A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events

Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders—Niemann-Pick type C, mucolipidosis type IV, and Sandhoff’s disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.     

长白山国家级自然保护区两栖类动物道路致死特征分析

为了解旅游道路交通导致的两栖类致死效应,于2007~2014年在环绕长白山国家级自然保护区的环长白山旅游公路(环区公路)、头道环区公路以及白山公路分别选取1条样线,采用直接计数法统计了交通致死的两栖类种类、数量,以及致死个体处于交通路段的位置,共统计257次,累计调查样带总长2 441 km。道路致死两栖类种类有5种,累计致死个体数为7 910只,平均致死率为3.34只/km。最容易致死的两栖类动物是中国林蛙(Rana chensinensi)、东方铃蟾(Bombina orientalis)、中华蟾蜍(Bufo gargariza),这3种动物占到致死总数的96.36%;经Moses Test检验,白山公路的致死率、头道公路的致死率及环区公路致死率均与对照土路致死率差异显著(白山公路:n=67,P=0.000;头道公路:n=76,P=0.000;环区公路:n=164,P=0.000)。白山公路与头道公路两栖类致死率差异显著(n=93,P=0.010),白山公路与环区公路的两栖类道路致死率差异显著(n=181,P=0.000);头道公路与环区公路的两栖类道路致死率差异显著(n=190,P=0.000)。两栖类道路致死高峰期在8月份,其次较集中出现在4~6月的繁殖期。从公路年度致死变化来看,2007~2014年期间除了中国林蛙和中华蟾蜍致死率呈小幅波动外,其他种类致死率总体呈下降趋势,只有日本树蟾(Hyla japonica)年度致死率差异显著(Kruskal Wallis Test:χ2=18.031,df=7,P=0.012);不同月份中华蟾蜍(Pearson:R=0.939,n=7,P=0.002)、中国林蛙(Pearson:R=0.912,n=7,P=0.004)、日本树蟾(Pearson:R=0.904,n=7,P=0.005)致死率与交通量显著相关。本研究认为,两栖类的道路致死率与两栖类种群大小、种群密度、生活史及交通量等都存在显著相关性。     

DNA damage,repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans

This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident.Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products.     

Fallout from the Chernobyl accident and overall cancer incidence in Finland

Aim: We studied whether incidence of all cancer sites combined was associated with the radiation exposure due to fallout from the Chernobyl accident in Finland. An emphasis was on the first decade after the accident to assess the suggested “promotion effect”. Methods: The segment of Finnish population with a stable residence in the first post-Chernobyl year (2 million people) was studied. The analyses were based on a 250 m × 250 m grid squares covering all of Finland and all cancer cases except cancers of the breast, prostate and lung. Cancer incidence in four exposure areas (based on first-year dose due to external exposure <0.1 mSv, 0.1–1.3, 0.3–0.5, or ≥0.5 mSv) was compared before the Chernobyl accident (1981–1985) and after it (1988–2007) taking into account cancer incidence trends for a longer period prior to the accident (since 1966). Results: There were no systematic differences in the cancer incidence in relation to radiation exposure in any calendar period, or any subgroup by sex or age at accident. Conclusion: The current large and comprehensive cohort analysis of the relatively low levels of the Chernobyl fallout in Finland did not observe a cancer promotion effect.     

The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.     

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning

Mutations in cobalamin or B₁₂ trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B₁₂ processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.     

Self‐Awareness of Impairment and the Decision to Drive after an Extended Period of Wakefulness

Fatigue is an increasingly noted factor in road accidents. The ability to predict and be aware of impairment in terms of driving capability is important for potential legal liability and road safety. However, to date, there have been few studies that have investigated the accuracy of individuals in predicting how safely they could drive during conditions of sleep loss. Research has demonstrated that individuals rate themselves as better than the population average in a number of domains, including driving‐related skills. Therefore, this study also aimed to investigate self‐ratings of predicted driving ability during extended wakefulness and compare them to ratings made of a hypothetical other person under the same conditions. Thirty‐two participants remained awake for a period of 40 h. Every 2 h, they completed the Psychomotor Vigilance Task (PVT) and rated on a seven‐point scale how well they thought they could drive safely, react quickly in an emergency, and stay in their own lane. They were also asked to assess how they thought someone else in their own position could drive. The participants rated their driving ability as becoming significantly poorer at the same time that their PVT performance became significantly slower. Self‐ratings indicating a qualitative assessment of poorer than neutral driving occurred at 03:00 h for both the “drive safely” and “react quickly” questions, after 19 h of continuous wakefulness (starting at 08:00 h). This occurred at 05:00 h for the “keep in my lane” question. Previous studies with a similar protocol demonstrated that under these conditions, individuals exhibit a performance decrements equivalent to someone with a blood alcohol concentration of 0.05% (the legal driving limit in Australia). Participants consistently rated the ability of others to drive as poorer than their own. The main implication from this study for road safety and legal liability is that it is reasonable to focus on a person's perception of the situation, as it does align with objective reality to a certain extent. A concern in terms of road safety is potential overconfidence, indicated by rating others consistently poorer than themselves.     

工作倦怠与交通警察生活质量的关系

目的:探讨交通警察工作倦怠与生活质量的关系。方法:采用MBI-GS和生活质量问卷调查了某市185名交通警察,运用相关分析和回归分析处理数据。结果:交通警察的情绪衰竭与其生活质量显著负相关(P<0.01);玩世不恭也与生活质量显著负相关(P<0.01);职业效能与生活质量显著正相关(P<0.05)。结论:降低工作倦怠,可以有效提高交通警察的生活质量。     

A Comparative Analysis of Accident Risks in Fossil,Hydro, and Nuclear Energy Chains

This study presents a comparative assessment of severe accident risks in the energy sector, based on the historical experience of fossil (coal, oil, natural gas, and LPG [Liquefied Petroleum Gas]) and hydro chains contained in the comprehensive Energy-related Severe Accident Database (ENSAD), as well as Probabilistic Safety Assessment (PSA) for the nuclear chain. Full energy chains were considered because accidents can take place at every stage of the chain. Comparative analyses for the years 1969–2000 included a total of 1870 severe (≥ 5 fatalities) accidents, amounting to 81,258 fatalities. Although 79.1% of all accidents and 88.9% of associated fatalities occurred in less developed, non-OECD countries, industrialized OECD countries dominated insured losses (78.0%), reflecting their substantially higher insurance density and stricter safety regulations. Aggregated indicators and frequency-consequence (F-N) curves showed that energy-related accident risks in non-OECD countries are distinctly higher than in OECD countries. Hydropower in non-OECD countries and upstream stages within fossil energy chains are most accident-prone. Expected fatality rates are lowest for Western hydropower and nuclear power plants; however, the maximum credible consequences can be very large. Total economic damages due to severe accidents are substantial, but small when compared with natural disasters. Similarly, external costs associated with severe accidents are generally much smaller than monetized damages caused by air pollution.     

Rounding a corner of a bent termite tunnel and tunnel traffic efficiency

Subterranean termites construct underground tunnels, tens to hundreds of feet, to reach feeding sites and to transport food items to their nest. To ensure a high rate food return to the nest, an optimized tunnel should be constructed. We found that termites (Coptotermes formosanus Shiraki) fill the corner of a bent tunnel with soil particles excavated from tunnel tip where their digging behavior is activated. The corner-filling behavior, eventually, made a sharp corner smooth-rounded. In the present study, we showed that the corner-filling behavior could play an important role in improving the tunnel traffic efficiency. To do this, we compared the termites' time spent for passing corners between with a right-angled flat tip (RA-corner), corresponding to the sharp corner, and with a rounded tip (R-corner) corresponding to the smooth-rounded corner. As a result, the passing time in the R-corner was significantly shorter than in the RA-corner. In addition, tunnel width effect was discussed in terms of individual movement.