High-throughput cell-based screening using scintillation proximity assay for the discovery of inositol phosphatase inhibitors

Inositol monophosphatase is a potential drug target for developing lithium-mimetic agents for the treatment of bipolar disorder. Enzyme-based assays have been traditionally used in compound screening to identify inositol monophosphatase inhibitors. A cell-based screening assay in which the compound needs to cross the cell membrane before reaching the target enzyme offers a new approach for discovering novel structure leads of the inositol monophosphatase inhibitor. The authors have recently reported a high-throughput measurement of G-protein-coupled receptor activation by determining inositol phosphates in cell extracts using scintillation proximity assay. This cell-based assay has been modified to allow the determination of inositol monophosphatase activity instead of G-protein-coupled receptors. The enzyme is also assayed in its native form and physiological environment. The authors have applied this cell-based assay to the high-throughput screening of a large compound collection and identified several novel inositol monophosphatase inhibitors.     

中国鞘蛾研究（鳞翅目：鞘蛾科）：脉鞘蛾组研究及三新种记述

报道了鞘蛾科 (Coleophoridae) 脉鞘蛾组 (Colephora follicularis group) ５个分布于中国的种, 其中有3个新种和2个中国新记录种：矛尖脉鞘蛾(新种)Coleophra jaculatoria sp. nov. 分布于陕西(周至);奇脉鞘蛾Coleophora frankii Schmidt新记录于新疆(巩留),国外分布奥地利,捷克,斯洛伐克,匈牙利,南斯拉夫,德国和意大利;隐脉鞘蛾 Coleophora pseudociconiella Toll 新记录于新疆(塔城),国外分布奥地利,意大利,捷克和土耳其;四叉脉鞘蛾(新种) Coleophora quadrifurca sp. nov.分布陕西(杨陵、澄城);榆中脉鞘蛾(新种)Coleophora yuzhongensis sp. Nov. 分布甘肃(榆中)。文中提供了分种检索表和新种的外生殖器特征图。模式标本保存在南开大学生物系。     

Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in <Emphasis Type="Italic">Taxus yunnanensis</Emphasis> suspension cells

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells induced by a fungal-derived cerebroside and the signal role of NO in the elicitation of plant defense responses and taxol production. (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine at 10 μg/ml induced a rapid and dose-dependent NO production in the Taxus cell culture, reaching a maximum within 5 h of the treatment. The NO donor sodium nitroprusside (SNP) potentiated cerebroside-induced H₂O₂ production and cell death. Inhibition of nitric oxide synthase activity by phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide or scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide partially blocked the cerebroside-induced H₂O₂ production and cell death. Moreover, NO enhanced cerebroside-induced activation of phenylalanine ammonium-lyase and accumulation of taxol in cell cultures. These results are suggestive of a role for NO as a new signal component for activating the cerebroside-induced defense responses and secondary metabolism activities of plant cells. Taxol is a trademark of Bristol-Myers Squibb, Madison, NJ.     

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.     

Status of water environment pollution in the Xixi Wetland and its ecological treatment countermeasures

In this paper,the water quality of the Xixi Wetland was evaluated and the characteristics of water pollution were described according to the survey data.Based on the status of water quality and its functional requirements as an urban wetland,biological-ecological countermeasures were suggested.The experimental use of ecological technologies,such as artificial wetlands,ecological aquiculture and artificial floating island,were done in several fish ponds in the Xixi Wetland.Water monitoring results show that the quality of the treated water has improved significantly and the measures to purify the eutrophic water in the wetland have been effective.     

Subspecies-specific intron length polymorphism markers reveal clear genetic differentiation in common wild rice （Oryza rufipogon L.） in relation to the domestication of cultivated rice （O. sativa L.）

It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice （O. sativa）. However, how the two subspecies of O. sativa （indica and japonica） were domesticated has long been debated. To investigate the genetic differentiation in O. rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism （SSILP） markers by comparison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that the indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa. Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups： one group （Group I） comprises all the O. rufipogon accesions from tropical （South and Southeast） Asia as well as the standard indica rice; the other group （Group II） comprises all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have significantly higher frequencies of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domestication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. ruffpogon might occur after its speciation from other AA-genome species.     

广西竹叶青蛇伤的临床特征探讨

目的探讨广西竹叶青蛇伤后的临床特征,为临床诊治提供较合理依据。方法选择被竹叶青蛇咬伤的急诊患者55例的临床资料并分析临床特征。结果 55例竹叶青蛇伤患者分别按咬伤地点与临床分型比较(χ2=18.03,P0.01),竹叶青蛇属(竹叶青和白唇竹叶青)与临床分型比较(χ2=24.67,P0.01)及咬伤地点与竹叶青蛇属比较(χ2=24.80,P0.01),差异均有统计学意义,咬伤地点与竹叶青蛇属呈中度正相关(rs=0.67);竹叶青蛇咬伤按临床分型比较血液学变化有统计学意义(均P0.01),其中血小板(PLT)、白细胞(WBC)各型比较均有统计学意义(P0.05)。结论竹叶青蛇伤主要在城市花园草丛、田间地头等地点发生,伤后临床分型绝大多数为轻型,少数为重型和危重型;白唇竹叶青蛇伤地点主要在深山老林,伤后临床分型以危重型、重型为主,少数为轻型。咬伤地点与临床分型和蛇种属有很大差异性,可为临床诊治提供参考依据。竹叶青蛇伤致血液功能障碍,甚至出现类DIC;除分型标准外,患者WBC的增高程度可作为严重程度的参考指标。     

A Modified Delta-Shaped Gastroduodenostomy in Totally Laparoscopic Distal Gastrectomy for Gastric Cancer: A Safe and Feasible Technique

Background

The present study introduced a modified delta-shaped gastroduodenostomy (DSG) technique and assessed the safety, feasibility and clinical results of this procedure in patients undergoing totally laparoscopic distal gastrectomy (TLDG) for gastric cancer (GC).

Materials and Methods

A total of 102 patients with distal GC undergoing TLDG with modified DSG between January 2013 and December 2013 were enrolled. A retrospective study was performed using a prospectively maintained comprehensive database to evaluate the results of the procedure. Univariate and multivariate analyses were performed to estimate the predictive factors for postoperative morbidity.

Results

The mean operation time was 150.6±30.2 min, the mean anastomosis time was 12.2±4.2 min, the mean blood loss was 48.2±33.2 ml, and the mean times to first flatus, fluid diet, soft diet and postoperative hospital stay were 3.8±1.3 days, 5.0±1.0 days, 7.4±2.1 days and 12.0±6.5 days, respectively. Two patients with minor anastomotic leakage after surgery were managed conservatively; no patient experienced any complications around the anastomosis, such as anastomotic stricture or anastomotic hemorrhage. Univariate analysis showed that age, gastric cancer with hemorrhage and cardiovascular disease combined were significant factors that affected postoperative morbidity (P<0.05). Multivariate analysis found that gastric cancer with hemorrhage was the independent risk factor for the postoperative morbidity (P = 0.042). At a median follow-up of 7 months, no patients had died or experienced recurrent or metastatic disease.

Conclusions

The modified DSG was technically safe and feasible, with acceptable surgical outcomes, in patients undergoing TLDG for GC, and this procedure may be promising in these patients.     

Non-contiguous finished genome sequence of Anoxybacillus flavithermus subsp. yunnanensis type strain (E13T), a strictly thermophilic and organic solvent-tolerant bacterium

Anoxybacillus flavithermus subsp. yunnanensis is the only strictly thermophilic bacterium that is able to tolerate a broad range of toxic solvents at its optimal temperature of 55-60°C. The type strain E13^T was isolated from water-sediment slurries collected from a hot spring. This study presents the draft genome sequence of A. flavithermus subsp. yunnanensis E13^T and its annotation. The 2,838,393bp long genome (67 contigs) contains 3,035 protein-coding genes and 85 RNA genes, including 10 rRNA genes, and no plasmids. The genome information has been used to compare with the genomes from A. flavithermus subsp. flavithermus strains.     

RNA Graph Partitioning for the Discovery of RNA Modularity: A Novel Application of Graph Partition Algorithm to Biology

Graph representations have been widely used to analyze and design various economic, social, military, political, and biological networks. In systems biology, networks of cells and organs are useful for understanding disease and medical treatments and, in structural biology, structures of molecules can be described, including RNA structures. In our RNA-As-Graphs (RAG) framework, we represent RNA structures as tree graphs by translating unpaired regions into vertices and helices into edges. Here we explore the modularity of RNA structures by applying graph partitioning known in graph theory to divide an RNA graph into subgraphs. To our knowledge, this is the first application of graph partitioning to biology, and the results suggest a systematic approach for modular design in general. The graph partitioning algorithms utilize mathematical properties of the Laplacian eigenvector (µ2) corresponding to the second eigenvalues (λ2) associated with the topology matrix defining the graph: λ2 describes the overall topology, and the sum of µ2′s components is zero. The three types of algorithms, termed median, sign, and gap cuts, divide a graph by determining nodes of cut by median, zero, and largest gap of µ2′s components, respectively. We apply these algorithms to 45 graphs corresponding to all solved RNA structures up through 11 vertices (∼220 nucleotides). While we observe that the median cut divides a graph into two similar-sized subgraphs, the sign and gap cuts partition a graph into two topologically-distinct subgraphs. We find that the gap cut produces the best biologically-relevant partitioning for RNA because it divides RNAs at less stable connections while maintaining junctions intact. The iterative gap cuts suggest basic modules and assembly protocols to design large RNA structures. Our graph substructuring thus suggests a systematic approach to explore the modularity of biological networks. In our applications to RNA structures, subgraphs also suggest design strategies for novel RNA motifs.