Study of non-conventional Aspergilli and Penicillia Ochratoxin A producers

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2019The continuous growth of the population is pushing the available natural resources to the limit, posing a threat to the balance of the planet. This increase in population means that more food is needed. Consequently, food safety is, nowadays, a matter of interest to the scientific community and world leaders. However, problems related to food/feed contamination have been frequently reported, including those related to fungi. Fungi are diverse, ubiquitous and their role and impact in food security is now an urgent concern. One thing that makes fungi so dangerous is their ability to resist the treatments during the food making process and their ability to produce harmful secondary metabolites like mycotoxins. Mycotoxins are fungal secondary metabolites that can cause severe health issues in either humans or domesticated animals when ingested, inhaled and/or absorbed. From all the mycotoxins reported, some are more studied due to the higher health risks. Ochratoxin A (OTA) is among those and is present in several food/feed products. Aspergillus and Penicillium species are commonly associated with food spoilage and both genera contain species that can produce this toxin. The major OTA producers are P. verrucosum, P. nordicum and A. carbonarius, but more than 20 species of Aspergillus can produce this toxin. However, recent studies reported the presence of OTA in food matrices where known OTA producers are not present. This triggered the scientists involved to try to find the origin of OTA. Based on previous evidence other species like P. crustosum and A. fumigatus are now being considered. Therefore, the main goal of this work was to search for potential OTA producers among P. crustosum and A. fumigatus strains, with different geographic origins, and try to find potential genetic differences at the sub-species level. A set of 28 Penicillium crustosum strains and 7 Aspergillus fumigatus strains, kindly supplied by Micoteca da Universidade do Minho (MUM), and 16 Penicillium crustosum strains, by Colección Chilena de Cultivos Tipo (CCCT), were studied. The Penicillium isolates are from four different countries: 16 from Italy, 16 from Chile, 6 from Portugal and 4 from Tunisia. The Aspergillus strains are all from Portugal, Spain or with unknown origin. Mycotoxin production was analysed by HPLC-FLD and results were compared with a standard sample. In addition, genes associated with OTA production [two ochratoxin polyketide synthase (Penicillium and Aspergillus related), ochratoxin non-ribosomal peptide synthetase and an ochratoxin transport protein] were tested. RAPD-PCR fingerprinting [M13 and (GACA)4] and beta-tubulin gene (BenA) sequencing were used to perform a wide molecular characterisation. Under the studied conditions, and with a HPLC-FLD detection limit of 7.6 ng/ml, preliminary results showed that OTA was not detected for all studied strains. However, regarding the genes associated with OTA production, there were 4 positive strains of P. crustosum for the 3 genes. Genetic differences, based on RAPD fingerprints, between P. crustosum isolates were found allowing the clustering of strains from the same geographic region, except for isolates from Europe. The low number of A. fumigatus strains did not allowed to draw conclusions, although they also presented genetic differences. Sequencing with BenA did not revealed any SNPs. Nevertheless, further studies with a broader array of conditions needs to be considered.O crescimento exponencial da população mundial está a levar ao limite os recursos naturais disponíveis representando um risco para o equilíbrio do planeta. O aumento da população tem como consequência o aumento da necessidade de mais alimentos. Consequentemente, a segurança alimentar é, hoje em dia, uma preocupação para a comunidade científica e para os líderes mundiais. Contudo, problemas relacionados com comida e rações animais contaminadas têm sido frequentemente reportados, incluindo aqueles com fungos. Os fungos são um grupo de organismos eucarióticos, diversos, ubíquos e têm um grande impacto na segurança alimentar. A capacidade de os fungos resistirem aos processos da indústria alimentar, como tratamentos térmicos, e a sua capacidade de produzirem metabolitos secundários prejudiciais, torna-os perigosos contaminantes. Um exemplo desses metabolitos produzidos são as micotoxinas. As micotoxinas quando ingeridas, inaladas e/ou absorvidas podem causar problemas severos de saúde a pessoas e animais. A preocupação com este assunto iniciou-se aquando da morte de cerca de 10000 perus no reino unido, que se deveu a uma contaminação com aflatoxinas. Aspergillus e Penicillium são dois grupos de fungos filamentosos que têm um grande impacto na contaminação alimentar particularmente pela capacidade de produzirem micotoxinas. As micotoxinas mais associadas a estes géneros são aflatoxinas, ocratoxina A, patulina e citrinina. A maior preocupação com estas toxinas é a sua toxicidade aguda e/ou crónica, que pode levar à morte ou a outras patologias. A maioria das micotoxinas são estáveis, resistentes ao calor e podem permanecer mesmo em produtos tratados (por exemplo, produtos pasteurizados). De todas as micotoxinas, a ocratoxina A (OTA) é uma das mais estudadas, devido à sua presença em vários produtos alimentares e também pelos problemas de saúde que pode causar. A OTA foi descoberta em 1965 no Aspergillus ochraceus. É considerada neurotóxica, nefrotóxica, carcinogénica, hepatotóxica e teratogénica, para diversas espécies. Está classificada com o grupo 2B - possivelmente carcinogénica para humanos. Está presente em diversos produtos alimentares como cereais, vinho, queijo e café. De entre os cereais, o centeio, o trigo e a cevada são os que apresentam os maiores níveis de contaminação. Isto representa um problema pois estes cereais são muito usados no fabrico de farinhas e rações destinadas à alimentação de animais. Consequentemente, os animais domésticos são os mais suscetíveis a ter reações adversas devido à presença da toxina, uma vez que estão expostas a ela por mais tempo e com maior frequência. Os maiores produtores de OTA são P. verrucosum, P. nordicum e A. carbonarius, no entanto cerca de mais 20 espécies de Aspergillus são capazes de a sintetizar. A identificação e a quantificação da OTA são comummente feitas por cromatografia líquida de alta eficiência com um detetor de fluorescência (HPLC-FLD). Além disso há genes que estão associados com a ocratoxina A e que podem ser usados como possíveis marcadores genéticos da capacidade de um fungo a produzir. Recentemente, um estudo identificou a presença de OTA em diversas amostras de queijos Italianos. Contudo, não foi possível identificar nenhum dos conhecidos produtores da toxina. Assim, surgiu a dúvida de qual seria a origem da OTA. Baseados em estudos anteriores, P. crustosum e A. fumigatus estão a ser considerados como possíveis novos produtores. Estes fungos são ubíquos e são diversas vezes encontrados em produtos alimentares. São produtores de toxinas, mas apenas foram descritos uma (A. fumigatus) ou duas (P. crustosum) vezes como produtores de OTA. O objetivo principal deste trabalho é procurar potenciais produtores de OTA entre um conjunto de estirpes de P. crustosum e de A. fumigatus (com diferentes origens geográficas) e tentar encontrar potenciais diferenças ao nível da subespécie. Um conjunto de 28 estirpes de P. crustosum e 7 estirpes de A. fumigatus fornecidas pela Micoteca da Universidade do Minho (MUM), e 16 estirpes de P. crustosum, fornecidas pela Colección Chilena de Cultivos Tipo (CCCT), foram estudadas. Os isolados de Penicillium provieram de quatro países e matrizes diferentes: 16 foram isolados de queijos italianos, 16 de merkén do Chile, 6 de diferentes origens em Portugal e 4 de maçãs da Tunísia. As estirpes de Aspergillus são, na sua maioria, de Portugal ou Espanha, no entanto algumas têm origem desconhecida. A produção de ocratoxina A foi avaliada por HPLC-FLD e os resultados foram comparados com um padrão de concentração conhecida. As estirpes de P. crustosum foram cultivadas em meio enriquecido com sal de forma a terem um stress externo que conduza à produção de micotoxinas. Genes relacionados com a produção de OTA (PKS, NRPS e um transportador) foram também procurados. Adicionalmente foi feito um estudo genético complementar. Apesar de todas as estirpes terem origem em coleções de culturas, o que à partida valida a sua identificação, o gene da beta tubulina foi amplificado. Uma vez que o estudo envolve uma panóplia de estirpes da mesma espécie, principalmente em P. crustosum, com origens geográficas diferentes, há uma possibilidade de nessas estirpes terem ocorrido processos de especiação e haver espécies crípticas por revelar. Espécies crípticas são espécies que são morfologicamente idênticas, no entanto apresentam diferenças genéticas. Para complementar este estudo foram feitas análises de perfis genómicos (fingerprinting) usando pequenos primers: M13 e (GACA)4. Nas condições testadas, tendo a HPLC um limite de deteção de 7,6 ng/ml, os resultados preliminares mostraram que não se detetou produção de OTA para nenhuma das 51 estirpes. Os cromatogramas obtidos de cada estirpe foram comparados com o cromatograma padrão, onde foi possível observar um pico aos 13 min. No entanto, em relação aos genes relacionados com a biossíntese de OTA os resultados foram mais promissores, exceto para o gene relacionado com a produção de OTA em Aspergillus (Acpks). Para esse gene apenas o controlo positivo foi amplificado e não em nenhuma das estirpes em estudo. Os restantes genes estavam relacionados com Penicillium [otapks (≈500 bp); otanps (≈700bp) e otatra (≈420 bp)]. Para estes genes, 8% estirpes amplificaram todos os genes (sendo todas do Chile), 16% amplificaram somente otapks; 10% otapks e otatra, 8% otanps e otatra; 45% apenas otatra; e 14% não amplificaram nenhum gene. O facto de haver espécies a amplificarem os três genes testados é um indicador que estas podem ter a capacidade de produzir OTA. Além disso a grande diversidade de padrões verificados mostra que as estirpes são distintas entre si. O estudo com os primers de fingerprinting de onde se obtiveram perfis genéticos e com os quais foram construídos dendrogramas mostraram a mesma distinção. Diferentes padrões foram obtidos podendo individualizar cada indivíduo. Além disso, fazendo uma análise aos dendrogramas foi possível verificar que as estirpes chilenas formam um cluster assim como as estirpes da Tunísia. Isto pode demonstrar que a distância geográfica poderá criar processos de especiação. Por outro lado, as estirpes Italianas e Portuguesas não formam nenhum cluster. Estes dois países encontram-se na Europa onde não há muitas restrições de fronteiras. Em relação a A. fumigatus, o baixo número de estirpes, e a falta de informação de algumas delas, não permitiu a formação de clusters, apenas se verificando que também apresentam perfis diferentes. Este foi um estudo preliminar em que não foi possível a quantificação de OTA nem em P. crustosum nem em A. fumigatus. No entanto, os resultados dos genes biossintéticos mostram que algumas das estirpes podem ter a capacidade de a produzir. Também foi possível verificar que RAPD é uma boa técnica de tipificação e que espécies afastadas geograficamente apresentam perfis genéticos diferentes. Não obstante, estudos com métodos adicionais e com diferentes condições precisam ser considerados

Mitigation of Ochratoxin A in the food chain, from prevention to remediation

Mycotoxins are metabolites produced by a few filamentous fungi, that are ubiquitous in Nature, being found in many food crops. Their toxicity to humans demands a very strict control under a properly designed food safety program. Also, food losses due to fungal deterioration raise food security concerns. Mitigation actions to avoid or reduce human exposure to mycotoxins start in the field, where most mycotoxin producing fungi are active and mycotoxin accumulation starts. These actions include strategies to prevent mycotoxin-producing fungi from proliferating in the food or feed, to prevent these same fungi to produce the toxins, and to either remove, segregate or degrade the mycotoxins that have been produced. Using the case of ochratoxin A in our food, different strategies to mitigate contamination, from the screening of mycotoxigenic strains, integrated in a preventive approach, to the use of enzymes, as a remediation approach, will be discussed in this presentation. The screening of mycotoxin-producing strains will be discussed based on a microbiome approach, where the producing fungi may be spotted without their isolation, while the use of enzymes will be discussed along with a molecular modelling approach to elucidate enzymatic activity. The authors are grateful for the PhD support grants from the Portuguese Foundation for Science and Technology (FCT): 2020.05849.BD. (Teresa Vale Dias) and UI/BD/152286/2021 (Joana Santos).The authors are grateful for the PhD support grants from the Portuguese Foundation for Science and Technology (FCT): 2020.05849.BD. (Teresa Vale Dias) and UI/BD/152286/2021 (Joana Santos).info:eu-repo/semantics/publishedVersio

Mycotoxigenic potential of fungi isolated from highly cured Portuguese cheese

The cheese industry in Portugal offers a plethora of high-quality products. The São Jorge cheese is an example and has obtained the Protected Designation of Origin (PDO) certification in 1986. This cheese has long ripening periods of up to 36 months, which raises concerns regarding food losses and health risks due to fungal proliferation. In this study, the mycobiota of three São Jorge cheese samples with different ripening periods (five, nine and thirty months) was studied to predict the associated mycotoxigenic risk. From the three cheese samples, 76 fungal isolates were identified through molecular methods (analysis of ITS and/or partial benA). Penicillium spp. ser. Camembertiorum, mainly P. solitum and P. echinulatum, were present in all the analyzed cheeses. Scopulariopsis spp. and some yeasts (predominantly Saccharomyces cerevisiae) were also part of the mycobiota of the cheeses. Although none of these species is a common producer of mycotoxins, an analysis of the cheese by mass spectrometry will be carried out. Furthermore, the overall mycobiota will be studied through metabarcoding to uncover the presence of potential mycotoxigenic species that have not been isolated through the culturomics approach.Teresa Vale Dias thanks for the Ph.D. scholarship given by the Foundation for Science and Technology (FCT) - 2020.05849.BD. This study was also supported by FCT under the scope of the strategic funding of CEB (UIDB/04469/2020), LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems (LA/P/0029/2020), CIMO (UIDB/00690/2020) and SusTEC (LA/P/0007/2020).info:eu-repo/semantics/publishedVersio

Is hand disinfectant a public health problem? A case study in the Centre of Biological Engineering

For about three years, the world was faced with the CoVID-19 pandemic that showed us the threats of unknown microorganisms. At the beginning of the pandemic several behavioural rules for prevention and protection were imposed. Besides the use of masks, ethanol solutions for hand disinfection were one of the most common measures. Public recipients of alcohol were seen and accessible in every public and private institutions and commercial places. However, with the official end of the pandemic this year (and even before that), it was possible to observe the abandoning of these recipients. Some stay in place empty and others still with the ethanol-glycerol solution. In the Centre of Biological Engineering building, we came to notice that some of these recipients were colonized with fungi. The aim of this work was to collect the fungal colonies and try to assess which fungi are present and their taxonomical variation. Fugal colonies were collected from 4 different recipients. The number of isolates from each recipient ranged from 2 to 10. Based on morphological characteristics it was possible to identify some of the isolates as Aspergillus sp., Penicillium sp. and Cladosporium sp. To complement this information molecular identification is being carried out to try to get identification at species level. We acknowledge that culture collections have a leading role in the search and preservation of microorganisms as well as public warning of their potential risks. Consequently, we intend to expand the sampling spots and make recommendations to the university staff (and the population in general) to assure that this is not a potential public health problem.Teresa Vale Dias thanks for the Ph.D. scholarship given by the Foundation for Science and Technology (FCT) - 2020.05849.BD. This study was also supported by FCT under the scope of the strategic funding of CEB (UIDB/04469/2020).info:eu-repo/semantics/publishedVersio

Mycobiota of São Jorge cheeses with different ripening periods

The growth of filamentous fungi in the cheese surface makes the product undesirable (and therefore disposable) and can even present a health risk due to the production of secondary metabolites, such as mycotoxins. The São Jorge cheese is a highly appreciated product from São Jorge Island, Azores, Portugal. It is made with raw cow milk and has long ripening periods, up to 36 months. This product obtained the Protected Designation of Origin (PDO) certification in 1986. Considering that the mycobiota of traditional Portuguese cheeses is understudied, the main goal of this work was to unveil the mycobiota of three São Jorge cheeses with different ripening periods (five, nine and thirty months). Direct inoculation of the cheese in three different culture media was used and the isolates were identified through molecular methods (analysis of ITS and/or partial benA). A total of 32 isolates were identified from the cheese with the lowest period of ripening, mainly Penicillium spp. ser. Camembertiorum (23 isolates), but also Aspergillus sp. (1 isolates), Scopulariopsis sp. (1 isolate), and several yeasts (7 isolates). The mycobiota of cheese with the seven months of ripening was mostly composed of Penicillium spp. ser. Camembertiorum and Saccharomyces cerevisiae, with 8 and 9 isolates, respectively. In the 30 months cheese Penicillium spp. ser. Camembertiorum were also isolated, but Scopulariopsis spp. was predominant, with 20 out of 24 isolates. Although the mycobiota was largely composed of Ascomycota, two Basidiomycota were found in the cheeses with the longest periods of ripening. Future studies will be conducted using metabarcoding techniques to disclose the uncultured mycobiota. These culture-independent techniques are less time consuming and more sensitive. They have shown to be a powerful tool to gain a better and faster understanding of the influence of the microorganisms in the cheese ripening process.Teresa Vale Dias thanks for the Ph.D. scholarship given by the Foundation for Science and Technology (FCT) - 2020.05849.BD. This study was also supported by FCT under the scope of the strategic funding of CEB (UIDB/04469/2020), LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020, CIMO (UIDB/00690/2020) and SusTEC (LA/P/0007/2020)info:eu-repo/semantics/publishedVersio

Isolation of filamentous fungi from different food matrices from Angola and Mozambique

Agriculture remains the main economic activity in most African countries. However, crops are often contaminated with fungi that can cause diseases or produce mycotoxins, which is a major concern to food safety and security. Little is known about the mycotoxigenic fungi contaminating the most relevant staples in Mozambique and Angola. The aim of this work was to isolate and identify fungi from three food commodities corn, peanuts and beans and understand if they are a source of mycotoxin exposure to the populations, as these products are fundamental to the local food diet, and important to the economy. Samples of corn from Mozambique, and samples of peanuts and beans from Angola (four samples of each) were analysed for fungal contamination. Samples were also surveyed for aflatoxins using the AgraStrip® Pro WATEX® (Romer) method. Twenty-five grains of each sample were directly plated onto DRBC, and filamentous fungi were isolated after 5 to 7 days of incubation at 25 °C. A total of 56 fungal isolates representing the various fungal morphotypes were molecularly identified by Sanger sequencing of the ITS region. The microbiota of all samples was mainly composed of Aspergillus sp., Fusarium sp. and Penicillium sp., many of them belonging to mycotoxigenic species. Phytopathogenic fungi of four genera Lasiodiplodia sp., Macrophomina sp., Nigrospora sp. and Pseudocercospora sp. were also identified. Most species were common to all types of samples. Aflatoxins were detected in all samples.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and to the Aga Khan Development Network for the financial support to the project Ref. FCT AGA-KHAN / 541590696 / 2019 “MYCOTOX-PALOP – Multi-actor partnership for the risk assessment of MYCOTOXins along the food chain in African Portuguese-speaking countries (PALOP)”. Teresa Dias and Cláudio Matusse thank FCT for the PhD grants 2020.05849.BD and PRT/BD/15483/2022, respectively. This study was also supported by FCT under the scope of CEB (UIDB/04469/2020), LABBELS (LA/P/0029/2020), CIMO (UIDB/00690/2020) and SusTEC (LA/P/0007/2020).info:eu-repo/semantics/publishedVersio

Phytoplankton dynamics in relation to seasonal variability and upwelling and relaxation patterns at the mouth of Ria de Aveiro (West Iberian Margin) over a four-year period

From June 2004 to December 2007, samples were weekly collected at a fixed station located at the mouth of Ria de Aveiro (West Iberian Margin). We examined the seasonal and inter-annual fluctuations in composition and community structure of the phytoplankton in relation to the main environmental drivers and assessed the influence of the oceano-graphic regime, namely changes in frequency and intensity of upwelling events, over the dynamics of the phytoplankton assemblage. The samples were consistently handled and a final subset of 136 OTUs (taxa with relative abundance > 0.01%) was subsequently submitted to various multivariate analyses. The phytoplankton assemblage showed significant changes at all temporal scales but with an overriding importance of seasonality over longer-(inter-annual) or shorter-term fluctuations (upwelling-related). Sea-surface temperature, salinity and maximum upwelling index were retrieved as the main driver of seasonal change. Seasonal signal was most evident in the fluctuations of chlorophyll a concentration and in the high turnover from the winter to spring phytoplankton assemblage. The seasonal cycle of production and succession was disturbed by upwelling events known to disrupt thermal stratification and induce changes in the phytoplankton assemblage. Our results indicate that both the frequency and intensity of physical forcing were important drivers of such variability, but the outcome in terms of species composition was highly dependent on the available local pool of species and the timing of those events in relation to the seasonal cycle. We conclude that duration, frequency and intensity of upwelling events, which vary seasonally and inter-annually, are paramount for maintaining long-term phytoplankton diversity likely by allowing unstable coexistence and incorporating species turnover at different scales. Our results contribute to the understanding of the complex mechanisms of coastal phytoplankton dynamics in relation to changing physical forcing which is fundamental to improve predictability of future prospects under climate change.Portuguese Foundation for Science and Technology (FCT, Portugal) [SFRH/BPD/ 94562/2013]; FEDER funds; national funds; CESAM [UID/AMB/50017]; FCT/MEC through national funds; FEDERinfo:eu-repo/semantics/publishedVersio

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

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

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements