical inertness, high porosity, and swelling property, the [Zn3-Al-Cl]/ChOx modified electrode exhibited an enhanced analytical performance. The biosensor provided a linear response to choline over a concentration range from 3.7 × 10−6 to 6.3 × 10−4 M with a low detection limit of 3 × 10−7 M based on S/N=3. The apparent Michaelis-Menten constant was calculated to be 1.38 mM. In addition, the interaction between ChOx and LDHs has also been investigated using FT-IR spectroscopy.

We developed a new cell surface display system in Corynebacterium glutamicum based on the C-terminally truncated NCgl1221 anchor protein to increase L-glutamate production from starch directly. The C-terminally truncated NCgl1221 protein is a mutant NCgl1221 and leads to the constitutive export of L-glutamate. The N terminus of alpha-amylase (AmyA) was fused to truncated NCgl1221, and the resulting fusion protein was expressed on the cell surface by IPTG induction. Localization of the fusion protein was confirmed by immunofluorescence microscopy and flow cytometric analysis. The results of L-glutamate fermentation showed that the soluble starch was utilized to grow and produce L-glutamate by the recombinant strain displaying AmyA. The amount of soluble starch was reduced from 30.0 +/- 2.8 to 4.5 +/- 0.7 g/l under non-inducing condition and from 50.0 +/- 2.4 to 12.5 +/- 1.1 g/l under biotin limitation in 36 h. The glutamate concentration in the medium was transiently increased in 14 h under no induction, while under biotin-limiting condition, glutamate production was continuously elevated during fermentation. The amount of glutamate reached 19.3 +/- 2.1 g/l after 26 h of fermentation with biotin limitation, which was greater than that produced by the strain using PgsA, one of the poly-gamma-glutamate synthetase complexes, as the anchor protein under the same condition. Therefore, the truncated NCgl1221 anchor protein has more advantages than the PgsA anchor protein in glutamate fermentation because truncated NCgl1221 leads to the constitutive export of L-glutamate without any treatments.

Corynebacterium glutamicum strains are used for the fermentative production of L-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of L-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and DeltadtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain DeltadtsR1Deltapyc was more than that of the mutant DeltadtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the DeltadtsR1Deltapyc strain than in the DeltadtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in DeltadtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (DeltadtsR1Deltappc and DeltadtsR1Deltapyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.

An efficient and novel one-pot synthesis of 3,4,5-trisubstituted-3,6-dihydro-2H-1,3-oxazine from alkynoates, anilines, and formaldehyde is described. The six-membered N,O-heterocyclic skeleton was constructed via Bronsted acid-promoted domino hydroamination/Prins reaction/cyclization/dehydration reactions.

An efficient and general one-pot procedure for the synthesis of pyrazoles from acid chlorides, terminal alkynes and hydrazines was described via a coupling and cyclocondensation sequence. Acid chlorides coupled with terminal alkynes to give α,β-unsaturated ynones, and in situ converted into pyrazoles by the cycloaddition of hydrazines. The desired pyrazoles were obtained with 15–85% isolated yields.

The coabsorptions of ethylene and iso-pentane, and ethylene and n-hexane were measured by the use of a pressure decay method. The solubility data of ethylene-iso-pentane and ethylene-n-hexane in semicrystalline polyethylene (PE) of crystallinity of 48.6% were obtained at temperatures 70, 80, and 90 °C, and the total pressure 2 MPa, iso-pentane partial pressure 80–190 KPa, n-hexane partial pressure 20–90 KPa. The presence of iso-pentane or n-hexane in the corresponding ternary system leads to increase the solubility of ethylene, while the solubility of iso-pentane or n-hexane remains unchanged with an increase of the ethylene partial pressure, even slightly decreases. Assumed that the presence of iso-pentane or n-hexane decreases the crystallinity of the polymer sample, a coabsorption model was built to model the solubility of each gas in the ternary systems. The relative root mean square errors of the coabsorption model for ethylene-iso-pentane-PE system and ethylene-n-hexane-PE system are 5.13% and 4.64%, respectively. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 3654–3662, 2007

Vascular smooth muscle cell (VSMC) migration triggered by TNF-α is an important event that occurs during the development of atherosclerosis. 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside (TSG) has been proven to exhibit significant anti-atherosclerotic activity. Herein we investigate the inhibitory effect of TSG on TNF-α-induced VSMC migration and explore the underlying mechanisms. TSG pretreatment markedly inhibited TNF-α-induced cell migration. The inhibition of vimentin redistribution and expression was involved in the inhibitory effect of TSG on VSMC migration. The suppression of vimentin expression by shRNA in VSMCs significantly inhibited TNF-α-induced cell migration. Furthermore, TSG inhibited the TNF-α-induced expression of TGFβ1 and TGFβR1, and phosphorylation of TGFβR1 and Smad2/3. TSG also suppressed the nuclear translocation of Smad4 induced by TNF-α. These results suggest that TSG inhibits VSMC migration induced by TNF-α through inhibiting vimentin rearrangement and expression. The interruption of TGFβ/Smad pathway appears to be responsible for the suppression of TSG on vimentin expression.

Maspin, a non-inhibitory member of serine protease family, acts as an effective tumor suppressor by inhibiting cell inhesion and mobility. We found that exogenous wild-type rMaspin had a low effect on tumor growth in vivo. However, when the peptide Arg-Gly-Asp-hexahistidine (RGD-6His) was introduced into rMaspin, the modified rMaspin showed significant inhibitory activity in angiogenic assays and tumor-bearing animal models. Overall, our data suggested that both the RGD and hexahistidine fragments contributed to improve the fusion protein activity and polyhistidine peptide could be considered as flexible linker to separate RGD and Maspin moieties to avoid function interference. Besides, it is an efficient tag to achieve purified recombinant proteins. Furthermore, rMaspin fusing with RGD and hexahistidine could be a viable anticancer candidate.

A novel cheap and simple amperometric biosensor, based on the immobilization of glucose oxidase (GOD) into anionic clay; layered double hydroxides (LDHs) [Zn3-Al-Cl] is presented. GOD can be entrapped in the LDHs gel via electrostatic interaction. Amperometric detection of glucose with an unmediated sensor at 0.6 V (vs. SCE) results in a rapid response (5 s), a wide linear range of 0.001–12 mM, as well as good operational stability. The low detection limit was 0.1 μM at 3σ. The apparent Michaelis-Menten constant (K is 4.4 mM. The general interferences that coexisted in blood serum do not affect glucose determination, except for uric acid. In addition, optimization of the biosensor construction and the effects of the applied potential on the amperometric response of the sensor were investigated and discussed herein.

An absorption model of gases in semicrystalline polymer was built that was based on the activity coefficient theory in polymer solution and associated with crystallinity dependent on temperature. The solubility of ethylene, isopentane, and n-hexane in three types of polyethylene (PE) were obtained by the use of a pressure-decay method at temperatures of 333–363 K and pressures of up to 2 MPa, 80–300 KPa, and 19–100 KPa, respectively. Experimental data from three gases in each PE sample were used for the single-parameter fitting, and fitting error was within about 12%. It was found that a single parameter was merely dependent on the properties of PE used. It was shown that, unlike with the Flory–Huggins model and the UNIFAC–M-H method, correlation between the crystallinity of the semicrystalline polymer and temperature had to be taken into account in order for the solubility data of alkane, olefin, and aromatic hydrocarbons in polyethylene to fit well, especially in the temperature range near the melting point of the polymer. The four free-energy contributions to the total gas activity were experimentally determined to be about 47%–60% combined, the free-volume contribution about 12%–25%, and the elastic effect about 22%–35%, but the interactional contribution was zero. The contributions changed with the size of the gas molecules. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1737–1744, 2007

A novel and efficient method for the regiospecific synthesis of polysubstituted furan aldehydes/ketones has been developed via a copper(I)-catalyzed rearrangement/dehydrogenation oxidation/carbene oxidation sequence of 1,5-enynes in situ formed from alkynols and diethyl but-2-ynedioate under atmospheric pressure. The domino reaction proceeds smoothly under mild conditions with commercially available catalysts and affords highly functionalized furans in moderate to good yields.

Background Induction of pro-inflammatory factors is one of the characteristics of microglia activation and can be regulated by numerous active components of Chinese traditional herbs. Suppression of pro-inflammatory factors is beneficial to alleviate microglia-mediated cell injury. The present study aims to investigate the effect and possible mechanism of 2,3,4’,5-tetrahydroxystilbene 2-O-β-D-glucoside (TSG) on LPS-mediated induction of pro-inflammatory factors in microglia. Methods Western blot, ELISA, and Hoechst 33258 were used to measure the protein expression, TNF-α/IL-6 content, and apoptotic nuclei, respectively. The mRNA level was measured by real time-PCR. Nitric oxide (NO) content, lactate dehydrogenase (LDH) content, and NF-κB binding activity were assayed by commercial kits. Results TSG reduced iNOS protein expression as well as TNF-α, IL-6, and NO content in LPS-stimulated BV-2 cells. TSG attenuated the increase in apoptotic nuclei, caspase-3 cleavage, and LDH content induced by BV-2 cell-derived conditioned medium in primary hippocampal neurons. Mechanistic studies showed that TSG reduced the mRNA level of iNOS, TNF-α, and IL-6. TSG failed to suppress IκB-α degradation, NF-κB phosphorylation and nuclear translocation, and ERK1/2, JNK, and p38 phosphorylation. TSG, however, markedly reduced the binding of NF-κB to its DNA element. Chromatin immunoprecipitation (ChIP) assays confirmed that TSG reduced NF-κB binding to the iNOS promoter. These findings were ascertained in primary microglia where the LPS-induced increase in iNOS expression, NO content, apoptotic nuclei, and NF-κB binding to its DNA element were diminished by TSG. Conclusions These studies demonstrate that TSG attenuates LPS-mediated induction of pro-inflammatory factors in microglia through reducing the binding activity of NF-κB. This might help us to further understand the pharmacological role of TSG in inflammatory response in the central nervous system.

We report the fabrication of a highly sensitive dopamine biosensor based on the entrapment of tyrosinase into CaCO3 nanoparticles at Multiwalled Carbon Nanotube (MWCNT) electrodes. CaCO3 acts as host matrix for tyrosinase and MWCNT provides a highly porous conductive network enhancing the enzyme immobilization and the electrochemical transduction of the enzyme reaction by boosting the amplification phenomenon involved in the biosensing of catechol and dopamine. The comparison of the performance of CaCO3-tyrosinase electrodes with and without MWCNT film clearly indicates the improvement in sensitivity and maximum current brought by the combination of MWCNTs and inorganic nanomaterials. These nanostructured hybrid bioelectrodes exhibit a high sensitivity for the detection of catechol and dopamine, namely 35.7 A mol−1 L cm−2, the detection limit for dopamine being 15 nmol L−1 with no influence of the presence of interferents, i.e. uric acid and ascorbic acid.

The Fe(ClO4)3-catalyzed intramolecular rearrangement/cyclization/oxidation reaction sequence for the synthesis of α-carbonyl furan derivatives from electron-deficient alkynes and 2-yn-1-ols is reported.

The Fe(ClO(4))(3)-catalyzed intramolecular rearrangement/cyclization/oxidation reaction sequence for the synthesis of alpha-carbonyl furan derivatives from electron-deficient alkynes and 2-yn-1-ols is reported.

A novel and efficient method for the regiospecific synthesis of polysubstituted furan aldehydes/ketones has been developed via a copper(I)-catalyzed rearrangement/dehydrogenation oxidation/carbene oxidation sequence of 1,5-enynes in situ formed from alkynols and diethyl but-2-ynedioate under atmospheric pressure. The domino reaction proceeds smoothly under mild conditions with commercially available catalysts and affords highly functionalized furans in moderate to good yields.

An efficient and general one-pot procedure for the synthesis of pyrazoles from acid chlorides, terminal alkynes and hydrazines was described via a coupling and cyclocondensation sequence. Acid chlorides coupled with terminal alkynes to give α,β-unsaturated ynones, and in situ converted into pyrazoles by the cycloaddition of hydrazines. The desired pyrazoles were obtained with 15–85% isolated yields.

This paper aimed at showing the interest of the composite material based on layered double hydroxides (LDHs) and chitosan (CHT) as suitable host matrix likely to immobilize enzyme onto electrode surface for amperometric biosensing application. This hybrid material combined the advantages of inorganic LDHs and organic biopolymer, CHT. Glucose oxidase (GOD) immobilized in the composite material maintained its activity well as the usage of glutaraldehyde was avoided. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, applied potential and temperature, were explored for optimum analytical performance of the enzyme electrode. The enzyme electrode provided a linear response to glucose over a concentration range of 1 x 10(-6) to 3 x 10(-3) M with a high sensitivity of 62.6 mA M(-1) cm(-2) and a detection limit of 0.1 microM based on the signal-to-noise ratio of 3.