Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris

The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate‐screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N‐methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing “jackpot” clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in‐culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.     

Condition‐dependent expression of pre‐ and postcopulatory sexual traits in guppies

Female choice can impose persistent directional selection on male sexually selected traits, yet such traits often exhibit high levels of phenotypic variation. One explanation for this paradox is that if sexually selected traits are costly, only the fittest males are able to acquire and allocate the resources required for their expression. Furthermore, because male condition is dependent on resource allocation, condition dependence in sexual traits is expected to underlie trade‐offs between reproduction and other life‐history functions. In this study we test these ideas by experimentally manipulating diet quality (carotenoid levels) and quantity in the guppy (Poecilia reticulata), a livebearing freshwater fish that is an important model for understanding relationships between pre‐ and post‐copulatory sexually selected traits. Specifically, we test for condition dependence in the expression of pre‐ and postcopulatory sexual traits (behavior, ornamentation, sperm traits) and determine whether diet manipulation mediates relationships among these traits. Consistent with prior work we found a significant effect of diet quantity on the expression of both pre‐ and postcopulatory male traits; diet‐restricted males performed fewer sexual behaviors and exhibited significant reductions in color ornamentation, sperm quality, sperm number, and sperm length than those fed ad libitum. However, contrary to our expectations, we found no significant effect of carotenoid manipulation on the expression of any of these traits, and no evidence for a trade‐off in resource allocation between pre‐ and postcopulatory episodes of sexual selection. Our results further underscore the sensitivity of behavioral, ornamental, and ejaculate traits to dietary stress, and highlight the important role of condition dependence in maintaining the high variability in male sexual traits.     

Cell perforation mediated by plasmonic bubbles generated by a single near infrared femtosecond laser pulse

We report on transient membrane perforation of living cancer cells using plasmonic gold nanoparticles (AuNPs) enhanced single near infrared (NIR) femtosecond (fs) laser pulse. Under optimized laser energy fluence, single pulse treatment (τ = 45 fs, λ = 800 nm) resulted in 77% cell perforation efficiency and 90% cell viability. Using dark field and ultrafast imaging, we demonstrated that the generation of submicron bubbles around the AuNPs is the necessary condition for the cell membrane perforation. AuNP clustering increased drastically the bubble generation efficiency, thus enabling an effective laser treatment using low energy dose in the NIR optical therapeutical window.

Schematic representation of single femtosecond laser pulse plasmonic bubble generation in the vicinity of a cell.     

Laboratory rearing of solitary bees and wasps

Ecological experiments often require standardized methods that exclude natural variation and allow manipulation of a single parameter. It has been shown that domesticated honey bee larvae are raisable in a controlled environment. Here we demonstrate that this approach is also transferable to wild solitary bees and wasps without inducing negative effects on their development. Wells may also be supplemented with the antibiotic substance oxytetracycline to control the presence of bacteria. The method thus provides a useful tool to investigate offspring recruitment and larval development in solitary bees and wasps, plus their responses to manipulation of factors as for example diets, toxins and microbiota.     

The effect of diet and time after bacterial infection on fecundity,resistance, and tolerance in Drosophila melanogaster

Mounting and maintaining an effective immune response in the face of infection can be costly. The outcome of infection depends on two host immune strategies: resistance and tolerance. Resistance limits pathogen load, while tolerance reduces the fitness impact of an infection. While resistance strategies are well studied, tolerance has received less attention, but is now considered to play a vital role in host–pathogen interactions in animals. A major challenge in ecoimmunology is to understand how some hosts maintain their fitness when infected while others succumb to infection, as well as how extrinsic, environmental factors, such as diet, affect defense. We tested whether dietary restriction through yeast (protein) limitation affects resistance, tolerance, and fecundity in Drosophila melanogaster. We predicted that protein restriction would reveal costs of infection. Because infectious diseases are not always lethal, we tested resistance and tolerance using two bacteria with low lethality: Escherichia coli and Lactococcus lactis. We then assayed fecundity and characterized bacterial infection pathology in individual flies at two acute phase time points after infection. As expected, our four fecundity measures all showed a negative effect of a low‐protein diet, but contrary to predictions, diet did not affect resistance to either bacteria species. We found evidence for diet‐induced and time‐dependent variation in host tolerance to E. coli, but not to L. lactis. Furthermore, the two bacteria species exhibited remarkably different infection profiles, and persisted within the flies for at least 7 days postinfection. Our results show that acute phase infections do not necessarily lead to fecundity costs despite high bacterial loads. The influence of intrinsic variables such as genotype are the prevailing factors that have been studied in relation to variation in host tolerance, but here we show that extrinsic factors should also be considered for their role in influencing tolerance strategies.     

Behavioral and functional strategies during tool use tasks in bonobos

Different primate species have developed extensive capacities for grasping and manipulating objects. However, the manual abilities of primates remain poorly known from a dynamic point of view. The aim of the present study was to quantify the functional and behavioral strategies used by captive bonobos (Pan paniscus) during tool use tasks. The study was conducted on eight captive bonobos which we observed during two tool use tasks: food extraction from a large piece of wood and food recovery from a maze. We focused on grasping postures, in‐hand movements, the sequences of grasp postures used that have not been studied in bonobos, and the kind of tools selected. Bonobos used a great variety of grasping postures during both tool use tasks. They were capable of in‐hand movement, demonstrated complex sequences of contacts, and showed more dynamic manipulation during the maze task than during the extraction task. They arrived on the location of the task with the tool already modified and used different kinds of tools according to the task. We also observed individual manual strategies. Bonobos were thus able to develop in‐hand movements similar to humans and chimpanzees, demonstrated dynamic manipulation, and they responded to task constraints by selecting and modifying tools appropriately, usually before they started the tasks. These results show the necessity to quantify object manipulation in different species to better understand their real manual specificities, which is essential to reconstruct the evolution of primate manual abilities.     

Is the fracto‐mechanoluminescence of ZnS:Mn phosphor dominated by charged dislocation mechanism or piezoelectrification mechanism?

Mathematical approaches made for both the charged dislocation model and piezoelectrically induced electron bombardment model of fracto‐mechanoluminescence (FML), the luminescence induced by fracture of solids, in ZnS:Mn phosphor indicate that the piezoelectrically induced electron bombardment model provides a dominating process for the FML of ZnS phosphors. The concentration of 3000 ppm Mn²⁺ is optimal for ML intensity of ZnS:Mn phosphor. The decay time of ML gives the relaxation time of the piston used to deform the sample and the time t_m of maximum of ML is controlled by both the relaxation time of the piston and decay time of charges on the newly created surfaces of crystals. As the product of the velocity of dislocations and pinning time of dislocations gives the mean free path of a moving dislocation. Both factors play an important role in the ML excitation of impurity doped II–VI semiconductors. The linear increase of total ML intensity I_T with the impact velocity indicates that the damage increases linearly with impact velocity of the load. Thus, the ML measurement can be used remotely to monitor the real‐time damage in the structures, and therefore, the ML of ZnS:Mn phosphor has also the potential for a structural health monitoring system. Copyright © 2015 John Wiley & Sons, Ltd.