5‐HT1B autoreceptor regulation of serotonin transporter activity in synaptosomes

Serotonin‐1B (5‐HT_1B) autoreceptors are located in serotonin (5‐HT) terminals, along with serotonin transporters (SERT), and play a critical role in autoregulation of serotonergic neurotransmission and are implicated in disorders of serotonergic function, particularly emotional regulation. SERT modulates serotonergic neurotransmission by high‐affinity reuptake of 5‐HT. Alterations in SERT activity are associated with increased risk for depression and anxiety. Several neurotransmitter receptors are known to regulate SERT K_m and V_max, and previous work suggests that 5‐HT_1B autoreceptors may regulate 5‐HT reuptake, in addition to modulating 5‐HT release and synthesis. We used rotating disk electrode voltammetry to investigate 5‐HT_1B autoreceptor regulation of SERT‐mediated 5‐HT uptake into synaptosomes. The selective 5‐HT_1B antagonist SB224289 decreased SERT activity in synaptosomes prepared from wild‐type but not 5‐HT_1B knockout mice, whereas SERT uptake was enhanced after pretreatment with the selective 5‐HT_1B agonist CP94253. Furthermore, SERT activity varies as a function of 5‐HT_1B receptor expression—specifically, genetic deletion of 5‐HT_1B decreased SERT function, while viral‐mediated overexpression of 5‐HT_1B autoreceptors in rat raphe neurons increased SERT activity in rat hippocampal synaptosomes. Considered collectively, these results provide evidence that 5‐HT_1B autoreceptors regulate SERT activity. Because SERT clearance rate varies as a function of 5‐HT_1B autoreceptor expression levels and is modulated by both activation and inhibition of 5‐HT_1B autoreceptors, this dynamic interaction may be an important mechanism of serotonin autoregulation with therapeutic implications. Synapse, 2012. © 2012 Wiley Periodicals, Inc.     

苦参煎与SB203580对p38MAPK信号传导通路的调控影响

目的:探讨苦参煎与SB203580对p38MAPK信号传导通路的调控影响。利用苦参煎与SB203580抑制p38 MAPK信号传导通路,来证明中药对基因转录表达具有一定的调控作用。方法:模拟血管内膜损伤术,采用ELISA方法检测188只Webster大鼠血清中P38MAPK的水平含量。将其随机平均分为正常对照组、假手术组、生理盐水组、苦参煎组和SB203580组。结果与结论:中药苦参煎有着与SB203580相同的功效,即对P38MAPK的显著抑制作用。所以对血管内皮有比较明确的保护作用,能干扰MAPK信号传导通路,既能减轻内皮损伤后的炎性细胞释放反应,也能使再生的内皮某些功能得到恢复,有利于内皮细胞结构和功能恢复,防止炎性因子对细胞的损害。     

Small-molecule GSK-3 inhibitor rescued apoptosis and neurodegeneration in anesthetics-injured dorsal root ganglion neurons

BackgroundApplication of general anesthetics may induce neurotoxicity in dorsal root ganglia (DRG) neurons. In this study, we examined the possible protective mechanism and associated signaling pathways of small-molecule glycogen synthase kinase-3 (GSK-3) inhibitor, SB216763, in bupivacaine-injured mouse DRG neurons in vitro.MethodsIn vitro DRG explant of 6-week old mice was treated with 5 mM bupivacaine to induce neurotoxicity. The explants were also pre-treated with SB216763 for 72 h. Neural protection of SB216763 on bupivacaine-injured DRG neurons was investigated by TUNEL assay, neurite outgrowth assay and western blot assay, respectively. Possible downstream gene of GSK-3 signaling pathway, protein kinase C (PKC) was knocked down by siRNA in DRG explant. Its function in regulating GSK-3 inhibition induced DRG neural protection was also examined by TUNEL, neurite outgrowth and western blot assays.ResultsPre-treatment of SB216763 significantly ameliorated bupivacaine induced apoptosis and neurite loss in DRG neurons. Western blot showed that, in addition to the decrease of phosphorylated-GSK-3 α/β protein, SB216763 increased PKC and decreased caspase-3 (Casp-3) in bupivacaine-injured DRG neurons. SiRNA-mediated PKC knockdown was able to reverse the neural protection of SB216763 in bupivacaine-injured DRG neurons. Western blot showed that PKC knockdown increased phosphorylated-GSK-3 α/β and Casp-3 protein in DRG neurons, confirming that PKC was directly involved in GSK-3-inhibition induced neural protection in DRG.ConclusionsGSK-3 inhibitor SB216763, through PKC, is effective in protecting anesthetics-induced neurotoxicity in DRG.     

Protein folding kinetics and thermodynamics from atomistic simulation

Advances in simulation techniques and computing hardware have created a substantial overlap between the timescales accessible to atomic-level simulations and those on which the fastest-folding proteins fold. Here we demonstrate, using simulations of four variants of the human villin headpiece, how simulations of spontaneous folding and unfolding can provide direct access to thermodynamic and kinetic quantities such as folding rates, free energies, folding enthalpies, heat capacities, Φ-values, and temperature-jump relaxation profiles. The quantitative comparison of simulation results with various forms of experimental data probing different aspects of the folding process can facilitate robust assessment of the accuracy of the calculations while providing a detailed structural interpretation for the experimental observations. In the example studied here, the analysis of folding rates, Φ-values, and folding pathways provides support for the notion that a norleucine double mutant of villin folds five times faster than the wild-type sequence, but following a slightly different pathway. This work showcases how computer simulation has now developed into a mature tool for the quantitative computational study of protein folding and dynamics that can provide a valuable complement to experimental techniques.     

Learning curve for endoscopic submucosal dissection of early colorectal neoplasms with a monopolar scissor-type knife: use of the cumulative sum method

Abstract

Objective

Although scissor-type knives such as the Stag-Beetle (SB) Knife Jr are expected to result in a safe and easy colorectal endoscopic submucosal dissection (CR-ESD), information regarding the learning curve is lacking. Therefore, this study evaluated the learning curve with using SB Knife Jr.     

3-Year Outcomes of the DKCRUSH-V Trial Comparing DK Crush With Provisional Stenting for Left Main Bifurcation Lesions

ObjectivesThe present study aimed to investigate the difference in target lesion failure (TLF) at 3 years after double kissing (DK) crush stenting versus provisional stenting (PS) for unprotected left main distal bifurcation (UPLMb) lesions.BackgroundThe multicenter and randomized DKCRUSH-V (Double Kissing Crush versus Provisional Stenting for Left Main Distal Bifurcation Lesions: The DKCRUSH-V Randomized Trial) study showed fewer 1-year TLF after DK crush for UPLMb lesions compared with PS. The study reports the 3-year clinical outcome of the DKCRUSH-V study.MethodsA total of 482 patients with UPLMb lesions who were randomly assigned to either the DK crush group (DK group) or PS group in the DKCRUSH-V study were followed for 3 years. The primary endpoint was the occurrence of a TLF at 3 years. Stent thrombosis (ST) was the safety endpoint. Patients were classified by lesion’s complexity and NERS (New Risk Stratification) II or SYNTAX (Synergy Between Percutaneous Coronary Intervention With TAXUS and Cardiac Surgery) score.ResultsAt 3 years, TLF occurred in 41 (16.9%) patients in the PS group and in 20 (8.3%) patients in the DK group (p = 0.005), mainly driven by increased target vessel myocardial infarction (5.8% vs. 1.7%; p = 0.017) and target lesion revascularization (10.3% vs. 5.0%; p = 0.029). Definite or probable ST rate at 3 years was 4.1% in the PS group and 0.4% in the DK group (p = 0.006). Notably, DK crush was associated with a significant reduction in both primary and secondary endpoints for patients with complex lesions or at high risk.ConclusionsProvisional stenting for UPLMb lesions was associated with significantly increased rates of TLF and ST over 3 years of follow-up. Further randomized study is warranted to confirm the benefits of DK crush stenting for complex UPLMb lesions. (Double Kissing and Double Crush versus Provisional T Stenting Technique for the Treatment of Unprotected Distal Left Main True Bifurcation Lesions: A Randomized, International, Multi-center Clinical Trial; ChiCTR-TRC-11001213).     

Fractional Flow Reserve and Coronary Bifurcation Anatomy: A Novel Quantitative Model to Assess and Report the Stenosis Severity of Bifurcation Lesions

ObjectivesThe aim of this study was to develop a new model for assessment of stenosis severity in a bifurcation lesion including its core. The diagnostic performance of this model, powered by 3-dimensional quantitative coronary angiography to predict the functional significance of obstructive bifurcation stenoses, was evaluated using fractional flow reserve (FFR) as the reference standard.BackgroundDevelopment of advanced quantitative models might help to establish a relationship between bifurcation anatomy and FFR.MethodsPatients who had undergone coronary angiography and interventions in 5 European cardiology centers were randomly selected and analyzed. Different bifurcation fractal laws, including Murray, Finet, and HK laws, were implemented in the bifurcation model, resulting in different degrees of stenosis severity.ResultsA total of 78 bifurcation lesions in 73 patients were analyzed. In 51 (65%) bifurcations, FFR was measured in the main vessel. A total of 34 (43.6%) interrogated vessels had an FFR ≤0.80. Correlation between FFR and diameter stenosis was poor by conventional straight analysis (ρ = −0.23, p < 0.001) but significantly improved by bifurcation analyses: the highest by the HK law (ρ = −0.50, p < 0.001), followed by the Finet law (ρ = −0.49, p < 0.001), and the Murray law (ρ = −0.41, p < 0.001). The area under the receiver-operating characteristics curve for predicting FFR ≤0.80 was significantly higher by bifurcation analysis compared with straight analysis: 0.72 (95% confidence interval: 0.61 to 0.82) versus 0.60 (95% confidence interval: 0.49 to 0.71; p = 0.001). Applying a threshold of ≥50% diameter stenosis, as assessed by the bifurcation model, to predict FFR ≤0.80 resulted in 23 true positives, 27 true negatives, 17 false positives, and 11 false negatives.ConclusionsThe new bifurcation model provides a comprehensive assessment of bifurcation anatomy. Compared with straight analysis, identification of lesions with preserved FFR values in obstructive bifurcation stenoses was improved. Nevertheless, accuracy was limited by using solely anatomical parameters.     

Single String Technique for Coronary Bifurcation Stenting: Detailed Technical Evaluation and Feasibility Analysis

ObjectivesThe study aimed to evaluate the adequacy and feasibility of the single string bifurcation stenting technique.BackgroundDouble-stent techniques may be required for complex bifurcations. Currently applied methods all have their morphological or structural limitations with respect to wall coverage, multiple strut layers, and apposition rate.MethodsSingle string is a novel method in which, first, the side branch (SB) stent is deployed with a single stent cell protruding into the main branch (MB). Second, the MB stent is deployed across this protruding stent cell. The procedure is completed by final kissing balloon dilation. The single string technique was first tested in vitro (n = 20) and next applied in patients (n = 11) with complex bifurcation stenoses.ResultsAll procedures were performed successfully, crossing a single stent cell in 100%. Procedure duration was 23.0 ± 7.9 min, and the fluoroscopy time was 9.4 ± 3.5 min. The results were evaluated by optical coherence tomography, showing fully apposed struts in 83.0 ± 9.2% in the bifurcation area. Residual area obstruction in the MB was 6.4 ± 5.6% and 25.0 ± 16.9% in the SB, as evaluated by micro computed tomography. All the human cases were performed successfully with excellent angiographic results: the residual area stenosis was 27 ± 8% and 29 ± 10% in the MB and in the SB, respectively, by 3-dimensional quantitative coronary angiography. No relevant periprocedural enzyme increase was observed. During follow-up (6 ± 4 months), no adverse clinical events (death, myocardial infarction, target vessel revascularization) were noted.ConclusionsThe single string technique for complex bifurcation dilation was shown to be adequate in vitro and feasible in humans, with favorable results in terms of stent overlap, malapposition rate, and low residual obstruction in both the MB and SB.     

The current and future role of magnetic resonance imaging in prostate cancer detection and management

Purpose

Accurate detection of clinically significant prostate cancer (PC) and correct risk attribution are essential to individually counsel men with PC. Multiparametric MRI (mpMRI) facilitates correct localization of index lesions within the prostate and MRI-targeted prostate biopsy (TPB) helps to avoid the shortcomings of conventional biopsy such as false-negative results or underdiagnosis of aggressive PC. In this review we summarize the different sequences of mpMRI, characterize the possibilities of incorporating MRI in the biopsy workflow and outline the performance of targeted and systematic cores in significant cancer detection. Furthermore, we outline the potential of MRI in patients undergoing active surveillance (AS) and in the pre-operative setting.

Materials and methods

An electronic MEDLINE/PubMed search up to February 2015 was performed. English language articles were reviewed for inclusion ability and data were extracted, analyzed and summarized.

Results

Targeted biopsies significantly outperform conventional systematic biopsies in the detection of significant PC and are not inferior when compared to transperineal saturation biopsies. MpMRI can detect index lesions in app. 90% of cases as compared to prostatectomy specimen. The diagnostic performance of biparametric MRI (T2w + DWI) is not inferior to mpMRI, offering options to diminish cost- and time-consumption. Since app 10% of significant lesions are still MRI-invisible, systematic cores seem to be necessary. In-bore biopsy and MRI/TRUS-fusion-guided biopsy tend to be superior techniques compared to cognitive fusion. In AS, mpMRI avoids underdetection of significant PC and confirms low-risk disease accurately. In higher-risk disease, pre-surgical MRI can change the clinically-based surgical plan in up to a third of cases.

Conclusions

mpMRI and targeted biopsies are able to detect significant PC accurately and mitigate insignificant PC detection. As long as the negative predictive value (NPV) is still imperfect, systematic cores should not be omitted for optimal staging of disease. The potential to correctly classify aggressiveness of disease in AS patients and to guide and plan prostatectomy is evolving.     

TNF-α 通过 p38 丝裂原活化蛋白激酶信号通路对小胶质细胞HMGB1 表达的调控作用

目的：观察P38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)抑制剂SB203580对 TNF-α诱导的原代培养大鼠小胶质细胞高迁移率族蛋白B1(high mobility group protein B1,HMGB1)表达的影响。方法： 采用原代培养大鼠小胶质细胞,设立对照组、TNF-α组(25 ng/mL)、TNF-α(25 ng/mL)+SB 203580组(10 μmol/L)、SB 203580组(10 μmol/L),各组细胞经药物处理16 h后分别用ELISA法检测细胞培养上清中HMGB1的表达情况,Western 印迹检测细胞HMGB1和磷酸化p38MAPK表达情况,用反转录PCR检测HMGB1 mRNA表达。结果：经TNF-α刺激 后,大鼠小胶质细胞及其培养上清液中HMGB1蛋白表达增高;SB 203580可抑制TNF-α诱导的磷酸化p38 MAPK表达 (P<0.01),同时下调HMGB1mRNA和HMGB1蛋白的表达。结论：TNF-α可诱导小胶质细胞晚期炎症因子HMGB1的表 达,并且其诱导机制与p38 MAPK被快速激活相关。