KCNN3 SNP rs13376333 on Chromosome 1q21 Confers Increased Risk of Atrial Fibrillation.

To investigate the relationship between KCNN3 SNP (single-nucleotide polymorphism) rs13376333 and risk of atrial fibrillation (AF) and to provide evidence for prevention and treatment for AF.The PubMed, Embase, OVID, Cochrane library, CNKI, and Wan Fang databases were searched to identify studies on the relationship between KCNN3 SNP rs13376333 polymorphism and atrial fibrillation. Two authors performed independent article reviews and study quality assessment using the Newcastle-Ottawa Scale (NOS) checklist.Seven studies involving 24,339 individuals were included in the meta-analysis. The overall combined OR of rs13376333 polymorphism was observed for both lone AF (OR: 1.58 [95%CI: 1.37 to 1.82]; P < 0.001; I(2) = 47.0%) and total AF (OR: 1.33 [95%CI: 1.14 to 1.54]; P < 0.001; I(2) = 0). Further, when stratified by ethnicity, control sources, sample sizes, and genotyping method, similar results were observed in both subgroups. Sensitivity analysis revealed that the source of control was the source of the heterogeneity for lone AF. Omission of any single study had little effect on the combined risk estimate. No evidence of publication bias was found.This meta-analysis suggests that KCNN3 SNP rs13376333 polymorphism significantly increases the risk of lone AF and total AF, which suggests the rs13376333 polymorphism of the KCNN3 gene may play an important role in the pathogenesis of AF.

Neuroprotective effects of saffron on the late cerebral ischemia injury through inhibiting astrogliosis and glial scar formation in rats.

This study is to explore the neuroprotective effects and involved glial scar of saffron (Crocus sativus L.) on the late cerebral ischemia in rats. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in Sprague Dawley rats that were randomly divided into sham group, MCAO group, edaravone group (as a positive control) and saffron groups (saffron extract 30, 100, 300 mg/kg). Saffron was administered orally at 2 h at the first day and once daily from day 2 to 42 after ischemia. Behavioral changes were detected from day 43 to 46 after ischemia to evaluate the effects of saffron. Infarct volume, survival neuron density, activated astrocyte, and the thickness of glial scar were also detected. GFAP, neurocan, phosphocan, neurofilament expressions and inflammatory cytokine contents were detected by Western-blotting and ELISA methods, respectively. Saffron improved the body weight loss, neurological deficit and spontaneous activity. It also ameliorated anxiety-like state and cognitive dysfunction, which were detected by elevated plus maze (EPM), marble burying test (MBT) and novel object recognition test (NORT). Toluidine blue staining found that saffron treatment decreased the infarct volume and increased the neuron density in cortex in the ischemic boundary zone. The activated astrocyte number and the thickness of glial scar in the penumbra zone reduced after saffron treatment. Additionally, saffron decreased the contents of IL-6 and IL-1ß, increased the content of IL-10 in the ischemic boundary zone. GFAP, neurocan, and phosphocan expressions in ischemic boundary zone and ischemic core zone all decreased after saffron treatment. Saffron exerted neuroprotective effects on late cerebral ischemia, associating with attenuating astrogliosis and glial scar formation after ischemic injury.

Inter- and intra-observer variability for the assessment of coronary artery tree description and lesion EvaluaTion (CatLet©) angiographic scoring system in patients with acute myocardial infarction.

BACKGROUND: Previously, we developed a novel Coronary Artery Tree description and Lesion EvaluaTion (CatLet©) angiographic scoring system, which was capable of accounting for the variability in the coronary anatomy and assisting in the risk-stratification of patients with acute myocardial infarction (AMI). Our preliminary study revealed that the CatLet score better predicted clinical outcomes for AMI patients than the Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery score. However, the reproducibility of the CatLet score in both inter- and intra-observer remains to be evaluated. METHODS: A total of 30 consecutive AMI patients, admitted in September of 2015, were independently assessed by two experienced interventional cardiologists to evaluate the inter-observer reproducibility of the CatLet score. Another set of 49 consecutive AMI patients, admitted between September and October in 2014, were assessed by one of the two interventional cardiologists on two occasions 3 months apart to evaluate the intra-observer reproducibility of the CatLet score. The weighted kappa was used to express the degree of agreement. RESULTS: The weighted kappa values (95% confidence interval) for the intra- and inter-observer reproducibility of the CatLet Score were 0.82 (0.59-1.00, Z = 7.23, P < 0.001) and 0.86 (0.54-1.00, Z = 5.20, P < 0.001), respectively, according to the tertile analysis (≤14, 15-22, >22). Regarding the adverse characteristics pertinent to lesions and dominance parameters, the kappa values for the inter-observer variability were 0.80 (0.56-1.00, Z = 6.47, P < 0.001) for total number of lesions, 0.57 (0.28-0.85, Z = 3.03, P < 0.001) for bifurcation, 0.69 (0.43-0.96, Z = 5.06, P < 0.001) for heavy calcification, 1.00 (0.72-1.00, Z = 6.93, P < 0.001) for tortuosity, 0.54 (0.26-0.82, Z = 3.78, P < 0.001) for thrombus, 0.69 (0.48-0.91, Z = 6.29, P < 0.001) for right coronary artery dominance, 0.69 (0.41-0.96, Z = 4.91, P < 0.001) for left anterior descending artery length, and 0.22 (0.06-0.51, Z = 1.56, P = 0.06) for diagonal size. Equivalent values for the intra-observer variability were moderate to almost perfect (range 0.54-1.00). CONCLUSIONS: The reproducibility of the CatLet angiographic scoring system for evaluation of the coronary angiograms ranged from substantial to excellent. The high reproducibility of the CatLet angiographic scoring system will boost its clinical application to patients with AMI.

Kinetic assay for characterization of spleen tyrosine kinase activity and inhibition with recombinant kinase and crude cell lysates.

Spleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells. The assay is based on the chelation-enhanced fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred to as Sox), which has been incorporated into a peptide substrate selected for robust detection of Syk activity. This homogeneous assay is simple to use, provides considerably more information, and has been adapted to a 384-well, low-volume microtiter plate format that can be used for the high-throughput identification and kinetic characterization of Syk inhibitors. The assay can be performed with a wide range of adenosine triphosphate (ATP) concentrations and, therefore, can be used to analyze ATP-competitive and ATP-noncompetitive/allosteric kinase inhibitors. Measurement of Syk activity in RAMOS crude cell lysates or immunoprecipitation (IP) capture formats may serve as a physiologically more relevant enzyme source. These Sox-based continuous and homogeneous assays provide a valuable set of tools for studying Syk signaling and for defining inhibitors that may be more effective in controlling disease.

A comprehensive analysis and comparison between vacuum and electric oven drying methods on Chinese saffron (Crocus sativus L.).

The red stigmas of saffron are one of the most expensive spices in the world and serve as a traditional Chinese medicine. More saffron has been cultivated in China, and different drying technologies have been studied. However, a comprehensive and comparative analysis of different drying approaches has not been well studied. In this study, we compared electric oven and vacuum oven drying approaches on saffron. We found saffron was dried quicker under high vacuum drying mode with high temperature and the quicker drying rate provided, the more open microstructural interstices on the saffron surface. Both methods were best fit to Midilli and Kucuk model. Besides, the coloring, aroma and bitterness strength after drying showed the similar results. In sum, our data suggested the optimal drying temperature was 100 °C for 20 min for two evaluated methods, however considering the machine cost, the electric oven drying would be the first choice.

Crocin induces autophagic apoptosis in hepatocellular carcinoma by inhibiting Akt/mTOR activity.

BACKGROUND: Autophagy induction is a common mechanism for antitumor chemicals in induction of cancer cell death. However, the role of autophagy in crocin-induced apoptosis is barely studied in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The influence of crocin on growth, apoptosis, and autophagy and its mutual relations were analyzed by Cell Counting Kit-8 assay, flow cytometer, EGFP-LC3 puncta analysis, and Western blot in HCC cells. The activities of Akt/mTOR axis and its roles in autophagy regulation were also detected by Western blot in HCC cells treated with crocin. Finally, the roles of Akt/mTOR axis in crocin-induced autophagic apoptosis were analyzed by Western blot and flow cytometer in HCC cells. RESULTS: The results showed that crocin can induce growth inhibition in a does- and time-dependent pattern by apoptosis. Increased LC3 puncta and upregulated LC3-II accumulation was observed as early as at 6 hours in HepG2 and HCCLM3 cells treated with 3 mg/mL crocin. Moreover, apoptosis analysis using flow cytometer and cleaved poly (ADP-ribose) polymerase detection revealed that autophagy initiation was prior to apoptosis activation in HCC cells treated with crocin. When autophagy was blocked with 3-methyladenine, crocin-induced apoptosis was inhibited in HCC cells. Furthermore, crocin treatment constrained the activities of key proteins in Akt/mTOR signaling, such as p-Akt (S473), p-mTOR (S2448), and p-p70S6K (T389), suggesting that crocin could induce autophagic apoptosis in HCC cells in an Akt/mTOR-dependent mechanism. Indeed, when autophagy was suppressed by forced expression of Akt, the crocin-induced apoptosis was also impaired in HCC cells. CONCLUSION: The results suggested that crocin could induce autophagic apoptosis in HCC cells by inhibiting Akt/mTOR activity. This study originally revealed that autophagic apoptosis is a novel cytotoxic function of crocin, which lays the theoretical foundation for clinical application of crocin in HCC.

Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury.

Traumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects against ischemic brain injuries with anti-oxidant and anti-inflammatory properties, and by scavenging reactive species. However, whether caffeic acid has protective effects against traumatic brain injury is unknown. Therefore, we determined the effect of caffeic acid on the lesion in the early (1 day) and late phases (7 to 28 days) of cryoinjury in mice. We found that caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days, but attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and glial fibrillary acidic protein (GFAP) protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduction of superoxide dismutase activity and the increase in malondialdehyde content in the brain 1 day after cryoinjury. These results indicate that caffeic acid exerts a protective effect in traumatic brain injury, especially on glial scar formation in the late phase, which at least is associated with its anti-oxidant ability.

Pranlukast, a cysteinyl leukotriene receptor 1 antagonist, protects mice against brain cold injury.

We have reported the neuroprotective effect of cysteinyl leukotriene receptor 1 (CysLT1) antagonists on cerebral ischemia. Here, we further determined the protective effect of pranlukast, a CysLT1 receptor antagonist, on brain cold injury in mice. Brains were injured by placing a cooled metal probe on the skull surface for 30 s. We found that pranlukast significantly reduced cold-induced lesion volume (0.3 mg/kg) and the percentage increase in lesioned hemisphere volume (0.03-0.3 mg/kg) 24 h after injury, but did not show any effect 72 h after injury. Pranlukast also significantly inhibited neuron loss 24 h (0.1 mg/kg) and 72 h (0.1-0.3 mg/kg) after injury, and decreased the density of degenerated neurons 24 h (0.01-0.3 mg/kg) and 72 h (0.03-0.3 mg/kg) after injury. In addition, pranlukast (0.1-0.3 mg/kg) significantly reduced endogenous IgG exudation both 24 h and 72 h after injury. Thus, this study indicates the protective effect of pranlukast on brain cold injury.

[Incomplete protective effects of minocycline on traumatic brain injury in rats and mice].

OBJECTIVE: To evaluate protective effect of minocycline,a semisynthetic tetracycline derivative on different traumatic brain injuries in rats and mice. METHODS: The opened brain trauma was induced in rats and the closed head injury and cold brain injury were induced in mice. In 3 brain trauma models, minocycline (45 mg/kg, ip) was administered twice daily for 2 d before the operation, at 30 min before and 1 h after the operation, and once daily for 2 d following the operation (totally 8 doses in 5 d). After the operation, the behavioral alteration was observed daily, lesion area and survival neuron density were measured at the end of the experiments (14 d after the injuries). RESULT: For rat opened traumatic injury, minocycline promoted the recovery of hindlimb motor activity (inclined board angle), but did not alter other indexes. For mouse closed head traumatic injury, minocycline reduced the neuron loss, but did not improve behavioral dysfunction. For mouse cold injury-induced trauma, minocycline reduced death rate and lesion area, but did not remarkably improve behavior and neuron loss. CONCLUSION: Minocycline only has an incomplete neuroprotective effect on different brain traumatic injuries in rats and mice.

Association between angiotensinogen M235T polymorphism and hypertrophic cardiomyopathy.

BACKGROUND: To investigate the relationship between angiotensinogen (AGT) gene M235T polymorphism and hypertrophic cardiomyopathy (HCM) to explore the potential role of the AGT polymorphism in HCM. METHODS: PubMed, Embase, OVID, Cochrane library, CNKI, Wan Fang Database were searched to identify the studies involving AGT M235T polymorphism and HCM. Two authors performed independent literature review and study quality assessment using the Newcastle-Ottawa Scale (NOS) checklist. A random-effects model was used to calculate the overall combined risk estimates. RESULTS: Nine studies involving 887 cases and 1407 controls were included in our meta-analysis. No significant associations were found between AGT M235T polymorphism and HCM (allele model T vs M: OR = 1.17, 95% CI = 0.95-1.45; dominant model TT vs (MM/MT): OR = 1.21, 95% CI = 1.00-1.45; recessive model (TT/MT) vs MM: OR = 1.12, 95% CI = 0.87-1.45; heterozygous comparison MT vs MM: OR = 1.07, 95% CI = 0.82-1.41; homozygous comparison TT vs MM OR = 1.19, 95% CI = 0.88-1.61. In subgroup analysis, the significant difference of association between AGT M235T polymorphism and HCM existed in Asian and sporadic hypertrophic cardiomyopathy (SHCM), but no significant difference was found in Europeans and familial hypertrophic cardiomyopathy (FHCM). CONCLUSIONS: There is no association between AGT M235T polymorphism and HCM in general populations, but such a relationship exists in Asians and SHCM.

Distinct pro-apoptotic properties of Zhejiang saffron against human lung cancer via a caspase-8-9-3 cascade.

Lung cancer is the leading cause of cancer-related death worldwide. Here we investigated the antitumor effect and mechanism of Zhejiang (Huzhou and Jiande) saffron against lung cancer cell lines, A549 and H446. Using high performance liquid chromatography (HPLC), the contents of crocin I and II were determined. In vitro, MTT assay and annexin-V FITC/PI staining showed cell proliferation activity and apoptosis to be changed in a dose- and time-dependent manner. The inhibition effect of Jiande saffron was the strongest. In vivo, when mice were orally administered saffron extracts at dose of 100mg/kg/d for 28 days, xenograft tumor size was reduced, and ELISA and Western blotting analysis of caspase-3, -8 and -9 exhibited stronger expression and activity than in the control. In summary, saffron from Zhejiang has significant antitumor effects in vitro and in vivo through caspase-8-caspase-9-caspase-3 mediated cell apoptosis. It thus appears to have more potential as a therapeutic agent.

Leukotriene D4 stimulates the migration but not proliferation of endothelial cells mediated by the cysteinyl leukotriene cyslt(1) receptor via the extracellular signal-regulated kinase pathway.

The actions of cysteinyl leukotrienes (CysLTs) are mediated by activating CysLT receptors, CysLT(1), and CysLT(2). The CysLT(1) receptor mediates vascular responses to CysLTs; however, its effect on the proliferation and migration of endothelial cells is not clarified. To determine this effect, we observed proliferation and migration in EA.hy926 cells, a human endothelial cell line, and the involvement of activation of mitogen-activated protein kinases (MAPKs). We found that LTD(4) did not affect the proliferation, but significantly stimulated the migration of endothelial cells. LTD(4) also induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, but not those of p38 or JNK. The LTD(4)-induced migration and ERK1/2 phosphorylation were blocked by the CysLT(1)-receptor antagonist montelukast and the dual antagonist Bay u9773, but not by the CysLT(2)-receptor antagonist Bay cysLT2; the migration was also inhibited by the ERK1/2 inhibitor U0126. Our findings indicate that LTD(4) stimulates the CysLT(1) receptor-mediated migration of endothelial cells; this may be regulated by the ERK1/2 pathway.

Homogenous fluorescent assays for characterizing small-molecule activators of AMP-activated protein kinase (AMPK).

AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the gamma-subunit of the kinase, which increases the activity of the catalytic alpha-subunit. Because increased AMPK activity in the liver and in skeletal muscle leads to increased fatty acid oxidation and decreased cholesterol and fatty acid biosynthesis, activators of AMPK are being sought for treatment of type-2 diabetes and other metabolic disorders. The unique mechanism of AMPK activation offers an opportunity to develop small molecules that directly upregulate AMPK activity, and there exists a need for simplified methods to identify and characterize small-molecules that show isoform-specific effects on AMPK. We have developed a suite of fluorescence-based assays to identify and characterize such compounds, and have used these to characterize and compare activity of recombinant AMPK alpha(1)beta(1)gamma(1) and alpha(2)beta(1)gamma(1) isoforms in response to small molecule activators and inhibitors.