Ammonium Accumulation and Cell Death in a Rat 3D Brain Cell Model of Glutaric Aciduria Type I

Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.     

Estimated Effects of Different Alcohol Taxation and Price Policies on Health Inequalities: A Mathematical Modelling Study

Introduction

While evidence that alcohol pricing policies reduce alcohol-related health harm is robust, and alcohol taxation increases are a WHO “best buy” intervention, there is a lack of research comparing the scale and distribution across society of health impacts arising from alternative tax and price policy options. The aim of this study is to test whether four common alcohol taxation and pricing strategies differ in their impact on health inequalities.

Methods and Findings

An econometric epidemiological model was built with England 2014/2015 as the setting. Four pricing strategies implemented on top of the current tax were equalised to give the same 4.3% population-wide reduction in total alcohol-related mortality: current tax increase, a 13.4% all-product duty increase under the current UK system; a value-based tax, a 4.0% ad valorem tax based on product price; a strength-based tax, a volumetric tax of £0.22 per UK alcohol unit (= 8 g of ethanol); and minimum unit pricing, a minimum price threshold of £0.50 per unit, below which alcohol cannot be sold. Model inputs were calculated by combining data from representative household surveys on alcohol purchasing and consumption, administrative and healthcare data on 43 alcohol-attributable diseases, and published price elasticities and relative risk functions. Outcomes were annual per capita consumption, consumer spending, and alcohol-related deaths. Uncertainty was assessed via partial probabilistic sensitivity analysis (PSA) and scenario analysis.The pricing strategies differ as to how effects are distributed across the population, and, from a public health perspective, heavy drinkers in routine/manual occupations are a key group as they are at greatest risk of health harm from their drinking. Strength-based taxation and minimum unit pricing would have greater effects on mortality among drinkers in routine/manual occupations (particularly for heavy drinkers, where the estimated policy effects on mortality rates are as follows: current tax increase, −3.2%; value-based tax, −2.9%; strength-based tax, −6.1%; minimum unit pricing, −7.8%) and lesser impacts among drinkers in professional/managerial occupations (for heavy drinkers: current tax increase, −1.3%; value-based tax, −1.4%; strength-based tax, +0.2%; minimum unit pricing, +0.8%). Results from the PSA give slightly greater mean effects for both the routine/manual (current tax increase, −3.6% [95% uncertainty interval (UI) −6.1%, −0.6%]; value-based tax, −3.3% [UI −5.1%, −1.7%]; strength-based tax, −7.5% [UI −13.7%, −3.9%]; minimum unit pricing, −10.3% [UI −10.3%, −7.0%]) and professional/managerial occupation groups (current tax increase, −1.8% [UI −4.7%, +1.6%]; value-based tax, −1.9% [UI −3.6%, +0.4%]; strength-based tax, −0.8% [UI −6.9%, +4.0%]; minimum unit pricing, −0.7% [UI −5.6%, +3.6%]). Impacts of price changes on moderate drinkers were small regardless of income or socioeconomic group. Analysis of uncertainty shows that the relative effectiveness of the four policies is fairly stable, although uncertainty in the absolute scale of effects exists. Volumetric taxation and minimum unit pricing consistently outperform increasing the current tax or adding an ad valorem tax in terms of reducing mortality among the heaviest drinkers and reducing alcohol-related health inequalities (e.g., in the routine/manual occupation group, volumetric taxation reduces deaths more than increasing the current tax in 26 out of 30 probabilistic runs, minimum unit pricing reduces deaths more than volumetric tax in 21 out of 30 runs, and minimum unit pricing reduces deaths more than increasing the current tax in 30 out of 30 runs). Study limitations include reducing model complexity by not considering a largely ineffective ban on below-tax alcohol sales, special duty rates covering only small shares of the market, and the impact of tax fraud or retailer non-compliance with minimum unit prices.

Conclusions

Our model estimates that, compared to tax increases under the current system or introducing taxation based on product value, alcohol-content-based taxation or minimum unit pricing would lead to larger reductions in health inequalities across income groups. We also estimate that alcohol-content-based taxation and minimum unit pricing would have the largest impact on harmful drinking, with minimal effects on those drinking in moderation.     

Anti-tumoral,anti-angiogenic and anti-metastatic efficacy of a tetravalent bispecific antibody (TAvi6) targeting VEGF-A and angiopoietin-2

Vascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.     

Is individual consistency in body mass and reproductive decisions linked to individual specialization in foraging behavior in a long‐lived seabird?

Individual specialization in diet or foraging behavior within apparently generalist populations has been described for many species, especially in polar and temperate marine environments, where resource distribution is relatively predictable. It is unclear, however, whether and how increased environmental variability – and thus reduced predictability of resources – due to global climate change will affect individual specialization. We determined the within‐ and among‐individual components of the trophic niche and the within‐individual repeatability of δ¹³C and δ¹⁵N in feathers and red blood cells of individual female southern rockhopper penguins (Eudyptes chrysocome) across 7 years. We also investigated the effect of environmental variables (Southern Annular Mode, Southern Oscillation Index, and local sea surface temperature anomaly) on the isotopic values, as well as the link between stable isotopes and female body mass, clutch initiation dates, and total clutch mass. We observed consistent red blood cell δ¹³C and δ¹⁵N values within individuals among years, suggesting a moderate degree of within‐individual specialization in C and N during the prebreeding period. However, the total niche width was reduced and individual specialization not present during the premolt period. Despite significant interannual differences in isotope values of C and N and environmental conditions, none of the environmental variables were linked to stable isotope values and thus able to explain phenotypic plasticity. Furthermore, neither the within‐individual nor among‐individual effects of stable isotopes were found to be related to female body mass, clutch initiation date, or total clutch mass. In conclusion, our results emphasize that the degree of specialization within generalist populations can vary over the course of 1 year, even when being consistent within the same season across years. We were unable to confirm that environmental variability counteracts individual specialization in foraging behavior, as phenotypic plasticity in δ¹³C and δ¹⁵N was not linked to any of the environmental variables studied.     

Flexible Strategies for Coping with Rainfall Variability: Seasonal Adjustments in Cropped Area in the Ganges Basin

One of the main manifestations of climate change will be increased rainfall variability. How to deal with this in agriculture will be a major societal challenge. In this paper we explore flexibility in land use, through deliberate seasonal adjustments in cropped area, as a specific strategy for coping with rainfall variability. Such adjustments are not incorporated in hydro-meteorological crop models commonly used for food security analyses. Our paper contributes to the literature by making a comprehensive model assessment of inter-annual variability in crop production, including both variations in crop yield and cropped area. The Ganges basin is used as a case study. First, we assessed the contribution of cropped area variability to overall variability in rice and wheat production by applying hierarchical partitioning on time-series of agricultural statistics. We then introduced cropped area as an endogenous decision variable in a hydro-economic optimization model (WaterWise), coupled to a hydrology-vegetation model (LPJmL), and analyzed to what extent its performance in the estimation of inter-annual variability in crop production improved. From the statistics, we found that in the period 1999–2009 seasonal adjustment in cropped area can explain almost 50% of variability in wheat production and 40% of variability in rice production in the Indian part of the Ganges basin. Our improved model was well capable of mimicking existing variability at different spatial aggregation levels, especially for wheat. The value of flexibility, i.e. the foregone costs of choosing not to crop in years when water is scarce, was quantified at 4% of gross margin of wheat in the Indian part of the Ganges basin and as high as 34% of gross margin of wheat in the drought-prone state of Rajasthan. We argue that flexibility in land use is an important coping strategy to rainfall variability in water stressed regions.     

Inhaled Methane Limits the Mitochondrial Electron Transport Chain Dysfunction during Experimental Liver Ischemia-Reperfusion Injury

Background

Methanogenesis can indicate the fermentation activity of the gastrointestinal anaerobic flora. Methane also has a demonstrated anti-inflammatory potential. We hypothesized that enriched methane inhalation can influence the respiratory activity of the liver mitochondria after an ischemia-reperfusion (IR) challenge.

Methods

The activity of oxidative phosphorylation system complexes was determined after in vitro methane treatment of intact liver mitochondria. Anesthetized Sprague-Dawley rats subjected to standardized 60-min warm hepatic ischemia inhaled normoxic air (n = 6) or normoxic air containing 2.2% methane, from 50 min of ischemia and throughout the 60-min reperfusion period (n = 6). Measurement data were compared with those on sham-operated animals (n = 6 each). Liver biopsy samples were subjected to high-resolution respirometry; whole-blood superoxide and hydrogen peroxide production was measured; hepatocyte apoptosis was detected with TUNEL staining and in vivo fluorescence laser scanning microscopy.

Results

Significantly decreased complex II-linked basal respiration was found in the normoxic IR group at 55 min of ischemia and a lower respiratory capacity (~60%) and after 5 min of reperfusion. Methane inhalation preserved the maximal respiratory capacity at 55 min of ischemia and significantly improved the basal respiration during the first 30 min of reperfusion. The IR-induced cytochrome c activity, reactive oxygen species (ROS) production and hepatocyte apoptosis were also significantly reduced.

Conclusions

The normoxic IR injury was accompanied by significant functional damage of the inner mitochondrial membrane, increased cytochrome c activity, enhanced ROS production and apoptosis. An elevated methane intake confers significant protection against mitochondrial dysfunction and reduces the oxidative damage of the hepatocytes.     

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.     

Propensity Scoring after Multiple Imputation in a Retrospective Study on Adjuvant Radiation Therapy in Lymph-Node Positive Vulvar Cancer

Propensity scoring (PS) is an established tool to account for measured confounding in non-randomized studies. These methods are sensitive to missing values, which are a common problem in observational data. The combination of multiple imputation of missing values and different propensity scoring techniques is addressed in this work. For a sample of lymph node-positive vulvar cancer patients, we re-analyze associations between the application of radiotherapy and disease-related and non-related survival. Inverse-probability-of-treatment-weighting (IPTW) and PS stratification are applied after multiple imputation by chained equation (MICE). Methodological issues are described in detail. Interpretation of the results and methodological limitations are discussed.