A quantitative assay measuring the function of lipase maturation factor 1

Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression.     

MAR-FISH技术及其在环境微生物群落与功能研究中的应用

对复杂环境中微生物群落结构和功能的研究是微生物生态学的重要任务。尽管现代分子生物学技术已经成功地用于解析环境中微生物的群落结构, 但是这些方法并不能提供微生物的原位生理学信息。而一种新的方法, 微观放射自显影和荧光原位杂交集成技术(MAR-FISH)则能够同时在单细胞水平上, 检测复杂环境中微生物的系统发育信息及其生理特性。本文总结了MAR-FISH方法的原理, 实验步骤及其在环境微生物群落与功能研究中的应用。     

Characterization and Mapping of Contaminated Sediments,Windermere Arm,Hamilton Harbour,Ontario, Canada

Windermere Arm is a 50-ha narrow channel located in the south-eastern portion of Hamilton Harbour, which is designated by the International Joint Commission (IJC) as one of the primary Areas of Concern in the Canadian waters of the Great Lakes. Sediments within the Arm have historically been contaminated with metals, PAHs, and to a lesser extent, PCBs. Sedimentological, geotechnical and geochemical investigations have been undertaken to determine the degree and the spatial extent of contamination. The thickness of contaminated sediment and their degree of contamination vary widely within the study site. Information on the thickness of contamination obtained from logging and chemical analysis of sediment cores agrees in general with the results of in situ penetrometer testing. Concentrations of Zn and Pb in some sub-samples are above the Severe Effect Level (SEL) for benthos set by the Ontario sediment quality guidelines. On average, surficial sediments tend to have lower metal concentrations than deeper sediments, probably due to the reduction of industrial point discharges. Apart from the two main boat slips, concentrations of PAHs are relatively low and in agreement with previously reported values. In contrast, new results yield higher values of PCB concentrations than previously reported, and 21 sub-samples of surficial sediment analyzed yielded values greater than 1 μg/g. Results suggest that the majority of recent PCB loadings are entering the study site from nearshore industrial spills or dumping, despite the detection of trace amounts of PCBs in a nearby creek. Pronounced sediment disturbance noted in several cores suggests that more contaminated historical sediments are at risk of being disturbed and re-exposed to the water column.     

Effects of Cadmium and Mixed Heavy Metals on Rice Growth in Liaoning,China

Joint effects of Cd and other heavy metals (Pb, Cu, Zn and As) on the growth and development of rice plants and the uptake of these heavy metals by rice were studied using the pot-culture method combined with chemical and statistical analyses. The results showed that the growth and development of rice plants were strongly influenced by the double-element combined pollution. There was an average decrease in the height of rice plants of 4.0–5.0 cm, and grain yield was decreased by 20.0–30.0%, compared with the control. The uptake of Cd by rice plants was promoted due to the interactions between Cd and the other heavy metals added to the soil. The Cd concentration in roots, stems/leaves and seeds increased 31.6–47.7, 16.7–61.5 and 19.6–78.6%, respectively. Due to interactions, uptake of Pb, Cu and Zn by roots and stems/leaves was inhibited, accumulation of Pb, Cu and Zn in seeds was increased, uptake of As by roots was promoted and uptake of As by stems/leaves was inhibited. In particular, the upward transporting ability of the heavy metals absorbed by rice plants was significantly increased.     

Are Seafood PCB Data Sufficient to Assess Health Risk for High Seafood Consumption Groups?

Some populations in Washington's Puget Sound area consume much more seafood than the general population. Mean consumption rates are 61?g/person/day for the Tulalip and Squaxin Island Tribes and 117.2?g/person/day for Asian and Pacific Islanders (API). There is concern about possible health risks from seafood PCB exposure for these groups, but exposure evaluation is difficult due to inadequacies of environmental data. Available seafood PCB data were matched to results from recent seafood consumption surveys. To rate quality of matches and identify data gaps, a ranking system based on species specificity, data quality, and location compatibility was developed. Sensitivity of total PCB and congenerspecific PCB testing (for use in TCDD toxic equivalency approaches) necessary for cancer risk assessment was explored and included in the ranking scheme. For the Squaxin Tribe, appropriate total PCB data for risk assessment were available for 58% of seafood consumed, which is dominated by local salmon. For API, appropriate total PCB data were identified for only 4% of seafood consumed, which is dominated by commercial shellfish. Insufficient sensitivity of commercial seafood PCB analysis and overall lack of sufficiently sensitive PCB congener analysis are major gaps in ability to characterize PCB exposure and risk for these groups.     

Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.     

Exploring the multiscale signaling behavior of phototropin1 from Chlamydomonas reinhardtii using a full‐residue space kinetic Monte Carlo molecular dynamics technique

Devising analysis tools for elucidating the regulatory mechanism of complex enzymes has been a challenging task for many decades. It generally requires the determination of the structural‐dynamical information of protein solvent systems far from equilibrium over multiple length and time scales, which is still difficult both theoretically and experimentally. To cope with the problem, we introduce a full‐residue space multiscale simulation method based on a combination of the kinetic Monte Carlo and molecular dynamics techniques, in which the rates of the rate‐determining processes are evaluated from a biomolecular forcefield on the fly during the simulation run by taking into account the full space of residues. To demonstrate its reliability and efficiency, we explore the light‐induced functional behavior of the full‐length phototropin1 from Chlamydomonas reinhardtii (Cr‐phot1) and its various subdomains. Our results demonstrate that in the dark state the light oxygen voltage‐2‐Jα (LOV2‐Jα) photoswitch inhibits the enzymatic activity of the kinase, whereas the LOV1‐Jα photoswitch controls the dimerization with the LOV2 domain. This leads to the repulsion of the LOV1‐LOV2 linker out of the interface region between both LOV domains, which results in a positively charged surface suitable for cell–membrane interaction. By contrast, in the light state, we observe that the distance between both LOV domains is increased and the LOV1‐LOV2 linker forms a helix–turn–helix (HTH) motif, which enables gene control through nucleotide binding. Finally, we find that the kinase is activated through the disruption of the Jα‐helix from the LOV2 domain, which is followed by a stretching of the activation loop (A‐loop) and broadening of the catalytic cleft of the kinase. Proteins 2014; 82:2018–2040. © 2014 Wiley Periodicals, Inc.     

Determination of the lysosomal hydrolase activity in blood collected on filter paper,an alternative to screen high risk populations

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.     

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers – OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 – in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3–5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.     

桃园中食心虫单一性诱芯及其复合配置的诱蛾效率比较

【目的】明确两种食心虫性诱芯复合配置的诱蛾效果,提高其监测或防治效率及其绿色环保化水平,为果树生产中食心虫的高效监测和绿色防控提供科学依据。【方法】田间系统调查研究了梨小食心虫(以下简称"梨小")、桃小食心虫(以下简称"桃小")单一性诱芯及其复合配置3种处理的诱蛾效率,并利用"Y"型嗅觉仪比较研究了其间梨小雄蛾趋向性的差异。【结果】(1)梨小和桃小性单一诱芯及其复合配置对梨小均具有引诱作用,其诱蛾总量依次为8 238.33、1 451.67、8 321.67头/诱捕器,其中第1、2、3代时复合配置诱蛾量最大,越冬代和第4代时梨小单一性诱芯诱蛾量最大,而各世代桃小单一性诱芯诱蛾量均最低。梨小单一性诱芯及其复合配置均监测到5个梨小发生高峰,且峰期基本一致,但复合配置的峰日诱蛾量均较高;桃小单一性诱芯仅监测到3个梨小发生高峰,且峰日诱蛾量亦较低。(2)桃小单一性诱芯及其复合配置对桃小均具有引诱作用,其诱蛾总量依次为4.00、2.33头/诱捕器,而梨小单一性诱芯对桃小无引诱作用。(3)"Y"型嗅觉仪研究发现,梨小食心虫对梨小和桃小各单一性诱芯及其复合配置均具有趋向作用,其趋向率依次为50.67%、8.67%、53.33%。【结论】梨小和桃小单一性诱芯复合配置对梨小诱捕量有微增效作用,而对桃小诱捕量有一定干扰作用,但影响均不显著。据此,该复合配置可用于桃园中梨小和桃小的监测与防控。