Single-cell Subcellular Protein Localisation Using Novel Ensembles of Diverse Deep Architectures

Unravelling protein distributions within individual cells is key to understanding their function and state and indispensable to developing new treatments. Here we present the Hybrid subCellular Protein Localiser (HCPL), which learns from weakly labelled data to robustly localise single-cell subcellular protein patterns. It comprises innovative DNN architectures exploiting wavelet filters and learnt parametric activations that successfully tackle drastic cell variability. HCPL features correlation-based ensembling of novel architectures that boosts performance and aids generalisation. Large-scale data annotation is made feasible by our "AI-trains-AI" approach, which determines the visual integrity of cells and emphasises reliable labels for efficient training. In the Human Protein Atlas context, we demonstrate that HCPL defines state-of-the-art in the single-cell classification of protein localisation patterns. To better understand the inner workings of HCPL and assess its biological relevance, we analyse the contributions of each system component and dissect the emergent features from which the localisation predictions are derived

Urinary Arsenic Species and Methylation Efficiency During Pregnancy: Concentrations and Associated Factors in Spanish Pregnant Women

Background: Arsenic (As) is considered to be toxic for humans, the main routes of exposure being through drinking water and the diet. Once ingested, inorganic arsenic can be methylated sequentially to monomethyl and dimethyl arsenicals. Several factors can affect both As exposure and methylation efficiency. Objectives: To describe the urinary concentrations of the different As species and evaluate the methylation effi-ciency during pregnancy, as well as their associated factors in a birth cohort of pregnant Spanish women. Methods: Participants in this cross-sectional study were 1017 pregnant women from two areas of Spain who had taken part in the INMA (Environment and Childhood) project (2003 & ndash;2008). Total As (organic and inorganic compounds) and its main metabolites (monomethylarsonic acid, [MMA], dimethylarsinic acid, [DMA], inorganic As [iAs]) and arsenobetaine [AB]) were measured in urine samples collected during the first trimester. Socio-demographic and dietary information was collected through questionnaires. Multivariate linear regression models were used to explore the association between As species concentrations and covariates. Arsenic methylation efficiency was determined through the percentages of the metabolites and using As methylation phenotypes, obtained from principal component analysis. Results: Median urine concentrations were 33.0, 21.6, 6.5, 0.35 and 0.33 lig/g creatinine for total As, AB, DMA, MMA and iAs, respectively. Daily consumption of rice and seafood during the first trimester of pregnancy were positively associated with the concentration of As species (i.e., beta [CI95%] = 0.36 [0.09, 0.64] for rice and iAs, and 1.06 [0.68, 1.44] for seafood and AB). TAs, AB and iAs concentrations, and DMA and MMA concentrations were associated with legume and vegetable consumption, respectively. The medians of the percentage of As metabolites were 89.7 for %DMA, 5.1 for %MMA and 4.7 for %iAs. Non-smoker women and those with higher body mass index presented a higher methylation efficiency (denoted by a higher %DMA and lower %MMA). Discussion: Certain dietary, lifestyle, and environmental factors were observed to have an influence on both As species concentrations and methylation efficiency in our population. Further birth cohort studies in low exposure areas are necessary to improve knowledge about arsenic exposure, especially to inorganic forms, and its potential health impact during childhood.This study was supported by grants from Instituto de Salud Carlos III, Spain [FIS-FEDER: 07/0314; FIS 11/01007, 13/1944, 16/1288, 19/1338; Miguel Servet-FEDER: CP15/0025, CPII20/00006; FIS-FSE: 17/00260; Miguel Servet-FSE: MS15/0025, MS20/0006; PI06/0867 and FIS-PI18/01142 incl. FEDER funds] , Generalitat Valenciana, Spain BEST/2020/059, Department of Health of the Basque Government, Spain (2005111093) , Provincial Government of Gipuzkoa (DFG06/002) , and annual agreements with the municipalities in the study area (Zumarraga, Urretxu, Legazpi, Azkoitia, Azpeitia and Beasain)

Pro-vegetarian dietary patterns and essential and heavy metal exposure in children of 4-5-years from the INfancia y medio Ambiente cohort (INMA)

Dietary patterns provide a comprehensive assessment of food consumption, including essential nutrients and potential exposure to environmental contaminants. While pro-vegetarian (PVG) dietary patterns have shown health benefits in adults, their effects on children are less well studied. This study aims to explore the association between children's adherence to the most common PVG dietary patterns and their exposure to metals, assessed through urine concentration. In our study, we included a population of 723 children aged 4-5-years from the INfancia y Medio Ambiente (INMA) cohort in Spain. We calculated three predefined PVG dietary patterns, namely general (gPVG), healthful (hPVG), and unhealthful (uPVG), using dietary information collected through a validated Food Frequency Questionnaire. Urinary concentrations of various essential and heavy metals (Co, Cu, Zn, Se, Mo, Pb, and Cd) were measured using mass spectrometry. Additionally, urinary arsenic speciation, including arsenobetaine (AsB), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), and inorganic arsenic (iAs), was measured. The sum of urinary MMA and iAs was used to assess iAs exposure. We estimated primary (PMI) and secondary iAs methylation (SMI) indices. To explore the association between PVG dietary patterns in quintiles and metal exposure, we utilized multiple-adjusted linear regression models and the quantile g-computation approach. Compared with the lowest quintile, participants in the highest quintile of gPVG showed a 22.7% lower urinary Co (95% confidence interval (CI): −38.7; −1.98) and a 12.6% lower Se (95%CI: −22.9; −1.00) concentrations. Second quintile of adherence to hPVG was associated with a 51.7% lower urinary iAs + MMA concentrations (95%CI: −74.3; −8.61). Second quintile of adherence to an uPVG was associated with a 13.6% lower Se levels (95%CI: −22.9; −2.95) while the third quintile to this pattern was associated with 17.5% lower Mo concentrations (95%CI: −29.5; −2.95). The fourth quintile of adherence to gPVG was associated with a 68.5% higher PMI and a 53.7% lower SMI. Our study showed that adherence to a gPVG dietary pattern in childhood may modestly reduce the intakes of some essential metals such as Co and Se. Further investigations are warranted to explore any potential health implications.</p

Pro-vegetarian dietary patterns and essential and heavy metal exposure in children of 4-5-years from the INfancia y medio Ambiente cohort (INMA)

Dietary patterns provide a comprehensive assessment of food consumption, including essential nutrients and potential exposure to environmental contaminants. While pro-vegetarian (PVG) dietary patterns have shown health benefits in adults, their effects on children are less well studied. This study aims to explore the association between children's adherence to the most common PVG dietary patterns and their exposure to metals, assessed through urine concentration. In our study, we included a population of 723 children aged 4-5-years from the INfancia y Medio Ambiente (INMA) cohort in Spain. We calculated three predefined PVG dietary patterns, namely general (gPVG), healthful (hPVG), and unhealthful (uPVG), using dietary information collected through a validated Food Frequency Questionnaire. Urinary concentrations of various essential and heavy metals (Co, Cu, Zn, Se, Mo, Pb, and Cd) were measured using mass spectrometry. Additionally, urinary arsenic speciation, including arsenobetaine (AsB), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), and inorganic arsenic (iAs), was measured. The sum of urinary MMA and iAs was used to assess iAs exposure. We estimated primary (PMI) and secondary iAs methylation (SMI) indices. To explore the association between PVG dietary patterns in quintiles and metal exposure, we utilized multiple-adjusted linear regression models and the quantile g-computation approach. Compared with the lowest quintile, participants in the highest quintile of gPVG showed a 22.7% lower urinary Co (95% confidence interval (CI): −38.7; −1.98) and a 12.6% lower Se (95%CI: −22.9; −1.00) concentrations. Second quintile of adherence to hPVG was associated with a 51.7% lower urinary iAs + MMA concentrations (95%CI: −74.3; −8.61). Second quintile of adherence to an uPVG was associated with a 13.6% lower Se levels (95%CI: −22.9; −2.95) while the third quintile to this pattern was associated with 17.5% lower Mo concentrations (95%CI: −29.5; −2.95). The fourth quintile of adherence to gPVG was associated with a 68.5% higher PMI and a 53.7% lower SMI. Our study showed that adherence to a gPVG dietary pattern in childhood may modestly reduce the intakes of some essential metals such as Co and Se. Further investigations are warranted to explore any potential health implications.</p

Folic Acid Supplementation during Pregnancy and Its Association with Telomere Length in Children at Four Years: Results from the INMA Birth Cohort Study

This study examined the association between folic acid supplements (FAs) during different periods of pregnancy and offspring telomere length (TL) at age four in 666 children from the INMA study. FAs were self-reported using food-structured questionnaires during three periods of pregnancy (the first three months of pregnancy, from month fourth onward, and the whole pregnancy). For each period, the average daily dosage of FAs was categorised into (i) <400 μg/d, (ii) ≥400 to 999 μg/d, (iii) ≥1000 to 4999 μg/d, and (iv) ≥5000 μg/d. Leucocyte TL at age four was measured using quantitative PCR methods. Multiple robust linear log-level regression models were used to report the % difference among FA categories. During the first period, and compared with children whose mothers were classified in the reference group (<400 μg/d), children whose mothers took higher dosages of FAs showed shorter TL at age four (≥5000 μg/d). When the first and the second periods were mutually adjusted, children whose mothers self-reported ≥5000 μg/d during the first period of pregnancy had a statistically significant shorter TL than their counterparts (% difference: −7.28% [95% CI: −14.42 to −0.13]). Similar trends were observed for the whole period of pregnancy. When the analysis was stratified by sex, the association was more evident in boys (% difference: −13.5% [95% CI: −23.0 to −4.04]), whereas no association was observed in girls. This study suggests that high dosages of FAs in the first pregnancy period may be associated with a shorter TL in children at age four, particularly among boys. Further studies should confirm these results.This research was funded by Instituto de Salud Carlos III/Agencia Estatal de Investigación, grant number PI18/00825 Project: “Dieta y actividad física en embarazo y tras el nacimiento y longitud del telómero en niños y adolescentes: Proyecto TeloDiPA” and Unión Europea (FEDER) “Una manera de hacer Europa”; PI07/0314, PI11/01007 incl. FEDER funds; Generalitat Valenciana (GVA/2021/191); Dries Martens holds a postdoctoral grant by the Flemish Scientific Fund (FWO grant 12X9620N). In Sabadell was funded by grants from Instituto de Salud Carlos III (Red INMA G03/176; CB06/02/0041; PI041436; PI081151 incl. FEDER funds; PI12/01890 incl. FEDER funds; CP13/00054 incl. FEDER funds, PI15/00118 incl. FEDER funds, CPII18/00018), CIBERESP, Generalitat de Catalunya-CIRIT 1999SGR 00241, Generalitat de Catalunya-AGAUR (2009 SGR 501, 2014 SGR 822), Fundació La marató de TV3 (090430), Spanish Ministry of Economy and Competitiveness (SAF2012-32991 incl. FEDER funds), Agence Nationale de Securite Sanitaire de l’Alimentation de l’Environnement et du Travail (1262C0010), EU Commission (261357, 308333, 603794 and 634453). We acknowledge support from the Spanish Ministry of Science and Innovation and the State Research Agency through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. In Asturias was funded by ISCIII: PI04/2018, PI09/02311, PI13/02429, PI18/00909 co-funded by FEDER, “A way to make Europe”/“Investing in your future”, Obra Social Cajastur/Fundación Liberbank, and Universidad de Oviedo. This study was funded by grants from Instituto de Salud Carlos III (FIS-PI06/0867 and FIS-PI09/00090), CIBERESP, Department of Health of the Basque Government (2005111093, 2009111069 and 2013111089), and the Provincial Government of Gipuzkoa (DFG06/002 and DFG08/001) and annual agreements with the municipalities of the study area (Zumarraga, Urretxu, Legazpi, Azkoitia y Azpeitia y Beasain). Jordi Julvez holds the Miguel Servet-II contract (CPII19/00015) awarded by the Instituto de Salud Carlos III (co-funded by the European Social Fund “Investing in your future”)

Serum levels of PCDDs, PCDFs and dl-PCBs in general population residing far and near from an urban waste treatment plant under construction in Gipuzkoa, Basque Country (Spain)

This research focused on investigating the basal serum concentrations of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in the general population residing in two urban-industrial zones near and far from an energy recovery plant under construction in Gipuzkoa, Basque Country (Spain). The study used a cross-sectional design and included 227 participants who were randomly selected from municipal censuses in both areas. The participants were stratified based on age (ranging from 18 to 70 years) and sex. Serum samples were collected from the participants and analysed following the established protocol to measure the concentrations of PCDD/Fs and dl-PCBs. The study used multiple linear regression models to assess the impact of various sociodemographic variables, lifestyle factors, reproductive history, and diet on the variability of the measured compounds in the participants' serum. The median total toxicity equivalent (TEQ) in serum, was 10.58 pg WHO-TEQ2005 g-1 lipid. Serum PCDD levels were lower in the population residing in the "far" zone than the "near" zone. Age was positively associated with both PCDD/F and dl-PCB levels, indicating that older participants had higher concentrations of these compounds in their serum. This finding might be attributed to cumulative exposure over time. In terms of sex differences, women exhibited lower levels of dl-PCBs compared to men. Among lifestyle factors, smokers showed lower levels of dl-PCBs compared to non-smokers. Furthermore, daily alcohol consumption was significantly associated with higher serum levels of these compounds, with daily drinkers showing higher levels than non-drinkers. Consumption of local poultry was associated with significantly higher serum levels and oil consumption with low levels of PCDD/Fs.This research, conducted between 2017 and 2019 before the operation of the Energy Recovery plant, was funded through a public tender (017/11-HH-ZE) by the Gipuzkoa Provincial Council. However, the funding source did not play any part in the study's design, data collection and analysis, or the interpretation and writing of the manuscript.Peer reviewe

Characterization of PCDD/F and dl-PCB levels in air in Gipuzkoa (Basque Country, Spain)

This research examines the levels and trends of pollutants, specifically 17 congeners of PCDD/Fs and 12 dl-PCBs, in the air measured in the province of Gipuzkoa (Basque Country, Spain). The study used PCDD/Fs, dl-PCB, and the sum of dioxin-like compounds as separate response variables. A total of 113 air samples were collected and analyzed using the method described in the European Standard (EN-1948:2006) from two industrial areas. The results were analyzed using non-parametric test to assess the variability of these pollutants based on different factors (year, season and day of the week) and General Linear Models to assess the weight of each factor. The study found that the toxic equivalents (TEQs) for PCDD/Fs were 12.29 fg TEQm-3 and for dl-PCBs were 1.63 fg TEQm-3, which were in a similar range or lower than those observed in other national and international studies in industrial areas. The results showed temporal variations, with higher levels of PCDD/Fs in autumn-winter than in spring-summer and higher levels of PCDD/Fs and dl-PCBs during weekdays than on weekends. The industrial area where the energy recovery plant (ERP) will be located had higher levels of air pollutants due to the presence of two PCDD/Fs emitting industries nearby, as indicated by the Spanish Registry of Polluting Emission Sources. Both industrial areas showed similar profiles of PCDD/Fs and dl-PCBs, with the PCDD/F profiles dominated by OCDD, 1,2,3,4,6,7,8-HpCDD, and 1,2,3,4,6,7,8-HpCDF in terms of concentrations and 1,2,3,7,8-PeCDD, 2,3,4,7,8-PeCDF, and 2,3,7,8-TCDD in terms of TEQs. The dl-PCB profiles were dominated by PCB 118, PCB 105, and PCB 77 in terms of concentrations and PCB 126 in terms of TEQs. The findings of this study can serve as an indicator of the potential impact of ERP on the health of the resident population and the environment.This research, conducted between 2017 and 2019 before the operation of the Energy Recovery plant, was funded through a public tender (017/11-HH-ZE) by the Gipuzkoa Provincial Council. However, the funding source did not play any part in the study's design, data collection and analysis, or the interpretation and writing of the manuscript.Peer reviewe

Influence of genetic polymorphisms on arsenic methylation efficiency during pregnancy: Evidence from a Spanish birth cohort

Background: Inorganic arsenic (iAs) is a widespread toxic metalloid. It is well-known that iAs metabolism and its toxicity are mediated by polymorphisms in AS3MT and other genes. However, studies during pregnancy are scarce. We aimed to examine the role of genetic polymorphisms in AS3MT, GSTO2, N6AMT1, MTHFR, MTR, FTCD, CBS, and FOLH1 in iAs methylation efficiency during pregnancy. Methods: The study included 541 pregnant participants from the INMA (Environment and Childhood) Spanish cohort. Using high-performance liquid chromatography coupled to inductively coupled plasma-tandem mass, we measured arsenic (iAs and the metabolites monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in urine samples collected during the first trimester. iAs methylation efficiency was determined based on relative concentrations of the As metabolites in urine (%MMA, %DMA, and %iAs). Thirty-two single nucleotide polymorphisms (SNPs) in nine genes were determined in maternal DNA; AS3MT haplotypes were inferred. We assessed the association between genotypes/haplotypes and maternal As methylation efficiency using multivariate linear regression models. Results: The median %MMA and %DMA were 5.3 %, and 89 %, respectively. Ancestral alleles of AS3MT SNPs (rs3740393, rs3740390, rs11191453, and rs11191454) were significantly associated with higher %MMA, %iAs, and lower %DMA. Pregnant participants with zero copies of the GGCTTCAC AS3MT haplotype presented a higher %MMA. Statistically significant associations were also found for the FOLH1 SNP rs202676 (β 0.89 95%CI: 0.24, 1.55 for carriers of the G allele vs. the A allele). Conclusions: Our study shows that ancestral alleles in AS3MT polymorphisms were associated with lower As methylation efficiency in early pregnancy and suggests that FOLH1 also plays a role in As methylation efficiency. These results support the hypothesis that As metabolism is multigenic, being a key element for identifying susceptible populations

Prenatal Metals Exposure and pre-adolescents’ Emotional and Behavioral Problems

Emotional and behavioral problems during childhood raise the risk of subsequent developmental of mental disorders. Our aim was to study the association between maternal metal and trace element concentrations during gestation and these problems in 9 year-old children. The study sample comprised Spanish mother-child pairs in the INMA project (n = 1003). Metals and trace elements (As, Cd, Co, Cu, Mo, Ni, Pb, Sb, Se, Tl and Zn) were measured in urine samples collected during pregnancy. Inorganic As metabolites were speciated in a subsample (n = 729). Emotional and behavioral problems were assessed using the Child Behavior Checklist (CBCL) composed of three scales: internalizing, externalizing and total problems. Sociodemographic, dietary and exposure to other environmental pollutants were obtained through questionnaires. Single nucleotide polymorphisms in brain- and metabolism-related genes APOE, BDNF, GSTP1, and PON1 were determined in cord blood. Multivariate negative binomial models were used. The interaction with sex and genotypes was evaluated including interaction terms. A multi-element analysis was carried out by a principal component analysis. Higher concentrations of Cu, monomethylarsonic acid, and Pb during pregnancy were associated with an increased incidence ratio risk (IRR) between 4.6 and 7.5% for internalizing and externalizing problems for all three CBCL scales in the children. Increasing Mo, Ni and Co concentrations were associated with higher IRR for internalizing problems (up to 8%), and Cd for externalizing problems (6.7%). Modifications by sex and genotypes were found for several associations. Multi-element analysis associated multiple metals and trace elements (Ni, Cu, Se, Cd and Pb) with higher internalizing problems.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. We are grateful to all the participants and their families for taking part in this study. We also appreciate the generous contribution in the study of all INMA members. A full roster of the INMA Project researchers can be found at http://www.proyectoinma.org/presentacion-inma/listado-investigadores/en_listado-investigadores.html. This study was supported by grants from Spanish government through the Ministry of Universities under the grant CAS21/00008 and grant for the requalification of the Spanish University, financed by the European Union, NextGeneration EU, Instituto de Salud Carlos III [FIS-FEDER: 13/1944, 16/1288, 17/00663 and 19/1338; FIS-FSE: 17/00260; Miguel Servet-FSE: MSII20/0006, FIS-PI06/0867, FIS-PI09/00090, CPII18/00018], CIBERESP, Department of Health of the Basque Government (2005111093, 2009111069, 2013111089, 2015111065), Generalitat Valenciana [BEST/2020/059, AICO/2020/285 and CIAICO/2021/132] and the Provincial Government of Gipuzkoa (DFG06/002, DFG08/001 and DFG15/221 and DFG 89/17). We also acknowledge support from the Spanish Ministry of Science and Innovation and the State Research Agency through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.Peer reviewe