Engineering bacteriocin‐mediated resistance against the plant pathogen Pseudomonas syringae

The plant pathogen, Pseudomonas syringae (Ps), together with related Ps species, infects and attacks a wide range of agronomically important crops, including tomato, kiwifruit, pepper, olive and soybean, causing economic losses. Currently, chemicals and introduced resistance genes are used to protect plants against these pathogens but have limited success and may have adverse environmental impacts. Consequently, there is a pressing need to develop alternative strategies to combat bacterial disease in crops. One such strategy involves using narrow‐spectrum protein antibiotics (so‐called bacteriocins), which diverse bacteria use to compete against closely related species. Here, we demonstrate that one bacteriocin, putidacin L1 (PL1), can be expressed in an active form at high levels in Arabidopsis and in Nicotiana benthamiana in planta to provide effective resistance against diverse pathovars of Ps. Furthermore, we find that Ps strains that mutate to acquire tolerance to PL1 lose their O‐antigen, exhibit reduced motility and still cannot induce disease symptoms in PL1‐transgenic Arabidopsis. Our results provide proof‐of‐principle that the transgene‐mediated expression of a bacteriocin in planta can provide effective disease resistance to bacterial pathogens. Thus, the expression of bacteriocins in crops might offer an effective strategy for managing bacterial disease, in the same way that the genetic modification of crops to express insecticidal proteins has proven to be an extremely successful strategy for pest management. Crucially, nearly all genera of bacteria, including many plant pathogenic species, produce bacteriocins, providing an extensive source of these antimicrobial agents.     

Making sense of ultrahigh‐resolution movement data: A new algorithm for inferring sites of interest

Decomposing the life track of an animal into behavioral segments is a fundamental challenge for movement ecology. The proliferation of high‐resolution data, often collected many times per second, offers much opportunity for understanding animal movement. However, the sheer size of modern data sets means there is an increasing need for rapid, novel computational techniques to make sense of these data. Most existing methods were designed with smaller data sets in mind and can thus be prohibitively slow. Here, we introduce a method for segmenting high‐resolution movement trajectories into sites of interest and transitions between these sites. This builds on a previous algorithm of Benhamou and Riotte‐Lambert (2012). Adapting it for use with high‐resolution data. The data’s resolution removed the need to interpolate between successive locations, allowing us to increase the algorithm’s speed by approximately two orders of magnitude with essentially no drop in accuracy. Furthermore, we incorporate a color scheme for testing the level of confidence in the algorithm's inference (high = green, medium = amber, low = red). We demonstrate the speed and accuracy of our algorithm with application to both simulated and real data (Alpine cattle at 1 Hz resolution). On simulated data, our algorithm correctly identified the sites of interest for 99% of “high confidence” paths. For the cattle data, the algorithm identified the two known sites of interest: a watering hole and a milking station. It also identified several other sites which can be related to hypothesized environmental drivers (e.g., food). Our algorithm gives an efficient method for turning a long, high‐resolution movement path into a schematic representation of broadscale decisions, allowing a direct link to existing point‐to‐point analysis techniques such as optimal foraging theory. It is encoded into an R package called SitesInterest , so should serve as a valuable tool for making sense of these increasingly large data streams.     

Prediction of mean adult survival rates of southern African birds from demographic and ecological covariates

Estimates of annual survival rates of birds are valuable in a wide range of studies of population ecology and conservation. These include modelling studies to assess the impacts of climatic change or anthropogenic mortality for many species for which no reliable direct estimates of survival are available. We evaluate the performance of regression models in predicting adult survival rates of birds from values of demographic and ecological covariates available from textbooks and databases. We estimated adult survival for 67 species using dead recoveries of birds ringed in southern Africa and fitted regression models using five covariates: mean clutch size, mean body mass, mean age at first breeding, diet and migratory tendency. Models including these explanatory variables performed well in predicting adult survival in this set of species, both when phylogenetic relatedness of the species was taken into account using phylogenetic generalized least squares (51% of variation in logit survival explained) and when it was not (48%). Two independent validation tests also indicated good predictive power, as indicated by high correlations of observed with expected values in a leave‐one‐out cross validation test performed using data from the 67 species (35% of variation in logit survival explained), and when annual survival rates from independent mark–recapture studies of 38 southern African species were predicted from covariates and the regression using dead recoveries (48%). Clutch size and body mass were the most influential covariates, both with and without the inclusion of phylogenetic effects, and a regression model including only these two variables performed well in both of the validation tests (39 and 48% of variation in logit survival explained). Our regression models, including the version with only clutch size and body mass, are likely to perform well in predicting adult survival rate for southern African species for which direct survival estimates are not available.     

Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non‐invasive determination of these characteristics. We present an image‐processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source‐code for MATLAB and ImageJ is freely available under a permissive open‐source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc.     

Improved expression of recombinant plant-made hEGF

Key message

The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF.

Abstract

Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.     

The influence of recovery duration after heavy resistance exercise on sprint cycling performance

ABSTRACT: Thatcher, R, Gifford, R, and Howatson, G. The influence of recovery duration after heavy resistance exercise on sprint cycling performance. J Strength Cond Res 26(11): 3089-3094, 2012-The aim of this study was to determine the optimal recovery duration after prior heavy resistance exercise (PHRE) when performing sprint cycling. On 5 occasions, separated by a minimum of 48 hours, 10 healthy male subjects (mean ± SD), age 25.5 ± 7.7 years, body mass 82.1 ± 9.0 kg, stature 182.6 ± 87 cm, deadlift 1-repetition maximum (1RM) 142 ± 19 kg performed a 30-second sprint cycling test. Each trial had either a 5-, 10-, 20-, or 30-minute recovery after a heavy resistance activity (5 deadlift repetitions at 85% 1RM) or a control trial with no PHRE in random order. Sprint cycling performance was assessed by peak power (PP), fatigue index, and mean power output over the first 5 seconds (MPO5), 10 seconds (MPO10), and 30 seconds (MPO30). One-way analysis of variance with repeated measures followed by paired t-tests with a Bonferroni adjustment was used to analyze data. Peak power, MPO5, and MPO10 were all significantly different during the 10-minute recovery trial to that of the control condition with values of 109, 112, and 109% of control, respectively; no difference was found for the MPO30 between trials. This study supports the use of PHRE as a strategy to improve short duration, up to, or around 10-second, sprint activity but not longer duration sprints, and a 10-minute recovery appears to be optimal to maximize performance.     

Inhaled adenosine A(2A) receptor agonists for the treatment of chronic obstructive pulmonary disease

COPD is a major cause of mortality in the western world. A(2A) agonists are postulated to reduce the lung inflammation that causes COPD. The cardiovascular effects of A(2A) agonists dictate that a compound needs to be delivered by inhalation to be therapeutically useful. A strategy of minimizing side-effect liability by maximizing systemic clearance was followed and pharmacological and pharmacokinetic SAR of a series of inhaled A(2A) agonists described. A sevenfold improvement in potency and 150-fold reduction in side-effect liability over the lead compound CGS-21680, were obtained.     

Performance of climate envelope models in retrodicting recent changes in bird population size from observed climatic change

Twenty-five-year population trends of 42 bird species rare as breeders in the UK were examined in relation to changes in climatic suitability simulated using climatic envelope models. The effects of a series of potential 'nuisance' variables were also assessed. A statistically significant positive correlation was found across species between population trend and climate suitability trend. The demonstration that climate envelope models are able to retrodict species' population trends provides a valuable validation of their use in studies of the potential impacts of future climatic changes.     

Potential impacts of climatic change on European breeding birds

Background

Climatic change is expected to lead to changes in species'' geographical ranges. Adaptation strategies for biodiversity conservation require quantitative estimates of the magnitude, direction and rates of these potential changes. Such estimates are of greatest value when they are made for large ensembles of species and for extensive (sub-continental or continental) regions.

Methodology/Principal Findings

For six climate scenarios for 2070–99 changes have been estimated for 431 European breeding bird species using models relating species'' distributions in Europe to climate. Mean range centroid potentially shifted 258–882 km in a direction between 341° (NNW) and 45° (NE), depending upon the climate scenario considered. Potential future range extent averaged 72–89% of the present range, and overlapped the present range by an average of 31–53% of the extent of the present range. Even if potential range changes were realised, the average number of species breeding per 50×50 km grid square would decrease by 6·8–23·2%. Many species endemic or near-endemic to Europe have little or no overlap between their present and potential future ranges; such species face an enhanced extinction risk as a consequence of climatic change.

Conclusions/Significance

Although many human activities exert pressures upon wildlife, the magnitude of the potential impacts estimated for European breeding birds emphasises the importance of climatic change. The development of adaptation strategies for biodiversity conservation in the face of climatic change is an urgent need; such strategies must take into account quantitative evidence of potential climatic change impacts such as is presented here.     

Effect of graded fructose coingestion with maltodextrin on exogenous 14C-fructose and 13C-glucose oxidation efficiency and high-intensity cycling performance.

The ingestion of solutions containing carbohydrates with different intestinal transport mechanisms (e.g., fructose and glucose) produce greater carbohydrate and water absorption compared with single-carbohydrate solutions. However, the fructose-ingestion rate that results in the most efficient use of exogenous carbohydrate when glucose is ingested below absorption-oxidation saturation rates is unknown. Ten cyclists rode 2 h at 50% of peak power then performed 10 maximal sprints while ingesting solutions containing (13)C-maltodextrin at 0.6 g/min combined with (14)C-fructose at 0.0 (No-Fructose), 0.3 (Low-Fructose), 0.5 (Medium-Fructose), or 0.7 (High-Fructose) g/min, giving fructose:maltodextrin ratios of 0.5, 0. 8, and 1.2. Mean (percent coefficient of variation) exogenous-fructose oxidation rates during the 2-h rides were 0.18 (19), 0.27 (27), 0.36 (27) g/min in Low-Fructose, Medium-Fructose, and High-Fructose, respectively, with oxidation efficiencies (=oxidation/ingestion rate) of 62-52%. Exogenous-glucose oxidation was highest in Medium-Fructose at 0.57 (28) g/min (98% efficiency) compared with 0.54 (28), 0.48 (29), and 0.49 (19) in Low-Fructose, High-Fructose, No-Fructose, respectively; relative to No-Fructose, only the substantial 16% increase (95% confidence limits +/-16%) in Medium-Fructose was clear. Total exogenous-carbohydrate oxidation was highest in Medium-Fructose at 0.84 (26) g/min. Although the effect of fructose quantity on overall sprint power was unclear, the metabolic responses were associated with lower perceptions of muscle tiredness and physical exertion, and attenuated fatigue (power slope) in the Medium-Fructose and High-Fructose conditions. With the present solutions, low-medium fructose-ingestion rates produced the most efficient use of exogenous carbohydrate, but fatigue and the perception of exercise stress and nausea are reduced with moderate-high fructose doses.