Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.     

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K_d = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.     

Mechanism of the Ca-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca²⁺ binds to the EF2 domain of S100A4 with micromolar affinity and that the K_d value for Ca²⁺ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K_d results from a reduced dissociation rate constant (from 16 s^− 1 to 0.3 s^− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca²⁺]. However, for Ca²⁺ transients to be effective in further promoting dissociation, the elevated Ca²⁺ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.     

Visualizing HIV-1 Assembly

The assembly of an HIV-1 particle is a complex, multistep process involving several viral and cellular proteins, RNAs and lipids. While many macroscopic and fixed-cell microscopic techniques have provided important insights into the structure of HIV-1 particles and the mechanisms by which they assemble, analysis of individual particles and their assembly in living cells offers the potential of surmounting many of the limitations inherent in other approaches. In this review, we discuss how the recent application of live-cell microscopic imaging techniques has increased our understanding of the process of HIV-1 particle assembly. In particular, we focus on recent studies that have employed total internal reflection fluorescence microscopy and other single-virion imaging techniques in live cells. These approaches have illuminated the dynamics of Gag protein assembly, viral RNA packaging and ESCRT (endosomal sorting complex required for transport) protein recruitment at the level of individual viral particles. Overall, the particular advantages of individual particle imaging in living cells have yielded findings that would have been difficult or impossible to obtain using macroscopic or fixed-cell microscopic techniques.     

The Copenhagen Oral Health Senior Cohort: design, population and dental health

Gerodontology 2010; doi: 10.1111/j.1741‐2358.2010.00383.x The Copenhagen Oral Health Senior Cohort: design, population and dental health Background: In order to study the way old age influence oral health, the Copenhagen Oral Health Senior Cohort (COHS) has been established. Objectives: To describe the design, measurement procedures, and baseline values for COHS including spatial distribution of restorations and dental caries as well as reasons for non‐participation. Materials and methods: Seven hundred and eighty‐three individuals aged 65 years or older, from a total of 1918 invited elderly people, underwent an interview regarding oral health‐related behaviour and a clinical oral examination including measurement of unstimulated whole saliva flow rate. Results: Twelve percent of the COHS was edentulous. The number of dental restorations was higher for women compared to men; however, men had more caries than women. Coronal caries was most frequent on mesial and distal surfaces and on the maxillary incisors and canines; root caries was most frequent on labial surfaces and evenly distributed within the dentition. Only 41% of all invited elderly people accepted the invitation, with old age and poor health being the primary reasons for non‐participation. Conclusion: The baseline values for COHS show that a substantial proportion of the participants had retained a natural dentition and that dental caries was prevalent with the anterior maxillary teeth being most affected.     

蚜虫新型预警网络的构建及其绿色防控技术研究

在公益性行业（农业）科研专项的支持下,项目组在我国大豆和小麦主产区进行了蚜虫监测预警及绿色防控技术的研究。构建了基于吸虫塔的蚜虫监测预警网络系统,在蚜虫基础生物学研究、天敌资源普查及其控蚜作用研究的基础上,研发了多项以生物防治为主体的蚜虫绿色防控技术,包括天敌人工助迁、人工饲养天敌释放、作物邻间作措施、物理防控、隐蔽性施药等。相关技术措施在我国的东北、华北等大豆蚜、麦蚜为害严重的大豆产区和小麦主产区共建立了4个规模较大的试验示范区,取得了较好的综合效益。     

基于LSMM与MSPA的深圳市绿色景观连通性研究

基于线性光谱混合模型(LSMM,Linear Spectral Mixture Model),引入形态学空间格局分析(MSPA,Morphological Spatial Pattern Analysis)进行城市地域绿色景观连通性评价。根据城市绿色景观特点和MSPA方法中的7种连通性类型的涵义,定义了城市绿色景观连通性功能类型。以深圳市1986年、1995年、2000年、2005年及2010年五期Landsat TM影像为数据源,应用线性光谱混合模型提取植被覆盖率,得到深圳市植被覆盖图。在此基础上,提取出高、全植被覆盖作为目标像元进行MSPA处理,分析植被覆盖状况与绿色景观功能类型的时序总体特征及空间梯度动态。结果表明:深圳市绿色景观破碎程度较高,表现为对结构连通性贡献最小的斑块类型总数最大。城市内部东西部连通性呈现出不同变化的趋势;右侧外圈层的大鹏半岛结构连通性最佳;在同一城市化发展梯度上,东部的样带连通性水平比西部要好。在城市化过程中,深圳市高、全覆被植被像元连通性大小受以下因素的影响:城市化程度,地形因素及区域定位。在同一城市化程度上,地形因素对景观连通性的影响较大。从整体的时间变化和空间梯度动态分析可知,在快速城市化过程中植被覆盖率和连通性功能均下降,而到稳定城市化阶段植被覆盖率和连通性均得到改善。研究表明线性光谱混合模型与形态学空间格局分析相结合可以较好的表征城市绿色景观连通性类型时空分布特征,进而明晰城市化过程与区域内绿色景观数量及连通性动态变化关系。     

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage.     

Correlated ab initio molecular dynamics simulations of the acetone–carbon dioxide complex: implications for solubility in supercritical CO2

Ab initio molecular dynamics simulations of the acetone–CO₂ complex (MP2/6-31G(d) level) were performed to investigate the effect of dynamics at finite temperature on the weak electron donor–acceptor intermolecular interactions. In addition, we carried out a study of the free energy of formation of the complex by means of umbrella sampling technique at the MP2 level with a perturbative CCSD(T) correction. The potential of mean force was obtained along a reaction coordinate describing the acetone–CO₂ interaction. The results obtained here support some hypothesis that we already explored in past works using static electronic calculations. In particular, when interacting with a molecule having a carbonyl function, carbon dioxide displays both Lewis acid and Lewis base behaviour. This property can be exploited to design molecular systems that are easily solubilised in supercritical CO₂.