Impact of Overhead Evaporative Cooling, Canopy Location, Sunlight Exposure, Inoculation Level, Region, and Growing Season on the Survival of Generic Escherichia coli on in-Field Fuji Apples.

AIMS: The survival of inoculated Escherichia coli on Fuji apples in Washington State orchards was studied, considering evaporative cooling, canopy location, year, and region, with the examination of sunlight exposure and inoculation levels in year 2. METHODS AND RESULTS: Rifampicin-resistant E. coli was applied to Fuji apples. Initial concentrations for the high-inoculation study were 7.4 ± 0.3 log10 CFU per apple and 3.4 ± 0.3 log10 CFU per apple for the low-inoculation study. Enumeration of E. coli was conducted at 0, 2, 10, 18, 34, 42, 58, 82, 106, and 154 h after inoculation. Results were analyzed using Tukey's honest significance difference test and a log-linear model. Log-linear, Weibull, and Biphasic models characterized E. coli die-off patterns for high and low inoculations. The application of evaporative overhead cooling water did not significantly influence E. coli survival on Fuji apples; inoculation level and sunlight exposure were significant factors in a log-linear model. E. coli decreased by 5.5 ± 1.3 and 3.3 ± 0.4 log10 CFU per apple for high and low inoculated apples, respectively, by 154 h. The Biphasic model best explained the die-off pattern for high and low-inoculated Fuji apples. CONCLUSIONS: Overhead evaporative cooling, a useful fruit quality practice, did not impact the survival of generic E. coli on Fuji apple surfaces. The significant impact of sunlight exposure and inoculation levels on die-off highlights the importance of ultraviolet radiation in risk reduction and the need for various inoculum concentrations in preharvest field studies.

Cytoskeletal organization through multivalent interactions.

The cytoskeleton consists of polymeric protein filaments with periodic lattices displaying identical binding sites, which establish a multivalent platform for the binding of a plethora of filament-associated ligand proteins. Multivalent ligand proteins can tether themselves to the filaments through one of their binding sites, resulting in an enhanced reaction kinetics for the remaining binding sites. In this Opinion, we discuss a number of cytoskeletal phenomena underpinned by such multivalent interactions, namely (1) generation of entropic forces by filament crosslinkers, (2) processivity of molecular motors, (3) spatial sorting of proteins, and (4) concentration-dependent unbinding of filament-associated proteins. These examples highlight that cytoskeletal filaments constitute the basis for the formation of microenvironments, which cytoskeletal ligand proteins can associate with and, once engaged, can act within at altered reaction kinetics. We thus argue that multivalency is one of the properties crucial for the functionality of the cytoskeleton.

Dihydropyrrolopyrazol-6-one MCHR1 antagonists for the treatment of obesity: Insights on in vivo efficacy from a novel FLIPR assay setup.

Our investigation of the structure-activity and structure-liability relationships for dihydropyrrolopyrazol-6-one MCHR1 antagonists revealed that off-rate characteristics, inferred from potencies in a FLIPR assay following a 2 h incubation, can impact in vivo efficacy. The in vitro and exposure profiles of dihydropyrrolopyrazol-6-ones 1b and 1e were comparable to that of the thienopyrimidinone counterparts 41 and 43 except for a much faster MCHR1 apparent off-rate. The greatly diminished dihydropyrrolopyrazol-6-one anti-obesity response may be the consequence of this rapid off-rate.

Non-basic azolotriazinone MCHR1 antagonists for the treatment of obesity: An empirical brain-exposures-driven candidate selection for in vivo efficacy studies.

Non-basic azolotriazinones were explored using an empirical free brain exposures-driven approach to identify potent MCHR1 antagonists for evaluation in in vivo efficacy studies. An optimized lead from this series, 1j (rMCHR1 Ki=1.8 nM), demonstrated a 6.9% reduction in weight gain relative to vehicle in a rat model at 30 mg/kg after 4 days of once-daily oral treatment as a glycine prodrug. Despite a promising efficacy profile, an assessment of the biliary toxicity risk of this compound rendered this compound non-progressible.

Overall avidity declines in TCR repertoires during latent CMV but not EBV infection.

Introduction: The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. Methods: To address these issues, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCRαß clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. The parameters involved during the long-term persistence of virus-specific T cell clonotypes were further evaluated by gene expression profiling, phenotype and functional analyses. Results: Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. Conclusions: These findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data further suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.

Comparing strategies for matching mortality forecasts to the most recently observed data: exploring the trade-off between accuracy and robustness.

BACKGROUND: Given the increased link between retirement age and payments to the development in life expectancy, a precise and regular forecast of life expectancy is of utmost importance. The choice of the jump-off rates, i.e. the rates in the last year of the fitting period, is essential for matching mortality forecasts to the most recently observed data. A general approach to the choice of the jump-off rates is currently lacking. OBJECTIVE: We evaluate six different options for the jump-off rates and examine their effects on the robustness and accuracy of the mortality forecast. DATA AND METHODS: Death and exposure numbers by age for eight European countries over the years 1960-2014 were obtained from the Human Mortality Database. We examined the use of model values as jump-off rates versus observed values in the last year or averaged over the last couple of years. The future life expectancy at age 65 is calculated for different fitting periods and jump-off rates using the Lee-Carter model and examined on accuracy (mean absolute forecast error) and robustness (standard deviation of the change in projected e65). RESULTS: The choice for the jump-off rates clearly influences the accuracy and robustness of the mortality forecast, albeit in different ways. For most countries using the last observed values as jump-off rates resulted in the most accurate method, which relates to the relatively high estimation error of the model in recent years. The most robust method is obtained by using an average of observed years as jump-off rates. The more years that are averaged, the better the robustness, but accuracy decreases with more years averaged. CONCLUSION: Carefully considering the best choice for the jump-off rates is essential when forecasting mortality. The best strategy for matching mortality forecasts to the most recently observed data depends on the goal of the forecast, the country-specific past mortality trends observed, and the model fit.

A short review of fecal indicator bacteria in tropical aquatic ecosystems: knowledge gaps and future directions.

Given the high numbers of deaths and the debilitating nature of diseases caused by the use of unclean water it is imperative that we have an understanding of the factors that control the dispersion of water borne pathogens and their respective indicators. This is all the more important in developing countries where significant proportions of the population often have little or no access to clean drinking water supplies. Moreover, and notwithstanding the importance of these bacteria in terms of public health, at present little work exists on the persistence, transfer and proliferation of these pathogens and their respective indicator organisms, e.g., fecal indicator bacteria (FIB) such as Escherichia coli and fecal coliforms in humid tropical systems, such as are found in South East Asia or in the tropical regions of Africa. Both FIB and the waterborne pathogens they are supposed to indicate are particularly susceptible to shifts in water flow and quality and the predicted increases in rainfall and floods due to climate change will only exacerbate the problems of contamination. This will be furthermore compounded by the increasing urbanization and agricultural intensification that developing regions are experiencing. Therefore, recognizing and understanding the link between human activities, natural process and microbial functioning and their ultimate impacts on human health are prerequisites for reducing the risks to the exposed populations. Most of the existing work in tropical systems has been based on the application of temperate indicator organisms, models and mechanisms regardless of their applicability or appropriateness for tropical environments. Here, we present a short review on the factors that control FIB dynamics in temperate systems and discuss their applicability to tropical environments. We then highlight some of the knowledge gaps in order to stimulate future research in this field in the tropics.