怀孕前后酒精摄入对雌鼠植入前胚胎DNA甲基化模式建立的影响

女性怀孕前后饮酒会对胎儿的发育及神经系统造成不利影响,称为“胎儿酒精综合征”（fetal alcohol spectrum disorders,FASD）。小鼠通常作为研究该病的动物模型。该实验采用体外培养技术及体内冲胚法研究雌鼠怀孕前后酒精摄入对各期植入前胚胎全基因组DNAT基化模式建立的影响。小鼠植入前胚胎体外培养实验发现,体外实验组I（怀孕前酒精处理组1,除8-cell外,其他各期胚胎的DNA甲基化水平明显低于体外对照组;体外实验组II（正常胚胎在含乙醇的培养基中培养）,各期植入前胚胎DNA甲基化水平均明显低于体外对照组。体内实验发现,体内实验组I（怀孕前酒精处理组）与体内的实验组II（怀孕后酒精处理组）,各期植入前胚胎DNA甲基化水平明显低于体内对照组。体内、外实验结果表明：受精前后酒精对各期植入前胚胎DNA甲基化模式的正确建立造成紊乱,该结果可为进一步揭示FSAD发病机制提供一定的实验基础。     

Combined activin A/LiCl/Noggin treatment improves production of mouse embryonic stem cell-derived definitive endoderm cells

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell-derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell-derived DE cells, which were defined as Cxcr4(+) c-Kit(+) , Cxcr4(+) E-cadherin(+) cells or Cxcr4(+) PDGFRa(-) cells, could be induced in the serum-free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A-mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell-derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell-derived hepatic cells and ES cell-derived pancreatic cells for future regenerative medicine.     

Spatiotemporal Transmission Dynamics of Hemorrhagic Fever with Renal Syndrome in China, 2005–2012

BackgroundHemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease caused by many serotypes of hantaviruses. In China, HFRS has been recognized as a severe public health problem with 90% of the total reported cases in the world. This study describes the spatiotemporal dynamics of HFRS cases in China and identifies the regions, time, and populations at highest risk, which could help the planning and implementation of key preventative measures.MethodsData on all reported HFRS cases at the county level from January 2005 to December 2012 were collected from Chinese Center for Disease Control and Prevention. Geographic Information System-based spatiotemporal analyses including Local Indicators of Spatial Association and Kulldorff''s space-time scan statistic were performed to detect local high-risk space-time clusters of HFRS in China. In addition, cases from high-risk and low-risk counties were compared to identify significant demographic differences.ResultsA total of 100,868 cases were reported during 2005–2012 in mainland China. There were significant variations in the spatiotemporal dynamics of HFRS. HFRS cases occurred most frequently in June, November, and December. There was a significant positive spatial autocorrelation of HFRS incidence during the study periods, with Moran''s I values ranging from 0.46 to 0.56 (P<0.05). Several distinct HFRS cluster areas were identified, mainly concentrated in northeastern, central, and eastern of China. Compared with cases from low-risk areas, a higher proportion of cases were younger, non-farmer, and floating residents in high-risk counties.ConclusionsThis study identified significant space-time clusters of HFRS in China during 2005–2012 indicating that preventative strategies for HFRS should be particularly focused on the northeastern, central, and eastern of China to achieve the most cost-effective outcomes.     

Structural and molecular interactions of CCR5 inhibitors with CCR5

We have characterized the structural and molecular interactions of CC-chemokine receptor 5 (CCR5) with three CCR5 inhibitors active against R5 human immunodeficiency virus type 1 (HIV-1) including the potent in vitro and in vivo CCR5 inhibitor aplaviroc (AVC). The data obtained with saturation binding assays and structural analyses delineated the key interactions responsible for the binding of CCR5 inhibitors with CCR5 and illustrated that their binding site is located in a predominantly lipophilic pocket in the interface of extracellular loops and within the upper transmembrane (TM) domain of CCR5. Mutations in the CCR5 binding sites of AVC decreased gp120 binding to CCR5 and the susceptibility to HIV-1 infection, although mutations in TM4 and TM5 that also decreased gp120 binding and HIV-1 infectivity had less effects on the binding of CC-chemokines, suggesting that CCR5 inhibition targeting appropriate regions might render the inhibition highly HIV-1-specific while preserving the CC chemokine-CCR5 interactions. The present data delineating residue by residue interactions of CCR5 with CCR5 inhibitors should not only help design more potent and more HIV-1-specific CCR5 inhibitors, but also give new insights into the dynamics of CC-chemokine-CCR5 interactions and the mechanisms of CCR5 involvement in the process of cellular entry of HIV-1.     

Rice cellulose synthase‐like D4 is essential for normal cell‐wall biosynthesis and plant growth

Cellulose synthase‐like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell‐wall polymers. The CSL D sub‐family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in‐depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map‐based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub‐family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C‐ or N‐terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype‐rescued transgenic plants expressing OsCSLD4–GUS under the control of its own promoter. Two phenotype‐altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell‐wall formation and plant growth.     

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK)

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.     

Establishment of a new hypotrichous genus,Heterotachysoma n. gen. and notes on the morphogenesis of Hemigastrostyla enigmatica (Ciliophora,Hypotrichia)

A marine hypotrich ciliate, Heterotachysoma multinucleatum (Gong and Choi, 2007) n. comb., found in coastal waters near Qingdao, China, was investigated. Heterotachysoma multinucleatum is characterized by its dorsal ciliature arranged in Gonostomum-pattern. Additionally, a new genus, Heterotachysoma n. gen., is established which is mainly characterized by: 18-cirri pattern; flexible body; three dorsal kineties with no dorsomarginal kineties nor kinety fragmentation; one right and one left row of marginal cirri; caudal cirri absent. The genus Tachysoma is redefined, and three new combinations, T. multinucleatum, T. ovatum and T. dragescoi, are proposed. The morphogenesis of Hemigastrostyla enigmatica (Dragesco and Dragesco-Kernéis, 1986) Song and Wilbert, 1997, is also described. Compared with that of its congeners, the differences are mainly in the dorsal ciliature: (1) the dorsal kinety anlagen are formed de novo in H. enigmatica (vs. intrakinetally in H. paraenigmatica and H. elongata); (2) the dorsal kineties anlagen develop in secondary mode in H. enigmatica (vs. primary mode in H. paraenigmatica); (3) the kinetal fragment anterior to the right marginal row in both filial product is absent in both H. enigmatica and H. elongata (vs. present in H. paraenigmatica). These findings suggest that morphogenesis is not uniform among members of the genus Hemigastrostyla.     

Urothelial hinge as a highly specialized membrane: detergent-insolubility, urohingin association, and in vitro formation

Urothelial surface is covered by numerous plaques (consisting of asymmetric unit membranes or AUM) that are interconnected by ordinary looking hinge membranes. We describe an improved method for purifying bovine urothelial plaques using 2% sarkosyl and 25 mM NaOH to remove contaminating membrane and peripheral proteins selectively. Highly purified plaques interconnected by intact hinge areas were obtained, indicating that the hinges are as detergent-insoluble as the plaques. These plaque/hinge preparations contained uroplakins, an as yet uncharacterized 18-kDa plaque-associated protein, plus an 85-kDa glycoprotein that is known to be hinge-associated in situ. Examination of the isolated, in vitro-resealed bovine AUM vesicles by quick-freeze deep-etch showed that each AUM particle consists of a 16-nm, luminally exposed "head" anchored to the lipid bilayer via a 9-mm transmembranous "tail", and that an AUM plaque can break forming several smaller plaques separated by newly formed particle-free, hinge-like areas. These data lend support to our recently proposed three-dimensional model of mouse urothelial plaques. In addition, our findings suggest that urothelial plaques are dynamic structures that can rearrange giving rise to new plaques with intervening hinges; that the entire urothelial apical surface (both plaque and hinge areas) is highly specialized; and that these two membrane domains may be equally important in fulfilling some of the urothelial functions.     

Effects of N- and C-terminal addition of oligolysines or native loop residues on the biophysical properties of transmembrane domain peptides from a G-protein coupled receptor.

Transmembrane domains (TMDs) of G-protein coupled receptors (GPCRs) have very low water solubility and often aggregate during purification and biophysical investigations. To circumvent this problem many laboratories add oligolysines to the N- and C-termini of peptides that correspond to a TMD. To systematically evaluate the effect of the oligolysines on the biophysical properties of a TMD we synthesized 21 peptides corresponding to either the second (TPIFIINQVSLFLIILHSALYFKY) or sixth (SFHILLIMSSQSLLVPSIIFILAYSLK) TMD of Ste2p, a GPCR from Saccharomyces cerevisiae. Added to the termini of these peptides were either Lys(n) (n = 1,2,3) or the corresponding native loop residues. The biophysical properties of the peptides were investigated by circular dichroism (CD) spectroscopy in trifluoroethanol-water mixtures, sodium dodecyl sulfate (SDS) micelles and dimyristoylphosphocholine (DMPC)-dimyristoylphosphoglycerol (DMPG) vesicles, and by attenuated total reflection Fourier transform infrared (ATR-FTIR) in DMPC/DMPG multilayers. The results show that the conformation assumed depends on the number of lysine residues and the sequence of the TMD. Identical peptides with native or an equal number of lysine residues exhibited different biophysical properties and structural tendencies.     

Site-directed mutagenesis and preliminary x-ray crystallographic studies of the tabtoxin resistance protein

Tabtoxin resistance protein (TTR) is an enzyme that catalyzes the acetylation of tabtoxin rendering tabtoxin-producing pathogens tolerant to their own phytotoxins. According to the structure based detoxification mechanism of TTR, three site-directed mutants Y141F, D130N and Y141F-D130N were constructed and overexpressed in E. coli. The products were then purified and their properties were analyzed by CD and DLS. Crystallization trials of two mutants Y141F andY141F-D130N were preformed.