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Perivascular adipose tissue (PVAT) is the connective tissue surrounding most of the systemic blood vessels. PVAT is now recognized as an important endocrine tissue that maintains vascular homeostasis. Healthy PVAT has anticontractile, anti-inflammatory, and antioxidative roles. Vascular oxidative stress is an important pathophysiological event in cardiometabolic complications of obesity, type 2 diabetes, and hypertension. Accumulating data from both humans and experimental animal models suggests that PVAT dysfunction is potentially linked to cardiovascular diseases, and associated with augmented vascular inflammation, oxidative stress, and arterial remodeling. Reactive oxygen species produced from PVAT can be originated from mitochondria, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, and uncoupled endothelial nitric oxide synthase. PVAT can also sense vascular paracrine signals and response by secreting vasoactive adipokines. Therefore, PVAT may constitute a novel therapeutic target for the prevention and treatment of cardiovascular diseases. In this review, we summarize recent findings on PVAT functions, ROS production, and oxidative stress in different pathophysiological settings and discuss the potential antioxidant therapies for cardiovascular diseases by targeting PVAT.

Arterial remodelling refers to the alteration in the structure of blood vessel that contributes to the progression of hypertension and other cardiovascular complications. Arterial remodelling is orchestrated by the crosstalk between the endothelium and vascular smooth muscle cells (VSMC). Vascular inflammation participates in arterial remodelling. Resveratrol is a natural polyphenol that possesses anti-oxidant and anti-inflammatory properties and has beneficial effects in both the endothelium and VSMC. Resveratrol has been studied for the protective effects in arterial remodelling and gut microbiota, respectively. Gut microbiota plays a critical role in the immune system and inflammatory processes. Gut microbiota may also regulate vascular remodelling in cardiovascular complications via affecting endothelium function and VSMC proliferation. Currently, there is new evidence showing that gut microbiota regulate the proliferation of VSMC and the formation of neointimal hyperplasia in response to injury. The change in population of the gut microbiota, as well as their metabolites (e.g., short-chain fatty acids) could critically contribute to VSMC proliferation, cell cycle progression, and migration. Recent studies have provided strong evidence that correlate the effects of resveratrol in arterial remodelling and gut microbiota. This review aims to summarize recent findings on the resveratrol effects on cardiovascular complications focusing on arterial remodelling and discuss the possible interactions of resveratrol and the gut microbiota that modulate arterial remodelling.

We reported here a turn-on fluorescent probe (1) for the detection of cysteine (Cys) by incorporating the recognition unit of 2,4-dinitrobenzenesulfonyl ester (DNBS) to a coumarin derivative. The structure of the obtained probe was confirmed by NMR and HRMS techniques. The probe shows a remarkable fluorescence off-on response (∼52-fold) by the reaction with Cys in 100% aqueous buffer. The sensing mechanism was verified by the HPLC test. Probe 1 also displays high selectivity towards Cys. The detection limit was calculated to be 23 nM. Moreover, cellular experiments demonstrated that the probe is highly biocompatible and can be used for monitoring intracellular Cys.

Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO), including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been proved in physiological test systems. Furthermore, only a few data exist on the antioxidant capacities of the enzymatic oxidation products of phenolic compounds generated by PPO. We report here first results about the antioxidant effects of phenolic substances, before and after oxidation by fungal model tyrosinase and laccase. In general, the common chemical 2,2-diphenyl-1-picrylhydrazyl assay and the biological tests using two different types of cell cultures (monocytes and endothelial cells) delivered similar results. The phenols tested showed significant differences with respect to their antioxidant activity in all test systems. Their antioxidant capacities after enzymatic conversion decreased or increased depending on the individual PPO used.

The large amount of pesticide residues in the environment is a threat to global health by inhibition of acetylcholinesterase (AChE). Biosensors for inhibition of AChE have been thus developed for the detection of pesticides. In line with the rapid development of nanotechnology, nanomaterials have attracted great attention and have been intensively studied in biological analysis due to their unique chemical, physical and size properties. The aim of this review is to provide insight into nanomaterial-based optical techniques for the determination of AChE and pesticides, including colorimetric and fluorescent assays and surface plasmon resonance.

We aimed to investigate the efficacy of four severity-of-disease scoring systems in predicting the 28-day survival rate among patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) requiring emergency care. Clinical data of patients with AECOPD who required emergency care were recorded over 2 years. APACHE II, SAPS II, SOFA, and MEDS scores were calculated from severity-of-disease indicators recorded at admission and compared between patients who died within 28 days of admission (death group; 46 patients) and those who did not (survival group; 336 patients). Compared to the survival group, the death group had a significantly higher GCS score, frequency of comorbidities including hypertension and heart failure, and age (P

Nitric oxide (NO) derived from the endothelial NO synthase (eNOS) has antihypertensive, antithrombotic, anti-atherosclerotic and antiobesogenic properties. Resveratrol is a polyphenol phytoalexin with multiple cardiovascular and metabolic effects. Part of the beneficial effects of resveratrol are mediated by eNOS. Resveratrol stimulates NO production from eNOS by a number of mechanisms, including upregulation of eNOS expression, stimulation of eNOS enzymatic activity and reversal of eNOS uncoupling. In addition, by reducing oxidative stress, resveratrol prevents oxidative NO inactivation by superoxide thereby enhancing NO bioavailability. Molecular pathways underlying these effects of resveratrol involve SIRT1, AMPK, Nrf2 and estrogen receptors.

MicroRNAs (miRNAs) play important functions in post-transcriptional regulation of gene expression. They have been regarded as reliable molecular biomarkers for many diseases including cancer. However, the content of miRNAs in cells can be low down to a few molecules per cell. Thus, highly sensitive analytical methods for miRNAs detection are desired. Recently, electrochemical biosensors have held great promise as devices suitable for point-of-care diagnostics and multiplexed platforms for fast, simple and low-cost nucleic acid analysis. Signal amplification by nanomaterials is one of the most popular strategies for developing ultrasensitive assay methods. This review surveys the latest achievements in the use of nanomaterials to detect miRNAs with a focus on electrochemical techniques.

Artichoke (Cynara scolymus L.) is one of the world’s oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects of artichoke on iNOS expression in human coronary artery smooth muscle cells (HCASMC). Incubation of HCASMC with a cytokine mixture led to an induction of iNOS mRNA expression. This iNOS induction was concentration- and time-dependently inhibited by an artichoke leaf extract (1–100 µg/mL, 6 h or 24 h). Consistently, the artichoke leaf extract also reduced cytokine-induced iNOS promoter activation and iNOS protein expression. In addition, treatment of HCASMC with four well-known artichoke compounds (cynarin > cyanidin > luteolin ≈ cynaroside) led to a downregulation iNOS mRNA and protein expression, with cynarin being the most potent one. In conclusion, artichoke contains both eNOS-upregulating and iNOS-downregulating compounds. Such compounds may contribute to the beneficial effects of artichoke and may per se have therapeutic potentials.

Visual impairment and blindness are often caused by retinal ischemia-reperfusion (I/R) injury. We aimed to characterize a new model of I/R in pigs, in which the intraocular pathways were not manipulated by invasive methods on the ocular system. After 12 min of ischemia followed by 20 h of reperfusion, reactivity of retinal arterioles was measured in vitro by video microscopy. Dihydroethidium (DHE) staining, qPCR, immunohistochemistry, quantification of neurons in the retinal ganglion cell layer, and histological examination was performed. Retinal arterioles of I/R-treated pigs displayed marked attenuation in response to the endothelium-dependent vasodilator, bradykinin, compared to sham-treated pigs. DHE staining intensity and messenger RNA levels for HIF-1α, VEGF-A, NOX2, and iNOS were elevated in retinal arterioles following I/R. Immunoreactivity to HIF-1α, VEGF-A, NOX2, and iNOS was enhanced in retinal arteriole endothelium after I/R. Moreover, I/R evoked a substantial decrease in Brn3a-positive retinal ganglion cells and noticeable retinal thickening. In conclusion, the results of the present study demonstrate that short-time ocular ischemia impairs endothelial function and integrity of retinal blood vessels and induces structural changes in the retina. HIF-1α, VEGF-A, iNOS, and NOX2-derived reactive oxygen species appear to be involved in the pathophysiology.

Crab sauce is a traditional umami seasoning in the coastal cities in South East China. The putative non-volatile taste-active components in crab sauce were measured, and their impacts on the taste were evaluated on the basis of the taste activity value (TAV), omission test, addition test and equivalent umami concentration (EUC). The EUC used to evaluate the synergistic effect of the flavor nucleotides and umami amino acids was 19.3 g monosodium glutamate (MSG)/100 mL, which illuminated that crab sauce had a very intense umami taste. The key non-volatile taste-active components in crab sauce demonstrated by the omission test and addition test were glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), alanine (Ala), lysine (Lys), histidine (His), 5′-inosine monophosphate (IMP), 5′-guanosine monophosphate (GMP), NaCl, KCl, serine (Ser) and valine (Val), and most of these components also had a higher TAV. So, the TAV could be a high-efficiency tool to predict the taste-active components, and the TAV combined with the omission test and addition test could be a very useful method to determine the taste-active components in crab sauce.

Resveratrol increases the production of nitric oxide (NO) in endothelial cells by upregulating the expression of endothelial NO synthase (eNOS), stimulating eNOS enzymatic activity, and preventing eNOS uncoupling. At the same time, resveratrol inhibits the synthesis of endothelin-1 and reduces oxidative stress in both endothelial cells and smooth muscle cells. Pathological stimuli-induced smooth muscle cell proliferation, vascular remodeling, and arterial stiffness can be ameliorated by resveratrol as well. In addition, resveratrol also modulates immune cell function, inhibition of immune cell infiltration into the vascular wall, and improves the function of perivascular adipose tissue. All these mechanisms contribute to the protective effects of resveratrol on vascular function and blood pressure in vivo. Sirtuin 1, AMP-activated protein kinase, and estrogen receptors represent the major molecules mediating the vascular effects of resveratrol.

The therapeutic value of inhibiting translation of the amyloid precursor protein (APP) offers the possibility to reduce neurotoxic amyloid formation, particularly in cases of familial Alzheimer’s disease (AD) caused by APP gene duplications (Dup⁻APP) and in aging Down syndrome individuals. APP mRNA translation inhibitors such as the anticholinesterase phenserine, and high throughput screened molecules, selectively inhibited the uniquely folded iron-response element (IRE) sequences in the 5’untranslated region (5’UTR) of APP mRNA and this class of drug continues to be tested in a clinical trial as an anti-amyloid treatment for AD. By contrast, in younger age groups, APP expression is not associated with amyloidosis, instead it acts solely as a neuroprotectant while facilitating cellular ferroportin-dependent iron efflux. We have reported that the environmental metallotoxins Lead (Pb) and manganese (Mn) cause neuronal death by interfering with IRE dependent translation of APP and ferritin. The loss of these iron homeostatic neuroprotectants thereby caused an embargo of iron (Fe) export from neurons as associated with excess unstored intracellular iron and the formation of toxic reactive oxidative species (ROS). We propose that APP 5’UTR directed translation activators can be employed therapeutically to protect neurons exposed to high acute Pb and/or Mn exposure. Certainly, high potency APP translation activators, exemplified by the Food and Drug Administration (FDA) pre-approved M1 muscarinic agonist AF102B and high throughput-screened APP 5’UTR translation activators, are available for drug development to treat acute toxicity caused by Pb/Mn exposure to neurons. We conclude that APP translation activators can be predicted to prevent acute metal toxicity to neurons by a mechanism related to the 5’UTR specific yohimbine which binds and targets the canonical IRE RNA stem loop as an H-ferritin translation activator.

Colorimetric immunoassays for tumor marker detection have attracted considerable attention due to their simplicity and high efficiency. With the achievements of nanotechnology and nanoscience, nanomaterials-based colorimetric immunoassays have been demonstrated to be promising alternatives to conventional colorimetric enzyme-linked immunoassays. This review is focused on the progress in colorimetric immunoassays with the signal amplification of nanomaterials, including nanomaterials-based artificial enzymes to catalyze the chromogenic reactions, analyte-induced aggregation or size/morphology change of nanomaterials, nanomaterials as the carriers for loading enzyme labels, and chromogenic reactions induced by the constituent elements released from nanomaterials.

The Global Burden of Disease Study identified cardiovascular risk factors as leading causes of global deaths and life years lost. Endothelial dysfunction represents a pathomechanism that is associated with most of these risk factors and stressors, and represents an early (subclinical) marker/predictor of atherosclerosis. Oxidative stress is a trigger of endothelial dysfunction and it is a hall-mark of cardiovascular diseases and of the risk factors/stressors that are responsible for their initiation. Endothelial function is largely based on endothelial nitric oxide synthase (eNOS) function and activity. Likewise, oxidative stress can lead to the loss of eNOS activity or even “uncoupling” of the enzyme by adverse regulation of well-defined “redox switches” in eNOS itself or up-/down-stream signaling molecules. Of note, not only eNOS function and activity in the endothelium are essential for vascular integrity and homeostasis, but also eNOS in perivascular adipose tissue plays an important role for these processes. Accordingly, eNOS protein represents an attractive therapeutic target that, so far, was not pharmacologically exploited. With our present work, we want to provide an overview on recent advances and future therapeutic strategies that could be used to target eNOS activity and function in cardiovascular (and other) diseases, including life style changes and epigenetic modulations. We highlight the redox-regulatory mechanisms in eNOS function and up- and down-stream signaling pathways (e.g., tetrahydrobiopterin metabolism and soluble guanylyl cyclase/cGMP pathway) and their potential pharmacological exploitation.

In this work, we designed a visual biosensor for dopamine (DA) detection using magnetic Fe3O4 particles and dithiobis(sulfosuccinimidylpropionate)-modified gold nanoparticles (DTSSP-AuNPs) as the recognition elements. Specifically, DA molecules were assembled onto the surface of DTSSP-AuNPs via the amine coupling reaction between the amino group of DA and activated carboxyl group of DTSSP. Accordingly, DA-anchored DTSSP-AuNPs were captured by Fe3O4 through the interaction of catechol and iron. In a magnetic field, the formed Fe3O4-DA-DTSSP-AuNPs conjugates were easily removed from the solution, leading to fading of the AuNPs suspension and decrease of the UV/Vis signal. As a result, a detection limit of 10 nM for DA was achieved. The theoretical simplicity and high selectivity demonstrated that the sandwich-type strategy based on Fe3O4 and AuNPs would lead to many colorimetric detection applications in clinical study by rationally designing the surface chemistry of AuNPs and Fe3O4.

Glycoproteins influence a broad spectrum of biological processes including cell-cell interaction, host-pathogen interaction, or protection of proteins against proteolytic degradation. The analysis of their glyco-structures and concentration levels are increasingly important in diagnosis and proteomics. Boronic acids can covalently react with cis-diols in the oligosaccharide chains of glycoproteins to form five- or six-membered cyclic esters. Based on this interaction, boronic acid-based ligands and materials have attracted much attention in both chemistry and biology as the recognition motif for enrichment and chemo/biosensing of glycoproteins in recent years. In this work, we reviewed the progress in the separation, immobilization and detection of glycoproteins with boronic acid-functionalized materials and addressed its application in sensing.

The present study was aimed at to demonstrate the antitumor effects of syringin in HeLa human cervical cancer cells. Its effects on apoptosis, cell cycle phase distribution as well as on cell migration were also examined. The effect on cell proliferation was evaluated by MTT assay, while as effects on colony formation were assessed using clonogenic assay. Syringin inhibited cancer cell growth in HeLa cells in a time-dependent as well as in a concentration-dependent manner. Syringin also led to inhibition of colony formation efficacy with complete suppression at 100 µM drug dose. Syringin could induce G2/M cell cycle arrest along with slight sub-G1 cell cycle arrest. HeLa cells began to emit red fluorescence as the dose of syringin increased from 0 µM in vehicle control to 100 µM. Syringin also inhibited cell migration in a dose-dependent manner with 100 µM dose of syringin leading to 100% inhibition of cell migration. Video Clip of Methodology: Clonogenic assay: 4 min 21 sec Full Screen Alternative

Autoantibodies have been widely used as markers of latent autoimmune diabetes in adults (LADA); however, the specificity and sensitivity of autoantibodies as markers of LADA are weak compared with those found in type 1 diabetes (T1DM). In this study, we aimed to identify other plasma proteins as potential candidates that can be used effectively to determine early stage LADA and type 2 diabetes (T2DM) to facilitate early diagnosis and treatment. These issues were addressed by studying new-onset 'classic' T1DM (n = 156), LADA (n = 174), T2DM (n = 195) and healthy cohorts (n = 166). Plasma samples were obtained from the four cohorts. We employed isobaric tag for relative and absolute quantitation (iTRAQ) together with liquid chromatography tandem mass spectrometry (LC-MS) to identify plasma proteins with significant changes in LADA. The changes were validated by Western blot and ELISA analyses. Among the four cohorts, 311 unique proteins were identified in three iTRAQ runs, with 157 present across the three data sets. Among them, 49/311 (16.0%) proteins had significant changes in LADA compared with normal controls, including glycoprotein phospholipase D (GPLD1), which was upregulated in LADA. Western blot and ELISA analyses showed that GPLD1 levels were higher in both LADA and T1DM cohorts than in both T2DM and healthy cohorts, while there were no significant differences in the plasma concentrations of GPLD1 between the LADA and T1DM cohorts. GPLD1 is implicated as a potential candidate plasma protein for determining early stage LADA and T2DM.

We report a simple, fast and selective colorimetric assay of adenosine triphosphate (ATP) using unmodified gold nanoparticles (AuNPs) as probes and metal ions as cross-linkers. ATP can be assembled onto the surface of AuNPs through interaction between the electron-rich nitrogen atoms and the electron-deficient surface of AuNPs. Accordingly, Cu2+ ions induce a change in the color and UV/Vis absorbance of AuNPs by coordinating to the triphosphate groups and a ring nitrogen of ATP. A detection limit of 50 nM was achieved, which is comparable to or lower than that achievable by the currently used electrochemical, spectroscopic or chromatographic methods. The theoretical simplicity and high selectivity reported herein demonstrated that AuNPs-based colorimetric assay could be applied in a wide variety of fields by rationally designing the surface chemistry of AuNPs. In addition, our results indicate that ATP-modified AuNPs are less stable in Cu2+, Cd2+ or Zn2+-containing solutions due to the formation of the corresponding dimeric metal-ATP complexes.

Patients suffering from chronic inflammatory diseases have an increased mortality risk resulting from cardiovascular disorders due to enhanced atherosclerotic and thrombotic events. Until now, it is not completely understood in which way an abnormal expression of pro-inflammatory mediators contributes to this elevated cardiovascular risk, but there is a need for new drugs that on the one hand suppress the expression of pro-inflammatory mediators and on the other hand inhibit arterial platelet adhesion. Thus, we analyzed the anti-inflammatory and anti-thrombotic capacity of the fungal metabolite Galiellalactone in atherosclerosis-prone apolipoprotein E-deficient mice. Treatment of the mice with Galiellalactone lowered the inflammatory expression profile and improved blood clotting times, as well as platelet adhesion to the injured common carotid artery. The results indicate that administration of Galiellalactone is able to reduce the extent of inflammation and arterial platelet adhesion in this mouse model.

The cardiovascular safety of inhaled long-acting β2-agonists (LABAs) in patients with chronic obstructive pulmonary disease (COPD) is a controversial problem. Certain studies have suggested that inhaled LABAs lead to an increased risk of cardiovascular events in patients with COPD. This meta-analysis aimed to assess the cardiovascular safety of inhaled LABAs in COPD.A meta-analysis of randomized, double-blind, parallel-group, placebo-controlled trials for LABA treatment of COPD with at least 3 months of follow-up was performed. The fixed-effects model was used to evaluate the effects of LABAs on fatal cardiovascular adverse events. Adverse events were collected for each trial, and the relative risk (RR) and 95% confidence intervals (CI) for LABA/placebo were estimated.There were 24 trials included in this meta-analysis. Compared with placebo, inhaled LABAs significantly decreased fatal cardiovascular adverse events in COPD patients (RR 0.65, 95% CI 0.50 to 0.86, P = 0.002). In sensitivity analysis, there was still no increased risk of fatal cardiovascular events (RR 0.68, 95%CI 0.46 to 1.01, P = 0.06) after excluding the trial with the largest weight. Among the different types of LABAs, only salmeterol had a significant effect (RR 0.64, 95% CI 0.46 to 0.90). In subgroup analyses, inhaled LABAs were able to significantly decrease fatal cardiovascular events in long-term trials (RR 0.64, 95% CI 0.47 to 0.87) and in trials with severe COPD patients (RR 0.69, 95% CI 0.50 to 0.96).Inhaled LABAs do not increase the risk of fatal cardiovascular events in COPD patients.

We presented the colorimetric and electrochemical methods for determination of the dipeptidyl peptidase-IV (DPP-IV) activity and screening of its inhibitor using gold nanoparticle (AuNP) as the probe. In the colorimetric assay, the substrate peptide with a sequence of Arg-Pro-Arg induced the aggregation and color change of AuNPs, whereas cleavage of the peptide by DPP-IV prevented the aggregation of AuNPs. Furthermore, the aggregation of AuNPs in the solution was easily initiated on a solid/liquid (electrode/electrolyte) surface, which induced a decrease in the electron-transfer resistance. However, once the peptide was clipped by DPP-IV, the assembly of AuNPs on electrode surface was prevented. Consequently, a higher electron-transfer resistance was observed. The colorimetric and electrochemical assays allowed for the determination of DPP-IV with the detection limits of 70 μU/mL and 0.55 μU/mL, respectively. Meanwhile, the proposed methods were used to determine DPP-IV inhibitor with satisfactory results. Both the colorimetric and electrochemical methods are simple, rapid and sufficiently sensitive for DPP-IV activity assay and inhibitor screening. The results also demonstrated that the AuNP-based colorimetric assay could be converted into an enhanced surface tethered electrochemical assay with improving sensitivity. The simple detection principle may be extended to the design of other peptidases biosensors with easy manipulation procedures.

Human chorionic gonadotropin (hCG) has been regarded as a biomarker for the diagnosis of pregnancy and some cancers. Because the currently used methods (e.g., disposable Point of Care Testing (POCT) device) for hCG detection require the use of many less stable antibodies, simple and cost-effective methods for the sensitive and selective detection of hCG have always been desired. In this work, we have developed a graphene oxide (GO)-based fluorescent platform for the detection of hCG using a fluorescein isothiocyanate (FITC)-labeled hCG-specific binding peptide aptamer (denoted as FITC-PPLRINRHILTR) as the probe, which can be manufactured cheaply and consistently. Specifically, FITC-PPLRINRHILTR adsorbed onto the surface of GO via electrostatic interaction showed a poor fluorescence signal. The specific binding of hCG to FITC-PPLRINRHILTR resulted in the release of the peptide from the GO surface. As a result, an enhanced fluorescence signal was observed. The fluorescence intensity was directly proportional to the hCG concentration in the range of 0.05–20 IU/mL. The detection limit was found to be 20 mIU/mL. The amenability of the strategy to hCG analysis in biological fluids was demonstrated by assaying hCG in the urine samples.

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with anti-inflammatory, antiviral and anti-cancer properties. Beneficial cardiovascular effects such as increased nitric oxide (NO) production through enhancement of endothelial NO synthase (eNOS) activity and upregulation of eNOS expression have been demonstrated for this compound. In the present study, immortalized human EA.hy 926 endothelial cells were incubated for up to 1 h with 1–100 µM BA and with the phosphatidylinositol-3-kinase (PI3K) inhibitors LY294002 and wortmannin, or the estrogen receptor (ER) antagonist ICI 182,780. Phosphorylation status of eNOS and total eNOS protein were analyzed by Western blotting using a serine 1177 phosphosite-specific antibody. Bioactive NO production was assessed by determination of cGMP content in rat lung fibroblasts (RFL-6) reporter cells. Short-term incubation of EA.hy 926 cells with BA resulted in eNOS phosphorylation at the serine 1177 residue in a concentration- and time-dependent manner with a half-maximal effective concentration of 0.57 µM. This was associated with an enhanced production of NO. BA-induced eNOS phosphorylation and NO production was completely blocked by pretreatment with ICI 182,780, and was attenuated by pretreatment with the PI3K inhibitors wortmannin and LY294002. These results indicate that fast non-genomic effects of ER with downstream signaling through the PI3K/Akt pathway and consecutive eNOS phosphorylation at serine 1177 are involved in BA-induced eNOS activation.

We presented a strategy for fabricating graphene oxide (GO)-based fluorescent biosensors to monitor the change of phosphorylation state and detect phosphatase activity. By regulating the interaction between the negatively charged phosphate group and the positively charged amino residue, we found that GO showed different quenching efficiency toward the phosphorylated and dephosphorylated dye-labeled peptides. To demonstrate the application of our method, alkaline phosphatase (ALP) was tested as a model enzyme with phosphorylated fluorescein isothiocyanate (FITC)-labeled short peptide FITC–Gly–Gly–Gly–Tyr(PO32−)–Arg as the probe. When the negatively charged phosphate group in the Tyr residue was removed from the peptide substrate by enzymatic hydrolysis, the resulting FITC–Gly–Gly–Gly–Tyr–Arg was readily adsorbed onto the GO surface through electrostatic interaction. As a result, fluorescence quenching was observed. Furthermore, the method was applied for the screening of phosphatase inhibitors.

Liver fibrosis is a worldwide clinical issue. It has been well established that activated hepatic stellate cells (HSCs) are responsible for excessive extracellular matrix (ECM) deposition in chronically damaged livers. The identification of key elements that control HSCs activation will help to further our understanding of liver fibrosis and improve the outcome of clinical treatment. This study demonstrates that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of liver fibrosis as NDRG2 mRNA and protein levels were reduced during HSCs activation. In addition, enhanced NDRG2 expression reduced Smad3 transcription and phosphorylation, which inhibited HSCs activation by blocking the TGF-β1 signal. Moreover, NDRG2 contributed to an increase in the ratio of matrix metalloproteinase 2 (MMP2) to tissue inhibitor of matrix metalloproteinase 2 (TIMP2), which may facilitate the degradation of the ECM. In dimethylnitrosamine (DMN)-induced fibrotic rat livers, adenovirus-mediated NDRG2 overexpression resulted in decreased ECM deposition and improved liver function compared with controls. In conclusion, the present findings indicate that the modulation of NDRG2 is a promising strategy for the treatment of liver fibrosis.