P2X7 receptor inhibition attenuated sympathetic nerve sprouting after myocardial infarction via the NLRP3/IL‐1β pathway

Mounting evidence supports the hypothesis that inflammation modulates sympathetic sprouting after myocardial infarction (MI). The myeloid P2X₇ signal has been shown to activate the nucleotide‐binding and oligomerization domain‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome, a master regulator of inflammation. We investigated whether P2X₇ signal participated in the pathogenesis of sympathetic reinnervation after MI, and whether NLRP3/interleukin‐1β (IL‐1β) axis is involved in the process. We explored the relationship between P2X₇ receptor (P2X₇R) and IL‐1β in the heart tissue of lipopolysaccharide (LPS)‐primed naive rats. 3′‐O‐(4‐benzoyl) benzoyl adenosine 5′‐triphosphate (BzATP), a P2X₇R agonist, induced caspase‐1 activation and mature IL‐1β release, which was further neutralized by a NLRP3 inhibitor (16673‐34‐0). MI was induced by coronary artery ligation. Following infarction, a marked increase in P2X₇R was localized within infiltrated macrophages and observed in parallel with an up‐regulation of NLRP3 inflammasome levels and the release of IL‐1β in the left ventricle. The administration of A‐740003 (a P2X₇R antagonist) significantly prevented the NLRP3/IL‐1β increase. A‐740003 and/or Anakinra (an IL‐1 receptor antagonist) significantly reduced macrophage infiltration as well as macrophage‐based IL‐1β and NGF (nerve growth factor) production and eventually blunted sympathetic hyperinnervation, as assessed by the immunofluorescence of tyrosine hydroxylase (TH) and growth‐associated protein 43 (GAP 43). Moreover, the use of Anakinra partly attenuated sympathetic sprouting. This indicated that the effect of P2X₇ on neural remodelling was mediated at least partially by IL‐1β. The arrhythmia score of programmed electric stimulation was in accordance with the degree of sympathetic hyperinnervation. In vitro studies showed that BzATP up‐regulated secretion of nerve growth factor (NGF) in M1 macrophages via IL‐1β. Together, these data indicate that P2X₇R contributes to neural and cardiac remodelling, at least partly mediated by NLRP3/IL‐1β axis. Therapeutic interventions targeting P2X₇ signal may be a novel approach to ameliorate arrhythmia following MI.     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.

     

8个禾本科草坪草品种遗传多样性的RAPD分析

利用RAPD技术,对禾本科4属4种8个品种的草坪草进行遗传多样性分析。15个有效引物用于PCR扩增,共获得RAPD谱带144条带,多态性带占63.2%。8个草坪草品种间的遗传距离在0.0857~0.2928之间,脱壳狗牙根与普通狗牙根间的遗传距离最小,交战2号与爱神特之间的遗传距离最大。用UPGMA和邻接法进行聚类分析,得到2个拓扑结构基本一致的树系图。同一种不同品种间的亲缘关系最近;不同属种间的遗传距离加大,同族不同属的草坪草基本构成一支;4个暖季型草坪草品种构成一个单系类群,但属于冷季型草种的黑麦草(爱神特)单独构成一支,并未与同族的其它3个冷季型草坪草品种(高羊茅)聚在一起。本研究不支持将黑麦草属与羊茅属放在同一族内的分类处理,并建议将高羊茅划分为过渡类型的草坪草。     

Comparison of bacterial diversity along the human intestinal tract by direct cloning and sequencing of 16S rRNA genes

Bacterial diversity of the mucosal biopsies from human jejunum, distal ileum, ascending colon and rectum were compared by analysis of PCR-amplified 16S rDNA clone libraries. A total of 347 clones from the mucosal biopsies were partially sequenced and assigned to six phylogenetic phyla of the domain Bacteria: Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Verrucomicrobia, and Actinobacteria. The jejunum sample had least microbial diversity compared to the other samples and a trend towards highest diversity in ascending colon was observed. The clone libraries of distal ileum, ascending colon and rectum were not significantly different from each other (P>0.0043), but they differed significantly from the jejunum library (P=0.001). The population of sequences retrieved from jejunal biopsies was dominated by sequences closely related to Streptococcus (67%), while the population of sequences derived from distal ileum, ascending colon and rectum were dominated by sequences affiliated with Bacteroidetes (27-49%), and Clostridium clusters XIVa (20-34%) and IV (7-13%). The results indicate that the microbial community in jejunum is different from those in distal ileum, ascending colon and rectum, and that the major phylogenetic groups are similar from distal ileum to rectum.     

Determination of an Optimal Standardized Uptake Value of Fluorodeoxyglucose for Positron Emission Tomography Imaging to Assess Pathological Volumes of Cervical Cancer: A Prospective Study

Purpose

To determine the optimal standardized uptake value (SUV) of ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) for positron emission tomography (PET) imaging, at which the PET-defined gross tumor volume (GTV_PET) best matches with the pathological volume (GTV_PATH) in the cervical cancer.

Materials and Methods

Ten patients with the cervical cancer who underwent surgery were enrolled in this study. The excised specimens were processed for whole-mount serial sections and H-E staining. The tumor borders were outlined in sections under a microscope, histopathological images were scanned and the GTV_PATH calculated. The GTV_PET was delineated automatically by using various percentages relative to the maximal SUV and absolute SUV. The optimal threshold SUV was further obtained as the value at which the GTV_PET best matched with the GTV_PATH.

Results

An average of 85±10% shrinkage of tissue was observed after the formalin fixation. The GTV_PATH was 13.38±2.80 cm³ on average. The optimal threshold on percentile SUV and absolute SUV were 40.50%±3.16% and 7.45±1.10, respectively. The correlation analysis showed that the optimal percentile SUV threshold was inversely correlated with GTV_PATH (p<0.05) and tumor diameter (p<0.05). The absolute SUV was also positively correlated with SUV_max (p<0.05).

Conclusion

The pathological volume could provide the more accurate tumor volume. The optimal SUV of FDG for PET imaging by use of GTV_PATH as standard for cervical cancer target volume delineation was thus determined in this study, and more cases are being evaluated to substantiate this conclusion.     

Diabetic Patients Could Do As Well as Non-Diabetic Patients without Inflammation on Peritoneal Dialysis

Background

Diabetic patients on peritoneal dialysis (PD) have lower survival and are more likely complicated with inflammation than their non-diabetic counterparts. Here, we explored the interaction effects between diabetes and inflammation on the survival of PD patients.

Methods

Overall, 2,264 incident patients were enrolled from a retrospective cohort study in China. Patients were grouped according to the baseline levels of high-sensitive C-reactive protein (hsCRP, ≤3 mg/L or >3 mg/L) or serum albumin (SA, ≥38 g/L or <38 g/L). Then, several multivariable adjusted stratified Cox regression models were constructed for these groups to explore the predicted role of diabetes on all-cause or cardiovascular death under inflammatory or non-inflammatory conditions.

Results

Diabetics on PD were more likely to have inflammation than non-diabetics on PD, and they presented with elevated hsCRP (52.7% vs. 47.3%, P = 0.03) or decreased SA (77.9% vs. 62.7%, P < 0.001) levels. After stratification by size of center and controlling for confounding factors, diabetes was found to predict all-cause death in patients with hsCRP >3 mg/L or SA <38 g/L but not in patients with hsCRP ≤3 mg/L or SA ≥38 g/L. Similarly, the presence of diabetes was an indication of cardiovascular death in patients with hsCRP >3 mg/L or SA <38 g/L. However, if further adjusted by baseline cardiovascular disease, the predicted role of diabetes on death related to cardiovascular disease in patients with SA <38 g/L disappeared.

Conclusion

Diabetic patients could do as well as non-diabetic patients without inflammation on peritoneal dialysis. Active strategies should be implemented to improve inflammation status in diabetic patients on PD.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Optimization of covalent immobilization of pectinase on sodium alginate support

Pectinase was immobilized on a sodium alginate support using glutaraldehyde and retained 66% activity. The optimal pH for activity shifted from 3.0 to 3.5 after immobilization; however, the optimum temperature remained unchanged at 40°C. The immobilized enzyme also had a higher thermal stability and reusability than the free enzyme, and retained 80% of initial activity after 11 batch reactions.     

持久性有机污染物γ-六六六生物降解研究进展

γ-六六六(γ-Hexachlorocyclohexane,γ-HCH)是一种有机氯杀虫剂,由于它具有持久性和很高的毒性,成为顽固性的世界性污染物。目前从世界上不同的受HCH污染的地区中已经分离出许多能够降解HCH的微生物,其中一些微生物对γ-HCH的降解途径得到了阐明,降解基因/酶也得到了鉴定。综述了γ-HCH降解菌的多样性、降解途径、降解基因和酶,为γ-HCH的生物修复提供了参考。