Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

<p>Abstract</p> <p>Background</p> <p>DNA polymerase γ (<it>POLG</it>) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the <it>POLG </it>gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes.</p> <p>Methods</p> <p>mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE.</p> <p>Results</p> <p>We characterise a novel splice site mutation in <it>POLG </it>found <it>in trans </it>with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities.</p> <p>Conclusions</p> <p>mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore<it>, POLG </it>analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.</p

Height and body-mass index trajectories of school-aged children and adolescents from 1985 to 2019 in 200 countries and territories: a pooled analysis of 2181 population-based studies with 65 million participants

Summary Background Comparable global data on health and nutrition of school-aged children and adolescents are scarce. We aimed to estimate age trajectories and time trends in mean height and mean body-mass index (BMI), which measures weight gain beyond what is expected from height gain, for school-aged children and adolescents. Methods For this pooled analysis, we used a database of cardiometabolic risk factors collated by the Non-Communicable Disease Risk Factor Collaboration. We applied a Bayesian hierarchical model to estimate trends from 1985 to 2019 in mean height and mean BMI in 1-year age groups for ages 5–19 years. The model allowed for non-linear changes over time in mean height and mean BMI and for non-linear changes with age of children and adolescents, including periods of rapid growth during adolescence. Findings We pooled data from 2181 population-based studies, with measurements of height and weight in 65 million participants in 200 countries and territories. In 2019, we estimated a difference of 20 cm or higher in mean height of 19-year-old adolescents between countries with the tallest populations (the Netherlands, Montenegro, Estonia, and Bosnia and Herzegovina for boys; and the Netherlands, Montenegro, Denmark, and Iceland for girls) and those with the shortest populations (Timor-Leste, Laos, Solomon Islands, and Papua New Guinea for boys; and Guatemala, Bangladesh, Nepal, and Timor-Leste for girls). In the same year, the difference between the highest mean BMI (in Pacific island countries, Kuwait, Bahrain, The Bahamas, Chile, the USA, and New Zealand for both boys and girls and in South Africa for girls) and lowest mean BMI (in India, Bangladesh, Timor-Leste, Ethiopia, and Chad for boys and girls; and in Japan and Romania for girls) was approximately 9–10 kg/m2. In some countries, children aged 5 years started with healthier height or BMI than the global median and, in some cases, as healthy as the best performing countries, but they became progressively less healthy compared with their comparators as they grew older by not growing as tall (eg, boys in Austria and Barbados, and girls in Belgium and Puerto Rico) or gaining too much weight for their height (eg, girls and boys in Kuwait, Bahrain, Fiji, Jamaica, and Mexico; and girls in South Africa and New Zealand). In other countries, growing children overtook the height of their comparators (eg, Latvia, Czech Republic, Morocco, and Iran) or curbed their weight gain (eg, Italy, France, and Croatia) in late childhood and adolescence. When changes in both height and BMI were considered, girls in South Korea, Vietnam, Saudi Arabia, Turkey, and some central Asian countries (eg, Armenia and Azerbaijan), and boys in central and western Europe (eg, Portugal, Denmark, Poland, and Montenegro) had the healthiest changes in anthropometric status over the past 3·5 decades because, compared with children and adolescents in other countries, they had a much larger gain in height than they did in BMI. The unhealthiest changes—gaining too little height, too much weight for their height compared with children in other countries, or both—occurred in many countries in sub-Saharan Africa, New Zealand, and the USA for boys and girls; in Malaysia and some Pacific island nations for boys; and in Mexico for girls. Interpretation The height and BMI trajectories over age and time of school-aged children and adolescents are highly variable across countries, which indicates heterogeneous nutritional quality and lifelong health advantages and risks

Results from a round robin test for the ecotoxicological evaluation of construction products using two leaching tests and an aquatic test battery

A European round robin test according to ISO 5725-2 was conceptually prepared, realised, and evaluated. The aim was to determine the inter-laboratory variability of the overall process for the ecotoxicological characterization of construction products in eluates and bioassays. To this end, two construction products BAM-G1 (granulate) and HSR-2 (roof sealing sheet), both made of EPDM polymers (rubber), were selected. The granular construction product was eluted in a one stage batch test, the planar product in the Dynamic Surface Leaching test (DSLT). A total of 17 laboratories from 5 countries participated in the round robin test: Germany (12), Austria (2), Belgium (1), Czech Republic (1) and France (1). A test battery of four standardised ecotoxicity tests with algae, daphnia, luminescent bacteria and zebrafish eggs was used. As toxicity measures, EC50 and LID values were calculated. All tests, except the fish egg test, were basically able to demonstrate toxic effects and the level of toxicity. The reproducibility of test results depended on the test specimens and the test organisms. Generally, the variability of the EC50 or LID values increased with the overall level of toxicity. For the very toxic BAM-G1 eluate a relative high variability of CV = 73%–110% was observed for EC50 in all biotests, while for the less toxic HSR-2 eluate the reproducibility of EC50 varied with sensitivity: it was very good (CV = 9.3%) for the daphnia test with the lowest sensitivity, followed by the algae test (CV = 36.4%). The luminescent bacteria test, being the most sensitive bioassay for HSR-2 Eluate, showed the highest variability (CV = 74.8%). When considering the complex overall process the reproducibility of bioassays with eluates from construction products was acceptable

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field