LEC–BiFC: a new method for rapid assay of protein interaction

Abstract

Protein–protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein–protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC–BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl–X_L and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC–BiFC can be applied easily in any lab equipped with a fluorescence microscope.     

Molecular dynamics simulations of the aggregation behaviour of overlapped graphene sheets in linear aliphatic hydrocarbons

Molecular dynamics simulations were used to investigate the aggregation of two partially overlapped graphene sheets in hexane, dodecane and eicosane. When partially overlapped graphene sheets are adjacent to one another, they will expel the adsorbed layers of the solvent molecules on the graphene surface, and the amount of overlap will increase. When the overlapped regions of the graphene sheets are separated by solvent molecules, they cannot expel the adsorption layers between them, and so the sheets remain separated. The driving force for aggregation is the van der Waals interaction between the two graphene sheets, while the van der Waals interaction between the graphene sheets and the solvent molecules inhibits graphene aggregation. The diffusion rate of the hydrocarbon molecules with shorter chain lengths is higher. Thus, they diffuse faster during graphene aggregation, which leads to a higher rate of graphene overlapping in the shorter hydrocarbons. This work provides useful insights into graphene aggregation in linear hydrocarbon solvents of varying lengths at the nanoscale.     

生理信号回归问题的一种解法

一自适应在线过程可通过Kalman滤波表示为时间变多变量的的自回归模型。时变模型的参数的估计能被用以计算线相关的瞬时测量,此方法对生理信号的分析非常有用。本文着重讨论了在呼吸运动,心率波动,血压的分析中的应用。     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin

Cancer-derived extracellular vesicles (EVs) promote tumorigenesis, premetastatic niche formation, and metastasis via their protein cargo. However, the proteins packaged by patient tumors into EVs cannot be determined in vivo because of the presence of EVs derived from other tissues. We therefore developed a cross-species proteomic method to quantify the human tumor-derived proteome of plasma EVs produced by patient-derived xenografts of four cancer types. Proteomic profiling revealed individualized packaging of novel protein cargo, and machine learning accurately classified the type of the underlying tumor.     

Coumarin constituents of Selinum tenuifolium

Three furocoumarins, bergapten, heraclenin and heraclenol have been isolated from the roots of Selinum tenuifolium (Umbelliferae). Another coumarin, m.p. 85–86°, isolated from the same source and provisionally designated as ST-1, has been proved to be mixture of imperatorin, and 8-geranyloxypsolaren by analyses of various reaction products and separation by preparative TLC on silica gel G impregnated with silver nitrate.     

Heterogeneity in the distribution of genetically modified and conventional oilseed rape within fields and seed lots

The implementation of co-existence in the commercialisation of GM crops requires GM and non-GM products to be segregated in production and supply. However, maintaining segregation in oilseed rape will be made difficult by the highly persistent nature of this species. An understanding of its population dynamics is needed to predict persistence and develop potential strategies for control, while to ensure segregation is being achieved, the production of GM oilseed rape must be accompanied by the monitoring of GM levels in crop or seed populations. Heterogeneity in the spatial distribution of oilseed rape has the potential to affect both control and monitoring and, although a universal phenomenon in arable weeds and harvested seed lots, spatial heterogeneity in oilseed rape populations remains to be demonstrated and quantified. Here we investigate the distribution of crop and volunteer populations in a commercial field before and during the cultivation of the first conventional oilseed rape (winter) crop since the cultivation of a GM glufosinate-tolerant oilseed rape crop (spring) three years previously. GM presence was detected by ELISA for the PAT protein in each of three morphologically distinguishable phenotypes: autumn germinating crop-type plants (3% GM), autumn-germinating 'regrowths' (72% GM) and spring germinating 'small-type' plants (17% GM). Statistical models (Poisson log-normal and binomial logit-normal) were used to describe the spatial distribution of these populations at multiple spatial scales in the field and of GM presence in the harvested seed lot. Heterogeneity was a consistent feature in the distribution of GM and conventional oilseed rape. Large trends across the field (50 x 400 m) and seed lot (4 x 1.5 x 1.5 m) were observed in addition to small-scale heterogeneity, less than 20 m in the field and 20 cm in the seed lot. The heterogeneity was greater for the 'regrowth' and 'small' phenotypes, which were likely to be volunteers and included most of the GM plants detected, than for the largely non-GM 'crop' phenotype. The implications of the volunteer heterogeneity for field management and GM-sampling are discussed.     

Distribution of badger latrines in a high-density population: habitat selection and implications for the transmission of bovine tuberculosis to cattle

Eurasian badgers Meles meles habitually deposit droppings and other scent marks at latrines, which may be associated with territorial defence, and communicate information related to group and individual identity and status, and food resources. Understanding patterns of latrine distribution contributes to our understanding of badger social behaviour, and may be relevant to managing the risks of transmission of bovine tuberculosis from badgers to cattle. We investigated the distribution of badger latrines relative to habitat composition in a high-density badger population occupying a 7 km² area of diverse landscape in south-west England. Results indicated that the frequency and density of badger latrines varied according to land use, with woodland and linear landscape features (particularly hedges and stone walls) being positively selected. The number of latrines decreased significantly with distance from linear features. Grassland was negatively selected given its availability, but contained the highest number of latrines. The tendency for latrines to be associated with particular habitat types covaried spatially across the study area. We present a habitat selection probability function, based on the output of our analyses, to allow comparison of observed versus expected latrine counts per habitat type at different sites. Habitat manipulation on farmland may offer opportunities to manage exposure of cattle to badger latrines. However, our analyses indicate that other factors (perhaps demographic or environmental) may also exert a substantial local influence on latrine location.     

Dangerous life at the edge: Implications of seed predation for roadside revegetation

Question

Anthropogenic edges caused by transport infrastructure such as dirt roads and trails (also known as Soft Linear Developments; SLD) are pervasive in almost every terrestrial ecosystem. Revegetating these edges may reduce some of their negative effects, such as their permeability to biological invasions and detrimental effects on wildlife, potentially becoming suitable habitat for a broad range of species. Selecting species with low post‐dispersal seed predation rates may improve the effectiveness of revegetation programmes.

Location

Mediterranean scrublands in SW Spain.

Methods

We made offerings of a total of 16,000 seeds of eight species of fleshy‐fruit shrubs both along SLD edges and scrubland interiors in two independent blocks in each of three distant locations. Using four types of selective enclosure, we assessed the relative contribution of three seed predator guilds (ants, rodents and birds) to seed predation rates both along SLD edges and scrubland interiors.

Results

The effects of anthropogenic edges on seed predation rates were species‐specific. The large and hard‐seeded species Chamaerops humilis was not predated at all. Juniperus phoenicea and Corema album seeds had higher predation rates in scrubland interiors than in edges. The small‐seeded Rubus ulmifolius experienced relatively low seed predation rates compared to the other species. Predation rates for this species were higher along SLD edges than in scrubland interiors. Ants were the main seed predators in the area, and showed marked preferences for J. macrocarpa and C. album seeds at both SLD edges and scrubland interiors.

Conclusions

Our results show the strong context‐dependency of seed predation rates in both SLD edges and scrubland interiors, and thus the importance of well spatially and temporally replicated studies. Species with large and hard seeds may be good candidates for roadside revegetation programmes. However, the relative suitability of plant species would depend on the seed predator community. Our findings confirm that studies on seed predation may help planning cost‐effective species selection for edge revegetation efforts worldwide.