Co-localization of antibiotic resistance genes is widespread in the infant gut microbiome and associates with an immature gut microbial composition.

BACKGROUND: In environmental bacteria, the selective advantage of antibiotic resistance genes (ARGs) can be increased through co-localization with genes such as other ARGs, biocide resistance genes, metal resistance genes, and virulence genes (VGs). The gut microbiome of infants has been shown to contain numerous ARGs, however, co-localization related to ARGs is unknown during early life despite frequent exposures to biocides and metals from an early age. RESULTS: We conducted a comprehensive analysis of genetic co-localization of resistance genes in a cohort of 662 Danish children and examined the association between such co-localization and environmental factors as well as gut microbial maturation. Our study showed that co-localization of ARGs with other resistance and virulence genes is common in the early gut microbiome and is associated with gut bacteria that are indicative of low maturity. Statistical models showed that co-localization occurred mainly in the phylum Proteobacteria independent of high ARG content and contig length. We evaluated the stochasticity of co-localization occurrence using enrichment scores. The most common forms of co-localization involved tetracycline and fluoroquinolone resistance genes, and, on plasmids, co-localization predominantly occurred in the form of class 1 integrons. Antibiotic use caused a short-term increase in mobile ARGs, while non-mobile ARGs showed no significant change. Finally, we found that a high abundance of VGs was associated with low gut microbial maturity and that VGs showed even higher potential for mobility than ARGs. CONCLUSIONS: We found that the phenomenon of co-localization between ARGs and other resistance and VGs was prevalent in the gut at the beginning of life. It reveals the diversity that sustains antibiotic resistance and therefore indirectly emphasizes the need to apply caution in the use of antimicrobial agents in clinical practice, animal husbandry, and daily life to mitigate the escalation of resistance. Video Abstract.

Vertical Metabolome Transfer from Mother to Child: An Explainable Machine Learning Method for Detecting Metabolomic Heritability.

Vertical transmission of metabolic constituents from mother to child contributes to the manifestation of disease phenotypes in early life. This study probes the vertical transmission of metabolites from mothers to offspring by utilizing machine learning techniques to differentiate between true mother-child dyads and randomly paired non-dyads. Employing random forests (RF), light gradient boosting machine (LGBM), and logistic regression (Elasticnet) models, we analyzed metabolite concentration discrepancies in mother-child pairs, with maternal plasma sampled at 24 weeks of gestation and children's plasma at 6 months. The propensity of vertical transfer was quantified, reflecting the likelihood of accurate mother-child matching. Our findings were substantiated against an external test set and further verified through statistical tests, while the models were explained using permutation importance and SHapley Additive exPlanations (SHAP). The best model was achieved using RF, while xenobiotics were shown to be highly relevant in transfer. The study reaffirms the transmission of certain metabolites, such as perfluorooctanoic acid (PFOA), but also reveals additional insights into the maternal influence on the child's metabolome. We also discuss the multifaceted nature of vertical transfer. These machine learning-driven insights complement conventional epidemiological findings and offer a novel perspective on using machine learning as a methodology for understanding metabolic interactions.

An Unhealthy Dietary Pattern during Pregnancy is Associated with Neurodevelopmental Disorders in Childhood and Adolescence.

Despite the high prevalence of neurodevelopmental disorders, there are a lack of clinical studies examining the impact of pregnancy diet on child neurodevelopment. This observational clinical study examined the associations between pregnancy dietary patterns and neurodevelopmental diagnoses, as well as their symptoms, in a prospective cohort of 10-year-old children (n=508). Data-driven dietary patterns were derived from self-reported food frequency questionnaires. An Unhealthy dietary pattern in pregnancy (per SD change) was significantly associated with attention deficit hyperactivity disorder (ADHD) OR 1.66 [1.21 - 2.27], p=0.002 and autism diagnosis OR 2.22 [1.33 - 3.74], p=0.002 and associated symptoms p<0.001. Findings for ADHD were validated in two large (n=656, n=348), independent mother-child cohorts via blood metabolome modelling. Objective metabolite scores, assessed at five timepoints in mothers and children in two independent mother-child cohorts, indicated that the strongest association with ADHD was during early-to mid-pregnancy. These results provide evidence for targeted prenatal dietary interventions to prevent neurodevelopmental disorders in children.

High-dose vitamin D3 supplementation in pregnancy and risk of neurodevelopmental disorders in the children at age 10: A randomized clinical trial.

BACKGROUND: Vitamin D deficiency in pregnancy may increase the risk of autism and attention deficit hyperactivity disorder (ADHD). OBJECTIVE: The objective of this study was to estimate the effect of vitamin D3 supplementation in pregnancy on risk of autism and ADHD. DESIGN: This randomized clinical trial was part of the COpenhagen Prospective Study on Neuro-PSYCHiatric Development (COPYCH) project nested within the Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC2010) cohort comprising a population-based sample of 700 healthy mother-child pairs enrolled at week 24 of pregnancy. Maternal 25-hydroxy-vitamin D (25(OH)D) was measured at inclusion and 623 mothers were randomized 1:1 to either high-dose (2800 IU/d) or standard dose (400 IU/d) vitamin D3 until 1 wk postpartum (315 received high-dose, 308 standard dose). At age 10, diagnoses and symptom load of autism and ADHD, respectively, were established using the Kiddie-Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version. RESULTS: The psychopathologic evaluation was completed by 591 children aged 10 y, and 16 children (2.7%) were diagnosed with autism and 65 (11.0%) with ADHD. Hereof, 496 children participated in the vitamin D3 trial (246 received high-dose, 250 standard dose). Of these, 12 children (2.4%) were diagnosed with autism and 58 (11.7%) with ADHD. Higher maternal preintervention 25(OH)D levels were associated with a decreased risk of autism [odd ratio (OR) per 10 nmol/L: 0.76 (0.59,0.97); P = 0.034], lower autistic symptom load [ß per 10 nmol/L: -0.03 (-0.05,0.00); P = 0.024), and decreased risk of ADHD diagnosis (OR per 10 nmol/L: 0.88 (0.78,0.99); P = 0.033]. High-dose vitamin D3 supplementation was not associated with risk of autism or ADHD. CONCLUSIONS: Higher maternal preintervention 25(OH)D was associated with a decreased risk of autism, lower autistic symptom load, and decreased risk of ADHD diagnosis, but high-dose vitamin D3 supplementation in pregnancy had no effect on risk of autism and ADHD. This trial was registered at clinicaltrials.gov as NCT00856947.

The infant gut virome is associated with preschool asthma risk independently of bacteria.

Bacteriophage (also known as phage) communities that inhabit the gut have a major effect on the structure and functioning of bacterial populations, but their roles and association with health and disease in early life remain unknown. Here, we analyze the gut virome of 647 children aged 1 year from the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) mother-child cohort, all deeply phenotyped from birth and with longitudinally assessed asthma diagnoses. Specific temperate gut phage taxa were found to be associated with later development of asthma. In particular, the joint abundances of 19 caudoviral families were found to significantly contribute to this association. Combining the asthma-associated virome and bacteriome signatures had additive effects on asthma risk, implying an independent virome-asthma association. Moreover, the virome-associated asthma risk was modulated by the host TLR9 rs187084 gene variant, suggesting a direct interaction between phages and the host immune system. Further studies will elucidate whether phages, alongside bacteria and host genetics, can be used as preclinical biomarkers for asthma.

Differential responses of the gut microbiome and resistome to antibiotic exposures in infants and adults.

Despite their crucial importance for human health, there is still relatively limited knowledge on how the gut resistome changes or responds to antibiotic treatment across ages, especially in the latter case. Here, we use fecal metagenomic data from 662 Danish infants and 217 young adults to fill this gap. The gut resistomes are characterized by a bimodal distribution driven by E. coli composition. The typical profile of the gut resistome differs significantly between adults and infants, with the latter distinguished by higher gene and plasmid abundances. However, the predominant antibiotic resistance genes (ARGs) are the same. Antibiotic treatment reduces bacterial diversity and increased ARG and plasmid abundances in both cohorts, especially core ARGs. The effects of antibiotic treatments on the gut microbiome last longer in adults than in infants, and different antibiotics are associated with distinct impacts. Overall, this study broadens our current understanding of gut resistome dynamics and the impact of antibiotic treatment across age groups.

A chemical structure and machine learning approach to assess the potential bioactivity of endogenous metabolites and their association with early-childhood hs-CRP levels.

Metabolomics has gained much attraction due to its potential to reveal molecular disease mechanisms and present viable biomarkers. In this work we used a panel of untargeted serum metabolomes in 602 childhood patients of the COPSAC2010 mother-child cohort. The annotated part of the metabolome consists of 493 chemical compounds curated using automated procedures. Using predicted quantitative-structure-bioactivity relationships for the Tox21 database on nuclear receptors and stress response in cell lines, we created a filtering method for the vast number of quantified metabolites. The metabolites measured in children's serums used here have predicted potential against the chosen target modelled targets. The targets from Tox21 have been used with quantitative structure-activity relationships (QSARs) and were trained for ~7000 structures, saved as models, and then applied to 493 metabolites to predict their potential bioactivities. The models were selected based on strict accuracy criteria surpassing random effects. After application, 52 metabolites showed potential bioactivity based on structural similarity with known active compounds from the Tox21 set. The filtered compounds were subsequently used and weighted by their bioactive potential to show an association with early childhood hs-CRP levels at six months in a linear model supporting a physiological adverse effect on systemic low-grade inflammation. The significant metabolites were reported.

Fungi and bacteria in the beds of rural and urban infants correlate with later risk of atopic diseases.

INTRODUCTION: Rural children have a lower risk of asthma and atopic diseases than urban children. However, whether indoor microbiota in non-farming rural homes provides protection is unclear. METHODS: Here, we examine if microbes in the beds of rural and urban infants are associated with later development of atopic diseases. We studied fungi and bacteria in the beds of 6-month-old infants (n = 514) in association with the risk of asthma, allergic rhinitis, eczema and aeroallergen sensitization at 6 years of age in the prospective COPSAC2010 cohort. RESULTS: Both fungal and bacterial diversity were lower in the beds of children, who later developed allergic rhinitis (-0.22 [-0.43,-0.01], padj = .04 and -.24 [-0.42,-0.05], padj = .01 respectively) and lower bacterial richness was discovered in beds of children later developing asthma (-41.34 [-76.95,-5.73], padj = .02) or allergic rhinitis (-45.65 [-81.19,-10.10], padj = .01). Interestingly, higher fungal diversity and richness were discovered in the beds of children developing eczema (0.23 [0.02,0.43], padj = .03 and 29.21 [1.59,56.83], padj = .04 respectively). We defined a limited set of fungal and bacterial genera that predicted rural/urban environment. Some rural-associated bacterial genera such as Romboutsia and Bacillus and fungal genera Spegazzinia and Physcia were also associated with reduced risk of diseases, including eczema. These fungal and bacterial fingerprints predicting the living environment were associated with asthma and allergic rhinitis, but not eczema, with rural compositions being protective. The bed dust bacteria mediated 27% of the protective association of a rural living environment for allergic rhinitis (p = .04). CONCLUSIONS: Bed dust microbes can be differentially associated with airway- and skin-related diseases. The differing bed dust microbiota between rural and urban infants may influence their later risk of asthma and allergic rhinitis.

COVID-19 lockdown, genetic ADHD susceptibility, and mental health in 10- year-old children.

Previous studies report that the COVID-19 lockdown had an impact on the mental health of the pediatric population. In this study, we harness the deep neuropsychiatric phenotyping of the population-based COPSAC2010 (n = 700) cohort at age 10 to study the impact of the COVID-19 lockdown on mental health outcomes with focus on the role of the genetic vulnerability to attention-deficit/hyperactivity disorder (ADHD), in the form of polygenic risk scores (PRS). A total of 593 children were examined between 2019 and 2021, resulting in two groups of different children, those evaluated before the lockdown (n = 230) and those evaluated after (n = 363). Children assessed after the lockdown presented higher odds of being diagnosed with ADHD and had significantly higher scores in most neuropsychiatric scales, particularly in subscales pertaining to behavior and attention problems. We observed a significant interaction between the lockdown and ADHD PRS on several neuropsychiatric dimensions, with a large post-lockdown increase in children with a high PRS, while there was little to no pre-post difference in children with low PRS. These results indicate mental health consequences of the lockdown in children and suggest that genetically susceptible individuals are more affected by such stressors in childhood.

Atopic and non-atopic effects of fish oil supplementation during pregnancy.

BACKGROUND: We recently conducted a double-blinded randomised controlled trial showing that fish-oil supplementation during pregnancy reduced the risk of persistent wheeze or asthma in the child by 30%. Here, we explore the mechanisms of the intervention. METHODS: 736 pregnant women were given either placebo or n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in the third trimester in a randomised controlled trial. Deep clinical follow-up of the 695 children in the trial was done at 12 visits until age 6 years, including assessment of genotype at the fatty acid desaturase (FADS) locus, plasma fatty acids, airway DNA methylation, gene expression, microbiome and metabolomics. RESULTS: Supplementation with n-3 LCPUFA reduced the overall risk of non-atopic asthma by 73% at age 6 (relative risk (RR) 0.27 (95% CI 0.06 to 0.85), p=0.042). In contrast, there was no overall effect on asthma with atopic traits (RR 1.42 (95% CI 0.63 to 3.38), p=0.40), but this was significantly modified by maternal FADS genotype and LCPUFA blood levels (interaction p<0.05), and supplementation did reduce the risk of atopic asthma in the subgroup of mothers with FADS risk variants and/or low blood levels of n-3 LCPUFA before the intervention (RR 0.31 (95% CI 0.11 to 0.75), p=0.016). Furthermore, n-3 LCPUFA significantly reduced the number of infections (croup, gastroenteritis, tonsillitis, otitis media and pneumonia) by 16% (incidence rate ratio 0.84 (95% CI 0.74 to 0.96), p=0.009). CONCLUSIONS: n-3 LCPUFA supplementation in pregnancy showed protective effects on non-atopic asthma and infections. Protective effects on atopic asthma depended on maternal FADS genotype and n-3 LCPUFA levels. This indicates that the fatty acid pathway is involved in multiple mechanisms affecting the risk of asthma subtypes and infections. TRIAL REGISTRATION NUMBER: NCT00798226.

Human blood plasma biomarkers of diet and weight loss among centrally obese subjects in a New Nordic Diet intervention.

Scope: The New Nordic Diet (NND) has been shown to promote weight loss and lower blood pressure amongst obese people. This study investigates blood plasma metabolite and lipoprotein biomarkers differentiating subjects who followed Average Danish Diet (ADD) or NND. The study also evaluates how the individual response to the diet is reflected in the metabolic differences between NND subjects who lost or maintained their pre-intervention weight. Methods: Centrally obese Danes (BMI >25) followed NND (90 subjects) or ADD (56 subjects) for 6 months. Fasting blood plasma samples, collected at three time-points during the intervention, were screened for metabolites and lipoproteins (LPs) using proton nuclear magnetic resonance spectroscopy. In total, 154 metabolites and 65 lipoproteins were analysed. Results: The NND showed a relatively small but significant effect on the plasma metabolome and lipoprotein profiles, with explained variations ranging from 0.6% for lipoproteins to 4.8% for metabolites. A total of 38 metabolites and 11 lipoproteins were found to be affected by the NND. The primary biomarkers differentiating the two diets were found to be HDL-1 cholesterol, apolipoprotein A1, phospholipids, and ketone bodies (3-hydroxybutyric acid, acetone, and acetoacetic acid). The increased levels of ketone bodies detected in the NND group inversely associated with the decrease in diastolic blood pressure of the NND subjects. The study also showed that body weight loss among the NND subjects was weakly associated with plasma levels of citrate. Conclusion: The main plasma metabolites associated with NND were acetate, methanol and 3-hydroxybutyrate. The metabolic changes associated with the NND-driven weight loss are mostly pronounced in energy and lipid metabolism.

Exposures to perfluoroalkyl substances and asthma phenotypes in childhood: an investigation of the COPSAC2010 cohort.

BACKGROUND: Exposure to perfluoroalkyl substances may affect offspring immune development and thereby increase risk of childhood asthma, but the underlying mechanisms and asthma phenotype affected by such exposure is unknown. METHODS: In the Danish COPSAC2010 cohort of 738 unselected pregnant women and their children plasma PFOS and PFOA concentrations were semi-quantified by untargeted metabolomics analyses and calibrated using a targeted pipeline in mothers (gestation week 24 and 1 week postpartum) and children (age ½, 1½ and 6 years). We examined associations between pregnancy and childhood PFOS and PFOA exposure and childhood infections, asthma, allergic sensitization, atopic dermatitis, and lung function measures, and studied potential mechanisms by integrating data on systemic low-grade inflammation (hs-CRP), functional immune responses, and epigenetics. FINDINGS: Higher maternal PFOS and PFOA exposure during pregnancy showed association with a non-atopic asthma phenotype by age 6, a protection against sensitization, and no association with atopic asthma or lung function, or atopic dermatitis. The effect was primarily driven by prenatal exposure. There was no association with infection proneness, low-grade inflammation, altered immune responses or epigenetic changes. INTERPRETATIONS: Prenatal exposure to PFOS and PFOA, but not childhood exposure, specifically increased the risk of low prevalent non-atopic asthma, whereas there was no effect on atopic asthma, lung function, or atopic dermatitis. FUNDING: All funding received by COPSAC are listed on www.copsac.com. The Lundbeck Foundation (Grant no R16-A1694); The Novo Nordic Foundation (Grant nos NNF20OC0061029, NNF170C0025014, NNF180C0031764); The Ministry of Health (Grant no 903516); Danish Council for Strategic Research (Grant no 0603-00280B); and The Capital Region Research Foundation have provided core support to the COPSAC research center. COPSAC acknowledges the National Facility for Exposomics (SciLifeLab, Sweden) for supporting calibration of the untargeted metabolomics PFAS data. BC and AS has received funding for this project from the European Union's Horizon 2020 research and innovation programme (BC: grant agreement No. 946228 DEFEND; AS: grant agreement No. 864764 HEDIMED).

Novel Connections of Common Childhood Illnesses Based on More Than 5 Million Diary Registrations From Birth Until Age 3 Years.

BACKGROUND: All children experience numerous episodes of illness during the first 3 years of life. Most episodes are mild and handled without medical attention but nevertheless burden the families and society. There is a large, and still unexplained, variation in the burden of illness between children. OBJECTIVE: To describe and provide a better understanding of the disease burden of common childhood diseases through a data-driven approach investigating the communalities between symptom patterns and predefined variables on predispositions, pregnancy, birth, environment, and child development. METHODS: The study is based on the prospectively followed clinical mother-child cohort COpenhagen Prospective Studies on Asthma in Childhood, which includes 700 children with daily symptom registration in the first 3 years of life, including symptoms of cough, breathlessness, wheeze, cold, pneumonia, sore throat, ear infections, gastrointestinal infections, fever, and eczema. First, we described the number of episodes of symptoms. Next, factor analysis models were used to describe the variation in symptom load in the second year of life (both based on n = 556, with >90% complete diary). Then we characterized patterns of similarity between symptoms using a graphical network model (based on n = 403, with a 3-year monthly compliance of >50%). Finally, predispositions and pregnancy, birth, environmental, and developmental factors were added to the network model. RESULTS: The children experienced a median of 17 (interquartile range, 12-23) episodes of symptoms during the first 3 years of life, of which most were respiratory tract infections (median, 13; interquartile range, 9-18). The frequency of symptoms was the highest during the second year of life. Eczema symptoms were unrelated to the other symptoms. The strongest association to respiratory symptoms was found for maternal asthma, maternal smoking during the third trimester, prematurity, and CDHR3 genotype. This was in contrast to the lack of associations for the well-established asthma locus at 17q21. CONCLUSIONS: Healthy young children are burdened by multiple episodes of symptoms during the first 3 years of life. Prematurity, maternal asthma, and CDHR3 genotype were among the strongest drivers of symptom burden.

Oropharyngeal Microbiota Clusters in Children with Asthma or Wheeze Associate with Allergy, Blood Transcriptomic Immune Pathways, and Exacerbation Risk.

Rationale: Children with preschool wheezing or school-age asthma are reported to have airway microbial imbalances. Objectives: To identify clusters in children with asthma or wheezing using oropharyngeal microbiota profiles. Methods: Oropharyngeal swabs from the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) pediatric asthma or wheezing cohort were characterized using 16S ribosomal RNA gene sequencing, and unsupervised hierarchical clustering was performed on the Bray-Curtis ß-diversity. Enrichment scores of the Molecular Signatures Database hallmark gene sets were computed from the blood transcriptome using gene set variation analysis. Children with severe asthma or severe wheezing were followed up for 12-18 months, with assessment of the frequency of exacerbations. Measurements and Main Results: Oropharyngeal samples from 241 children (age range, 1-17 years; 40% female) revealed four taxa-driven clusters dominated by Streptococcus, Veillonella, Rothia, and Haemophilus. The clusters showed significant differences in atopic dermatitis, grass pollen sensitization, FEV1% predicted after salbutamol, and annual asthma exacerbation frequency during follow-up. The Veillonella cluster was the most allergic and included the highest percentage of children with two or more exacerbations per year during follow-up. The oropharyngeal clusters were different in the enrichment scores of TGF-ß (transforming growth factor-ß) (highest in the Veillonella cluster) and Wnt/ß-catenin signaling (highest in the Haemophilus cluster) transcriptomic pathways in blood (all q values <0.05). Conclusions: Analysis of the oropharyngeal microbiota of children with asthma or wheezing identified four clusters with distinct clinical characteristics (phenotypes) that associate with risk for exacerbation and transcriptomic pathways involved in airway remodeling. This suggests that further exploration of the oropharyngeal microbiota may lead to novel pathophysiologic insights and potentially new treatment approaches.

Alterations of NMR-Based Lipoprotein Profile Distinguish Unstable Angina Patients with Different Severity of Coronary Lesions.

Non-invasive detection of unstable angina (UA) patients with different severity of coronary lesions remains challenging. This study aimed to identify plasma lipoproteins (LPs) that can be used as potential biomarkers for assessing the severity of coronary lesions, determined by the Gensini score (GS), in UA patients. We collected blood plasma from 67 inpatients with angiographically normal coronary arteries (NCA) and 230 UA patients, 155 of them with lowGS (GS ≤ 25.4) and 75 with highGS (GS > 25.4), and analyzed it using proton nuclear magnetic resonance spectroscopy to quantify 112 lipoprotein variables. In a logistic regression model adjusted for four well-known risk factors (age, sex, body mass index and use of lipid-lowering drugs), we tested the association between each lipoprotein and the risk of UA. Combined with the result of LASSO and PLS-DA models, ten of them were identified as important LPs. The discrimination with the addition of selected LPs was evaluated. Compared with the basic logistic model that includes four risk factors, the addition of these ten LPs concentrations did not significantly improve UA versus NCA discrimination. However, thirty-two selected LPs showed notable discrimination power in logistic regression modeling distinguishing highGS UA patients from NCA with a 14.9% increase of the area under the receiver operating characteristics curve. Among these LPs, plasma from highGS patients was enriched with LDL and VLDL subfractions, but lacked HDL subfractions. In summary, we conclude that blood plasma lipoproteins can be used as biomarkers to distinguish UA patients with severe coronary lesions from NCA patients.

Effect of perfluoroalkyl exposure in pregnancy and infancy on intrauterine and childhood growth and anthropometry. Sub study from COPSAC2010 birth cohort.

BACKGROUND: Perfluoroalkyl substances PFOS and PFOA are persistent and bioaccumulative exogenous chemicals in the human body with a range of suspected negative health effects. It is hypothesised that exposure during prenatal and early postnatal life might have particularly detrimental effects on intrauterine and childhood growth. In a Danish longitudinal mother-child cohort we investigate effect of PFOS and PFOA in pregnancy and infancy on intrauterine and childhood growth and anthropometry. METHODS: COPSAC2010 is an ongoing population based mother-child cohort of 738 pregnant women and their children followed from 24 week gestation with longitudinal deep clinical phenotyping until age 10 years. In this observational cohort sub study plasma PFOS and PFOA concentrations were semi-quantified by untargeted metabolomics in the mothers at week 24 and 1 week postpartum and in the children at ages 6 and 18 months and calibrated using a targeted pipeline. We examined associations to intrauterine and childhood growth and anthropometry, including interactions with child sex. Untargeted and targeted blood metabolomics profiles were integrated to investigate underlying mechanisms. FINDINGS: Pregnancy plasma PFOA concentrations were associated with lower birth size -0.19 [-0.33; -0.05] BMI z-score per 1-ng/mL and increased childhood height (z-scored) at age 6: 0.18 [0.05; 0.31], but there was no association between childs' own infancy plasma PFOA concentration and height. Pregnancy plasma PFOS concentrations were also associated with lower birth BMI (-0.04 [-0.08; -0.01]), but in childhood pregnancy plasma PFOS concentration interacted with child sex on BMI and fat percentage at 6 years with negative associations in girls and positive in boys. The effect of maternal plasma PFOS concentration on lower girl BMI was borderline mediated through increasing child plasma lactosyl-ceramide levels (p-mediation=0.08). Similarly the effect of maternal plasma PFOS concentration on higher boy fat percentage was borderline mediated through increasing child plasma lactosyl-ceramide levels (p-mediation=0.07). Infancy concentrations of plasma PFOS associated with lower height in childhood, -0.06 z-score at age 6 [-0.19; -0.03]. INTERPRETATION: Higher PFOS and PFOA plasma concentrations during pregnancy had detrimental effects on fetal growth. The effects on childhood growth were not similar as PFOA increased child height, opposite of PFOS in multipollutant models suggesting a differing fetal programming effect. Sex specific growth effects were borderline mediated through an altered lactosyl-ceramide metabolism, proposing a possible mechanism of PFOS that has long-lasting health consequences in this observational study. FUNDING: All funding received by COPSAC are listed on www.copsac.com. The Lundbeck Foundation (Grant no R16-A1694); The Novo Nordic Foundation (Grant nos NNF20OC0061029, NNF170C0025014, NNF180C0031764) The Ministry of Health (Grant no 903516); Danish Council for Strategic Research (Grant no 0603-00280B) and The Capital Region Research Foundation have provided core support to the COPSAC research center. Effort from JALS is supported by R01HL123915, R01HL141826, and R01HL155742 from NIH/NHLBI. CEW was supported by the Swedish Heart Lung Foundation (HLF 20180290, HLF 20200693). BC has received funding for this project from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 946228). The funding agencies did not have any role in design and conduct of the study; collection, management, and interpretation of the data; or preparation, review, or approval of the manuscript.

Asthma and Wheeze Severity and the Oropharyngeal Microbiota in Children and Adolescents.

Rationale: There is a major unmet need for improving the care of children and adolescents with severe asthma and wheeze. Identifying factors contributing to disease severity may lead to improved diagnostics, biomarkers, or therapies. The airway microbiota may be such a key factor. Objectives: To compare the oropharyngeal airway microbiota of children and adolescents with severe and mild/moderate asthma/wheeze. Methods: Oropharyngeal swab samples from school-age and preschool children in the European U-BIOPRED (Unbiased BIOmarkers in the PREDiction of respiratory disease outcomes) multicenter study of severe asthma, all receiving severity-appropriate treatment, were examined using 16S ribosomal RNA gene sequencing. Bacterial taxa were defined as amplicon sequence variants. Results: We analyzed 241 samples from four cohorts: A) 86 school-age children with severe asthma; B) 39 school-age children with mild/moderate asthma; C) 65 preschool children with severe wheeze; and D) 51 preschool children with mild/moderate wheeze. The most common bacteria were Streptococcus (mean relative abundance, 33.5%), Veillonella (10.3%), Haemophilus (7.0%), Prevotella (5.9%), and Rothia (5.5%). Age group (school-age vs. preschool) was associated with the microbiota in ß-diversity analysis (F = 3.32, P = 0.011) and in a differential abundance analysis (28 significant amplicon sequence variants). Among all children, we found no significant difference in the microbiota between children with severe and mild/moderate asthma/wheeze in univariable ß-diversity analysis (F = 1.99, P = 0.08, N = 241), but a significant difference in a multivariable model (F = 2.66, P = 0.035), including the number of exacerbations in the previous year. Age was also significant when expressed as a microbial maturity score (Spearman Rho, 0.39; P = 4.6 × 10-10); however, this score was not associated with asthma/wheeze severity. Conclusions: There was a modest difference in the oropharyngeal airway microbiota between children with severe and mild/moderate asthma/wheeze across all children but not in individual age groups, and a strong association between the microbiota and age. This suggests the oropharyngeal airway microbiota as an interesting entity in studying asthma severity, but probably without the strength to serve as a biomarker for targeted intervention.

The developing airway and gut microbiota in early life is influenced by age of older siblings.

BACKGROUND: Growing up with siblings has been linked to numerous health outcomes and is also an important determinant for the developing microbiota. Nonetheless, research into the role of having siblings on the developing microbiota has mainly been incidental. RESULTS: Here, we investigate the specific effects of having siblings on the developing airway and gut microbiota using a total of 4497 hypopharyngeal and fecal samples taken from 686 children in the COPSAC2010 cohort, starting at 1 week of age and continuing until 6 years of age. Sibship was evaluated longitudinally and used for stratification. Microbiota composition was assessed using 16S rRNA gene amplicon sequencing of the variable V4 region. We found siblings in the home to be one of the most important determinants of the developing microbiota in both the airway and gut, with significant differences in alpha diversity, beta diversity, and relative abundances of the most abundant taxa, with the specific associations being particularly apparent during the first year of life. The age gap to the closest older sibling was more important than the number of older siblings. The signature of having siblings in the gut microbiota at 1 year was associated with protection against asthma at 6 years of age, while no associations were found for allergy. CONCLUSIONS: Having siblings is one of the most important factors influencing a child's developing microbiota, and the specific effects may explain previously established associations between siblings and asthma and infectious diseases. As such, siblings should be considered in all studies involving the developing microbiota, with emphasis on the age gap to the closest older sibling rather than the number of siblings. Video abstract.

Vertical Transfer of Metabolites Detectable from Newborn's Dried Blood Spot Samples Using UPLC-MS: A Chemometric Study.

The pregnancy period and first days of a newborn's life is an important time window to ensure a healthy development of the baby. This is also the time when the mother and her baby are exposed to the same environmental conditions and intake of nutrients, which can be determined by assessing the blood metabolome. For this purpose, dried blood spots (DBS) of newborns are a valuable sampling technique to characterize what happens during this important mother-child time window. We used metabolomics profiles from DBS of newborns (age 2-3 days) and maternal plasma samples at gestation week 24 and postpartum week 1 from n=664 mother-child pairs of the Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC2010) cohort, to study the vertical mother-child transfer of metabolites. Further, we investigated how persistent the metabolites are from the newborn and up to 6 months, 18 months, and 6 years of age. Two hundred seventy two metabolites from UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) analysis of DBS and maternal plasma were analyzed using correlation analysis. A total of 11 metabolites exhibited evidence of transfer (R>0.3), including tryptophan betaine, ergothioneine, cotinine, theobromine, paraxanthine, and N6-methyllysine. Of these, 7 were also found to show persistence in their levels in the child from birth to age 6 years. In conclusion, this study documents vertical transfer of environmental and food-derived metabolites from mother to child and tracking of those metabolites through childhood, which may be of importance for the child's later health and disease.

Application of multivariate data analysis for food quality investigations: An example-based review.

These days, large multivariate data sets are common in the food research area. This is not surprising as food quality, which is important for consumers, and its changes are the result of a complex interplay of multiple compounds and reactions. In order to comprehensively extract information from these data sets, proper data analysis tools should be applied. The application of multivariate data analysis (MVDA) is therefore highly recommended. However, at present the use of MVDA for food quality investigations is not yet fully explored. This paper focusses on a number of MVDA methods (PCA (Principal Component Analysis), PLS (Partial Least Squares Regression), PARAFAC (Parallel Factor Analysis) and ASCA (ANOVA Simultaneous Component Analysis)) useful for food quality investigations. The terminology, main steps and the theoretical basis of each method will be explained. As this is an example-based review, each method was applied on the same experimental data set to give the reader an idea about each selected MVDA method and to make a comparison between the outcomes. Numerous MVDA methods are available in literature. Which method to select depends on the data set and objective. PCA should be the first choice for data exploration of two-dimensional data. For predictive purposes, PLS is the most appropriate method. Given an underlying experimental design, ASCA takes into account both the relation between the different variables and the design factors. In case of a multi-way data set, PARAFAC can be used for data exploration. While these methods have already proven their value in research, there is a need to further explore their potential to investigate the complex interplay of compounds and reactions contributing to food quality. With this work we would like to encourage food scientists with no or limited knowledge of MVDA to get some first insights into the selected methods.