Autophagy alleviates neurodegeneration caused by mild impairment of oxidative metabolism

Thiamine deficiency (TD) causes mild impairment of oxidative metabolism and region‐selective neuronal loss in the brain, which may be mediated by neuronal oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation. TD‐induced brain damage is used to model neurodegenerative disorders, and the mechanism for the neuronal death is still unclear. We hypothesized that autophagy might be activated in the TD brain and play a protective role in TD‐induced neuronal death. Our results demonstrated that TD induced the accumulation of autophagosomes in thalamic neurons measured by transmission electron microscopy, and the up‐regulation of autophagic markers LC3‐II, Atg5, and Beclin1 as measured with western blotting. TD also increased the expression of autophagic markers and induced LC3 puncta in SH‐SY5Y neuroblastoma cells. TD‐induced expression of autophagic markers was reversed once thiamine was re‐administered. Both inhibition of autophagy by wortmannin and Beclin1 siRNA potentiated TD‐induced death of SH‐SY5Y cells. In contrast, activation of autophagy by rapamycin alleviated cell death induced by TD. Intraperitoneal injection of rapamycin stimulated neuronal autophagy and attenuated TD‐induced neuronal death and microglia activation in the submedial thalamus nucleus (SmTN). TD inhibited the phosphorylation of p70S6 kinase, suggesting mTOR/p70S6 kinase pathway was involved in the TD‐induced autophagy. These results suggest that autophagy is neuroprotective in response to TD‐induced neuronal death in the central nervous system. This opens a potential therapeutic avenue for neurodegenerative diseases caused by mild impairment of oxidative metabolism.

     

A Comparison of Ship-Recycling Legislation Between Chinese Law and the 2009 Hong Kong Convention

This article discusses the Chinese legal framework in relation to ship recycling and suggests that the various legislation, standards, and opinions provide disorderly and nosystematic regulation. A uniform law specifically regulating the ship-recycling industry should be adopted by China, with a single competent authority empowered to supervise ship-recycling activities, that will effectively implement the 2009 Hong Kong International Convention for the Safe and Environmentally Sound Recycling of Ships.     

Introducing Titratable Water to All-Atom Molecular Dynamics at Constant?pH

Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pK_a values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λ_i} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pK_a values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:

• 1.Loss of a proton to give a negatively charged hydroxide,

H₂O ? OH^? + H⁺, (1)or

• 2.Gain of a proton to give a positively charged hydronium,

H₂O + H⁺ ? H₃O⁺.(2)We now couple the titration of hydroxide (Eq. 1) with that of an acidic site of the solute in the CpHMD simulation,HA+OH−⇌KaA−+H2O.(3)The use of hydronium is avoided here to prevent a potential artifact due to prolonged attraction with A⁻. Analogously, we couple the titration of hydronium (Eq. 2) with that of a basic site,BH++H2O⇌KbH3O++B.(4)Thus, effectively, a proton is transferred between the solute and solvent. However, we should note that in CpHMD simulations, titratable protons are represented by covalently attached dummies (2,4). Through varying the atomic charges and van der Waals interactions, they are seen by other atoms in the protonated state but not in the unprotonated state (see Table S1 in the Supporting Material). Furthermore, the solution proton concentration is implicitly modeled through a free energy term (2,4).In CpHMD, the reference potential of mean force (PMF) for titration is that of the model compound (blocked single amino acid in water) along λ (2,4). In the presence of cotitrating water molecules, it is necessary to add the PMF for the conversion of water to hydroxide or hydronium. One-nanosecond NPT simulations at ambient pressure and temperature were performed to calculate the average force, 〈dU/d,θ〉 at given θ-values, which are related to λ by λ = sin² θ (see Fig. S1 in the Supporting Material). Thermodynamic integration was then applied to calculate the PMF. We found that the average force can be accurately fit when assuming the PMF is quadratic in λ (Fig. 1). The same applies to the PMFs for titration of models Asp, Glu, and His. After testing on the titration of model compounds (see Table S2), we performed 10-ns all-atom CpHMD simulations with the pH replica-exchange protocol for three proteins: HP36, BBL and HEWL (see the Supporting Material for details). Most of the calculated pK_a values were converged in 10 ns per replica (see Fig. S3). Results are summarized in Fig. S4. Based on the 10-ns data, the root-mean-square (RMS) and average absolute errors are 0.9 and 0.7 pH units, respectively, while the largest absolute error is 2.5 (Glu³⁵ of HEWL). Linear regression of the calculation versus experiment gives R² of 0.8 and slope of 1.2.Open in a separate windowFigure 1Average force and potential of mean force for converting a water molecule to hydroxide (A) and hydronium. (B) (Data points) Average forces. (Dashed curves) Best fits using a linear function, 2A(λ − B). (Solid curves) Corresponding potential of mean force.

Table 1

Calculated and experimental pK_a values of three proteins

Actinoplanes hulinensis sp. nov., a novel actinomycete isolated from soybean root (Glycine max (L.) Merr)

A novel actinomycete, designated strain NEAU-M9^T, was isolated from soybean root (Glycine max (L.) Merr) and characterized using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain NEAU-M9^T belonged to the genus Actinoplanes, being most closely related to Actinoplanes campanulatus DSM 43148^T (98.85 %), Actinoplanes capillaceus DSM 44859^T (98.70 %), Actinoplanes lobatus DSM 43150^T (98.30 %), Actinoplanes auranticolor DSM 43031^T (98.23 %) and Actinoplanes sichuanensis 03-723^T (98.06 %); similarity to other type strains of the genus Actinoplanes ranged from 95.87 to 97.56 %. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that the isolate formed a distinct phyletic line with A. campanulatus DSM 43148^T and A. capillaceus DSM 44859^T. This branching pattern was also supported by the tree constructed with the maximum-likelihood method. However, the low level of DNA–DNA relatedness allowed the isolate to be differentiated from the above-mentioned two Actinoplanes species. Moreover, strain NEAU-M9^T could also be distinguished from the most closely related species by morphological, physiological and characteristics. Therefore, it is proposed that strain NEAU-M9^T represents a novel Actinoplanes species, Actinoplanes hulinensis sp. nov. The type strain of Actinoplanes hulinensis is NEAU-M9^T (= CGMCC 4.7036^T = DSM 45728^T).     

Phylogeography of Sophora davidii (Leguminosae) across the ‘Tanaka‐Kaiyong Line’, an important phytogeographic boundary in Southwest China

The ‘Tanaka‐Kaiyong Line’ (TKL) is a major phytogeographic boundary in Southwest China, separating East Asia's Sino‐Himalayan and Sino‐Japanese Floras. However, little is known about the importance of this boundary in promoting intraspecific phylogeographic subdivision and divergence. Using chloroplast (cpDNA) and nuclear‐intron (nDNA) sequence data, we reconstructed the population history of Sophora davidii, a drought‐tolerant riparian shrub widely distributed on either side of the TKL. Specifically, we aimed at testing two long‐standing explanations for possible vicariant events across the TKL: (i) Late Pliocene (c. 3 Ma) geological uplift of the eastern Qinghai‐Tibetan Plateau (QTP) or (ii) a sharp environmental gradient associated with the establishment of different monsoon regimes on either side of the TKL during the (Late) Pleistocene. Our genealogical analyses detected a major west–east split in cpDNA, geographically largely consistent with the TKL, and dated to c. 1.28 Ma (95% HPD: 0.21–2.96 Ma), hence postdating the latest phase of eastern QTP uplift. Furthermore, integrating cpDNA phylogeographic patterns with mismatch analyses, we found multiple refugial isolation and long‐term demographic stability of populations in the west (Hengduan Mountain Range) compared with extensive range expansions in the east, possibly during the last glacial period(s) and followed by differentiation into regional sublineages (southeast: Yunnan‐Guizhou Plateau vs. northeast: Qinling Mts./Loess Plateau). Although nuclear differentiation was less marked, the geographical pattern of nDNA haplotypes provided some further indication of the species' eastward expansion, possibly from source populations located just east of the TKL (lower Jinshajiang region). Overall, the present data reject the geological (tectonic) explanation for the TKL and, instead, provide supportive evidence for its role as a climatically driven barrier to present‐day plant dispersal. In addition, our study highlights changing temperatures and vegetation types during the last glacial period(s), along with aspects of regional topography, to be important determinants of the glacial eastward expansion of S. davidii. In consequence, our study lends support to a ‘glacial out‐of‐Hengduan Mts’. hypothesis for the xerophytic‐riparian flora of Southwest China, which in turn is inconsistent with the traditional view of the TKL as a ‘classical’ vicariant‐biogeographic boundary.     

Exploring pyrimidine-substituted curcumin analogues: Design,synthesis and effects on EGFR signaling

Epidermal growth factor receptor (EGFR) is an effective molecular target of anti-cancer therapies. Curcumin inhibits cancer cell growth in vitro by suppressing gene expression of EGFR and reduces tumor growth in various animal models. To overcome instable and insoluble properties of curcumin as therapeutics, we designed and synthesized six novel pyrimidine-substituted curcumin analogues with or without a hydroxyl group originally present in curcumin. The cell viability tests indicated that IC₅₀ of the analogues containing hydroxyl group were 3 to 8-fold lower than those of the analogues without hydroxyl group in two colon cancer cell lines tested. Western blot analysis indicates the analogues containing hydroxyl group inhibited expression and tyrosine phosphorylation of EGFR. Further protein analyses showed that the analogues had anti-cellular proliferation, pro-apoptosis, and cell cycle arrest properties associated with suppressed EGFR expression. These results indicate that the hydroxyl groups in curcumin and the analogues were critical for observed biological activities.     

分子马达生物传感器检测食源性轮状病毒

通过分子马达生物传感器技术建立一种特异、便捷、快速的食源性轮状病毒检测方法.以F0F1-ATPase为核心构建分子马达,以轮状病毒保守片段VP7设计各血清型通用探针,通过生物素-亲和素系统将探针与分子马达连接构建F0F1-ATPase分子马达检测装置.提取病毒RNA并将其与生物传感器结合的同时启动ATP合成,比较其荧光强度的差别,可以对样品中的RNA进行检测.此方法的病毒RNA检测灵敏度为0.005 ng/mL,对轮状病毒检测特异,与甲肝病毒、诺如病毒无交叉反应,在1h内即可完成检测.运用此方法随机检测15份样品,检测结果与RT-PCR一致.结果表明,分子马达生物传感器检测轮状病毒的方法灵敏、特异,可用于食源性轮状病毒的快速检测.     

稳定碳同位素在滨海湿地碳生物地球化学循环中的应用

碳作为滨海湿地中重要的生命元素,其生物地球化学循环过程是滨海湿地研究的核心内容之一.稳定同位素技术越来越多地被应用到滨海湿地碳生物地球化学循环过程的研究中,提高了其研究水平,并推动了其研究的进程.本文从有机物质生产、土壤有机质来源、食物链传递、温室气体排放以及可溶性有机碳输出5个方面,综述了滨海湿地碳生物地球化学循环过程的稳定同位素研究进展.通过植物及土壤δ13C值的测定进行有机质的生产机理研究及外源追溯,通过对比各生物种群的δ13C值分析碳在生态系统中的流动过程,通过湿地排放温室气体及可溶性有机碳δ13C值的测定揭示影响碳输出的环境因子.最后,文章总结了当前研究中存在的问题,并对其研究前景进行了展望.     

贵州省2011年急性胃肠炎诺如病毒的哨点监测及其基因特征分析

为了解贵州省2011年诺如病毒的基因型,监测了2011年5月至12月于贵州省人民医院就诊的急性胃肠炎病例,收集粪便标本,采用荧光定量逆转录-聚合酶链反应(Real-time RT-PCR)初步鉴定,并对阳性株的VP1基因区克隆及测序。检测标本70份,GⅠ型诺如病毒阳性1株,阳性率1.43%,GⅡ型诺如病毒阳性34株,阳性率48.57%,获得7份GⅡ型诺如病毒VP1基因序列,3株为GⅡ.4亚型,为新型变异株(GⅡ4 2011),与GⅡ4 2006b变异株的亲缘关系最近,有1个氨基酸位发生了回复突变;4株为GⅡ.3亚型,分为2个基因簇,1簇与2008～2009年韩国株(HM635118)及上海株(GU991355)的亲缘关系最近,1簇与2010年日本株(AB629943)及2007年印度株(EU921389)等的亲缘关系最近,有4个氨基酸位点易发生回复突变。