Anorexia nervosa (AN) is a severe mental disorder characterized by restrictive eating and disturbance in the way one's body weight or shape is experienced, often accompanied by depression and anxiety. Current evidence-based treatments for AN have limited efficacy, with less than half of the patients achieving full recovery in long-term follow-up studies. Recent findings have identified gut microbiota (GM) dysbiosis as a potential contributor to AN pathology through the gut-brain axis. This open-label, non-randomized, feasibility trial (Clinicaltrials.gov Identifier: NCT05834010) evaluated the feasibility of utilizing fecal microbiota transplantation (FMT) to modify the GM and GM-associated signaling in females with AN and to examine biological effects following a single FMT procedure. Adult female participants diagnosed with AN were recruited. FMT was administered either orally via capsules or as rectal enema. Stool and blood samples were collected pre- and one week post-FMT to assess GM composition, hormonal changes, and biomarkers. Primary endpoints: Feasibility of FMT in individuals with AN and preferred route of FMT. Secondary endpoints: A single FMT treatment can alter GM composition in individuals with AN short term and relevant gut brain signaling in serum. 18/22 participants (81%) completed FMT and sampling and 19/22 participants chose oral capsules, with no serious adverse effects reported. GM analysis showed significant shifts toward donor composition 1-week post-FMT, with improved stool consistency. No significant changes were observed in psychopathology measures or appetite-related biomarkers. Oral FMT is a feasible intervention for adult women with AN, leading to changes in GM profile. Future studies should focus on placebo-controlled trials to assess the efficacy of repeated oral treatments and explore long-term effects on GM, appetite, body weight, sex hormones, disorder-specific symptoms, and overall well-being

Bioinformatics tools are increasingly important for diagnostics in clinical care and precision medicine, but despite a very active bioinformatics research community, implementation and adaptation is slow. Drawing on multidisciplinary expertise, we have identified key systemic barriers on the journey from research to implementation, using the Danish healthcare ecosystem as the example. We find the main obstacles to be regulatory uncertainty, fragmented data access, and limited infrastructure for implementation. Cultural resistance to commercialization and workforce gaps further impedes progress. We believe that these challenges reflect broader international trends and could be generally applicable. Consensus recommendations include centralized data and regulatory resources, cross-sector collaboration models, and pilot initiatives to support scalable implementation. These findings offer a roadmap for translating bioinformatics innovation into clinical practice.

In observational studies, an unhealthy dietary pattern during pregnancy is associated with an increased likelihood of offspring ADHD. We investigated whether such associations may be partly attributable to genetic confounding. Polygenic scores (PGSs) for a healthy dietary pattern were calculated for mother, father, and child trios in the COPSAC2010 cohort. Diagnoses and trait scores of ADHD were assessed in the children at age 10. Using trio models, we tested whether the association between maternal genetic liability to a healthy dietary pattern and offspring ADHD could be accounted for by genetic confounding. In COPSAC2010 trio models (N-trio=437), a maternal healthy dietary pattern PGS was associated with reduced ADHD trait score after adjustment for child and paternal dietary pattern PGS, suggesting indirect genetic effects consistent with causal effects from maternal diet. However, this was not replicated in MoBa (N-trio=41 580) or ALSPAC (N-trio=1 211), where direct genetic effect estimates implied an important role for genetic confounding. Collectively, these genetic results indicate a potential pathway by which genetic confounding can inflate observed associations between maternal dietary pattern in pregnancy and offspring ADHD, and do not provide any robust evidence consistent with a causal pathway between the two. These findings should be interpreted in the light of both the limited predictive power of the dietary pattern PGS, which accounted for only a small proportion of pregnancy diet variance, and the multi-faceted nature of diet as an exposure.

Abstract Persistent SARS-CoV-2 infections have been hypothesized to play a key role in the emergence of variants of concern. However, the factors determining which individuals are at risk and their viral molecular signatures during infection remain poorly understood. Using Denmark’s extensive COVID-19 surveillance, comprising over 700,000 genomes, we identify 303 persistent infections and, critically, link them to health and sociodemographic data. Our analysis confirms the hypothesis that immunocompromised individuals are at the highest risk of experiencing persistent infections. Other disease groups associated with mortality, such as diabetes, show no such associations. Among these persistent infections, the viral sequences exhibit signs of positive selection, with recurrent mutations linked to treatment resistance. Our findings suggest that immunosuppression plays a key role in the emergence of novelty in persistent infections.

MOTIVATION:Temporal multivariate data is ubiquitous in many domains, for instance, being collected over time at planned visits (every few months/years) in longitudinal cohorts, or every few minutes/hours in challenge tests. The analysis of such data often focuses on revealing the underlying temporal patterns common across subjects. However, there are subject-specific differences in temporal patterns, which hold the promise to enhance our understanding of underlying mechanisms and facilitate personalized approaches. Nevertheless, extracting subject-specific temporal patterns from longitudinal multivariate data reliably is an open challenge. RESULTS:We introduce coupled matrix factorizations (CMF) as effective tools to capture subject-specific temporal patterns focusing on two novel applications: analysis of longitudinal metabolomics data and sensitization data. Our analysis shows that CMF models reliably capture subject-specific (shape) differences in temporal patterns with the promise to reveal further insights compared to the state of the art. In metabolomics, CMF models reveal differences in metabolic responses of individuals (in a postprandial meal challenge) according to anthropometric and insulin sensitivity measures. In sensitization data analysis, CMF-based methods capture differences in temporal trajectories of children according to delivery/birth mode and atopic disease diagnosis. We demonstrate the reliability of extracted patterns using reproducibility and replicability. AVAILABILITY:The code is available on github.com/cchatzis/Revealing-Subject-specific-Temporal-Patterns-from-Longitudinal-Data and doi.org/10.5281/zenodo.22084338. Clinical data is not publicly available due to privacy reasons. Data can be made available under a joint research collaboration by contacting COPSAC (administration@dbac.dk).

Sufficient cleansing of the colonic mucosa is of great importance during colonoscopy emphasizing the need for effective preparation agents. Studies evaluating the efficacy of a novel 1 L polyethylene glycol with ascorbate (1 L-PEG-Asc), Plenvu®, versus standard 2 L polyethylene glycol with ascorbate (2 L-PEG-Asc), Moviprep®, have shown promising results regarding bowel cleansing. Nonetheless superiority of 1 L-PEG-Asc has not been shown. In this single-center randomized controlled superiority trial, participants of the Danish national colorectal cancer screening program scheduled for colonoscopy were randomized to either 1 L-PEG-Asc or 2 L-PEG-Asc administered as evening/morning split-dose. Primary endpoint was efficacy of the bowel preparation assessed as bowel cleansing success according to Boston Bowel Preparation Scale (BBPS), adequate defined as BBPS ≥ 2 in each segment. Secondary outcomes included excellent bowel preparation defined as BBPS = 3 in each segment, nausea, vomiting, willingness to repeat the cleansing, and lesion detection defined as polyp, adenoma, sessile serrated lesion, and cancer detection. 1275 participants were randomized to 1 L-PEG-Asc (n = 629) or 2 L-PEG-Asc (n = 646). 1 L-PEG-Asc achieved superior frequencies for adequate (92.2

BACKGROUND:Faecal immunochemical test (FIT)-based colorectal cancer (CRC) screening was gradually implemented in Denmark from March 2014 to December 2017. From January 2018, the FIT screening became biennial and is offered to all citizens aged 50-74 years. Citizens with a positive FIT (≥20 μg haemoglobin/g faeces), are scheduled for colonoscopy within 14 days. We examined indicators of quality in FIT-based CRC screening over time and compared with European guidelines. Furthermore, the overall CRC incidence and mortality before, during and after the implementation of CRC screening were examined. METHODS:Nation-wide register data on CRC screening in Denmark were used to assess screening performance during prevalence screening (2014-2017) and incidence screening (2018-2022). For both prevalence and incidence screening we compared results with quality indicators from the European guidelines. Aggregated data on CRC incidence and mortality were obtained from surveillance data. RESULTS:During prevalence and incidence screening, participation in CRC screening was 61.7% and 60.5%, the proportion of positive FITs were 6.9% and 4.7%, respectively. The compliance to follow-up colonoscopy was approximately 90%. The adenoma detection rate increased slightly over time, while number of screen-detected CRC decreased over time. The 3-year post-colonoscopy CRC rate fluctuated around 7.1-8.9% and decreased to 6.8% in 2021. A CRC incidence peak was observed after the introduction of screening followed by a decrease, while mortality continued to decline. CONCLUSION:The performance of FIT-based CRC screening in Denmark is within the acceptable levels defined by the European guidelines; however, improvements are needed, particularly an increase in participation.

Introduction: Preeclampsia (PE) is a leading cause of maternal and neonatal morbidity and mortality, and low-dose aspirin (LDA) prophylaxis is the cornerstone of evidence-based prevention. Despite guideline recommendations, LDA adherence remains poor, with 10-25% of moderate-risk patients taking aspirin. Objective personalized risk stratification using biomarkers has been shown to motivate behavior change in other disease contexts. Survey data suggest that patients are more motivated to take aspirin if informed by an objective predictive test. Here, we report real-world LDA adherence among patients who received a high-risk result from a cell-free RNA (cfRNA) PE risk prediction test. Methods: This retrospective, observational survey study included asymptomatic patients of advanced maternal age (AMA; ≥ 35 years at delivery) with singleton pregnancies without USPSTF-defined preexisting high-risk conditions for PE who received the cfRNA PE risk prediction test. Patients who opted in to receive text message surveys were asked about LDA use following receipt of test results. High adherence was defined as reporting LDA use on at least 6 of 7 days per week at least 85% of the time surveyed. The primary analysis included patients with a high-risk test result and at least one LDA frequency survey response following receipt of test result. The observed proportion of adherent patients was compared to a baseline estimate of 25% using an exact binomial test. Results: Of 166 patients who received a cfRNA PE risk prediction test result, 48 (28.9%) received a high-risk result. Of these, 29 (60%) opted in and responded to at least one survey, constituting the primary analysis population. Twenty-seven of the 29 (93.1%; 95% CI: 78.0-98.1%) were classified as highly adherent, significantly higher than the 25% baseline adherence estimate for moderate-risk patients (p < 0.0001). Conclusion: Among surveyed patients who received a high-risk cfRNA PE test result, the proportion classified as highly adherent to LDA (93%) substantially exceeded published estimates of adherence in a similar patient population and met the clinically meaningful threshold of ≥ 80% associated with reduced risk of preterm preeclampsia. These findings indicate that objective and personalized biomarker risk testing may be a powerful driver of behavior change that current guidelines have failed to produce.

The host genetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have previously been studied based on cases from the earlier waves of the pandemic in 2020 and 2021, identifying 51 genomic loci associated with infection and/or severity. SARS-CoV-2 has shown rapid sequence evolution, increasing transmissibility, particularly for Omicron variants, which raises the question of whether this affected the host genetic factors. We performed a genome-wide association study of SARS-CoV-2 infection with Omicron variants, including more than 150,000 cases from four cohorts. We identified 13 genome-wide significant loci, of which only five were previously described as associated with SARS-CoV-2 infection. The strongest signal was a single nucleotide polymorphism in an intron of ST6GAL1, a gene affecting immune development and function, connected to three other associated loci (harboring MUC1, MUC5AC and MUC16) through O-glycan biosynthesis. Our study provides robust evidence for individual genetic variation related to glycosylation, translating into susceptibility to SARS-CoV-2 infections with Omicron variants. Genome-wide analyses identify 13 loci associated with susceptibility to infection with SARS-CoV-2 Omicron variants, including variation in ST6GAL1, previously associated with influenza susceptibility, and other genes involved in glycosylation processes.

Spore-forming Bacillus species, including pathogenic Bacillus cereus and spoilage-associated Bacillus subtilis, are major contributors to foodborne illness and product degradation. Understanding their metabolic behaviour in diverse food matrices is essential for improving risk assessment, spoilage prediction, and fermentation control. This study integrates isothermal microcalorimetry and targeted metabolomics to characterize the metabolic activity of B. cereus and B. subtilis in five nutrient sources: Brain Heart Infusion (BHI) medium, oat drink, milk, pea hydrolysate, and a combined oat-pea matrix. Metabolic heat production was monitored for 24 h at 30 °C. In BHI, B. cereus exhibited a shorter lag phase (mean ± sd: 4.3 h ± 0.8) than B. subtilis (7.9 h ± 1.0) but produced less total heat. Across all food matrices, B. subtilis consistently generated more heat. The oat-pea matrix supported the highest calorimetric growth rates, surpassing oat or pea alone, and showed sugar depletion and accumulation of organic acids, indicating enhanced carbohydrate metabolism. Free amino acid release was matrix- and species-specific: B. subtilis had increased levels in oat, while B. cereus did so in pea. While B. cereus was metabolically active in all matrices, cereulide levels were matrix-dependent: 47.3 ± 1.7 ng/mL in oat, 3.0 ± 0.1 ng/mL in oat-pea, and undetectable in pea. These findings reveal clade-specific and matrix-driven metabolic strategies. This is the first study to combine calorimetry and metabolomics to evaluate Bacillus activity in plant-based and dairy matrices. This approach enhances our understanding of microbial physiology in complex food systems and provides a foundation for developing targeted strategies to improve food safety, stability, and product design.

G-quadruplexes (G4s) are non-canonical DNA structures that have proved to play a pivotal role in various biological processes, including telomere maintenance and gene expression regulation. Owing to their prevalence in tumor cells, G4s have emerged as promising targets for cancer therapy, with a substantial body of research demonstrating the potential of G4 ligands as anti-cancer tools. Nonetheless, a comprehensive multi-omics study to fully elucidate the mode of action of G-quadruplex ligands is still lacking. Such an investigation would be crucial for advancing the development of potent G4-based therapies against cancer. Herein, we employed a multi-omics approach, integrating transcriptomics, proteomics, and metabolomics, to identify key signaling pathways that mediate the anti-cancer effects of well-characterized G4-binding agents (berberine, pyridostatin and RHPS4) on human cervical adenocarcinoma (HeLa) cells. Particularly, we analyzed gene expression changes using RNA sequencing, quantified proteins by liquid-chromatography tandem mass spectrometry and examined metabolite levels via nuclear magnetic resonance. Our results revealed that, under the investigated experimental conditions, berberine treatment had only negligible cellular effects. In contrast, pyridostatin induced significant changes at the transcriptomic, proteomic, and metabolomic levels, decreasing the abundance of enzymes involved in cellular energy production, reducing the availability of precursors for lipid and nucleotide biosynthesis, and depleting essential cofactors and enzymes required for redox balance. Notably, RHPS4 could selectively disrupt mitochondrial activity, possibly through the specific stabilization of mitochondrial G-quadruplex structures. Overall, our findings provide a valuable multi-omics perspective on the cellular changes driven by G-quadruplex binders, that may accelerate the development of effective anti-cancer G4-targeted therapies.

Environmental exposure to polycyclic aromatic hydrocarbons (PAHs) causes metabolic dysfunction, but reliable biomarkers are still needed to assess human health effects. This study used 21-day matured human HepG2 spheroids, a metabolically competent three-dimensional (3D) liver model, to assess metabolic responses to graded, non-cytotoxic concentrations of benzo[a]pyrene (BaP) and benzo[b]fluoranthene (BBF) after 24- and 96-h exposure. Untargeted liquid chromatography-mass spectrometry (LC–MS) metabolomics, combined with multivariate and network analyses, identified compound- and time-specific metabolic signatures. At 24 hours, no metabolites showed significant changes. In contrast, at 96 hours, both PAHs consistently altered seven robust metabolites linked to polyamine metabolism, membrane dynamics, mitochondrial energy, and DNA-repair pathways. Network analysis showed BBF caused broader and more connected changes than BaP, indicating distinct toxicodynamics. These findings underscore the importance of extended exposure in revealing metabolic disruption and support a set of candidate biomarkers for future low-dose studies and improved risk assessment of airborne toxicants. ### Competing Interest Statement The authors have declared no competing interest. European Commission, https://ror.org/00k4n6c32, 101057497, 10050740 Croatian Science Foundation, https://ror.org/03n51vw80, MOBODL-2023-12-5502, MOBDOK-2023-1077 Slovenian Research Agency, https://ror.org/059bp8k51, P1-0245

BACKGROUND:Early life is a critical period for neurodevelopment, where factors such as maternal nutrition and breastfeeding duration significantly impact the growth of head circumference and cognitive development in children. Our study aimed to explore the associations between maternal dietary patterns during pregnancy, duration of breastfeeding, and their impacts on child head circumference and cognitive outcomes. METHODS AND FINDINGS:Our study utilised data from the Copenhagen Prospective Studies on Asthma in Childhood 2010 cohort, which enrolled 700 mother-child pairs between 2008 and 2010 with 86% clinical follow-up at age 10. Pregnancy dietary patterns, described as 'Varied' and 'Western,' were derived from food frequency questionnaires and used to model quantitative metabolite scores via sparse partial least squares modelling of blood metabolome data. Cognitive development was assessed using the Bayley Scales of Infant Development at 2.5 years and the Wechsler Intelligence Scale for Children at age 10. Head circumference was measured from 20 weeks gestation to 10 years, and calibrated using related anthropometric measures. Growth trajectories were evaluated using linear mixed models and latent class trajectory models. Parental and child genetic influences for cognition and head circumference were controlled by including polygenic risk scores derived from genomic data. We found that a Western dietary pattern during pregnancy was associated with lower cognitive scores at age 2.5 (β -1.24 [-2.16, -0.32], p = 0.008) and reduced head circumference growth (p-interaction < 0.0001). We found that a Varied dietary pattern during pregnancy was associated with higher estimated intelligence quotient (IQ) at age 10 (β 1.29 [0.27, 2.3], p = 0.014). Additionally, head circumference growth was associated with higher cognitive scores at age 10 (β 3.40 [1.21, 5.60], p = 0.002), and it partly mediates the association between the Varied dietary pattern and estimated IQ (proportion mediated 13.5% [0.01, 0.71], p = 0.034). Extended breastfeeding duration was also independently associated with increased head circumference growth (p-interaction < 0.0001). These patterns and correlations were consistent even after adjusting for potential confounders and accounting for genetic influences. CONCLUSIONS:Our findings reveal that a Western dietary pattern during pregnancy is associated with lower cognitive scores at age 2.5 and decreased head circumference growth, suggesting potential adverse impacts on early neurodevelopment. Conversely, a Varied dietary pattern is linked with a higher estimated IQ at age 10, with head circumference growth contributing to this positive outcome. These findings highlight the critical role of maternal nutrition during pregnancy, and duration of breastfeeding, in promoting optimal neurodevelopmental outcomes. Effective public health strategies should therefore focus on enhancing maternal dietary practices to support better cognitive and physical development in children.

244 Background: Colorectal cancer (CRC) is the third most common cancer worldwide motivating national screening strategies utilizing fecal immunochemical tests (FIT). Blood-based biomarkers could be an alternative method to increase compliance in population-based screening programs for early detection of CRC. We aimed to identify new blood-based biomarkers that could be potential candidates for use in colorectal cancer screening. Methods: In a nested cohort study of 1967 FIT positive participants of the Danish CRC screening program serum levels of GDF-15, hepsin, IL-8, keratin1-10, L1CAM, MIA, monocyte MCP-1, NSE and OPG were measured using the Luminex xMAP immunoassay platform. Main outcomes were CRC vs non-CRC and CRC, high-risk adenomas (HRA) or medium-risk adenomas (MRA) vs low-risk adenomas (LRA) or clean colorectum. Odds ratios for associations between biomarker expressions and outcomes were calculated using logistic regression models and visualized by area under the receiver operating characteristic (ROC) curve (AUC). Analyses were made on the Luminex biomarkers alone and with addition of clinical and demographic information. Results: FIT-induced colonoscopies detected 240 CRCs and 625 HRA or MRA. Multivariate analyses using all biomarkers and age found hepsin, IL-8 and OPG significantly (p<0.001) associated in relation to the main outcome (CRC vs non-CRC) with odds ratios of 0.74 [0.59-0.92], 2.59 [2.12-3.15] and 0.90 [0.82-0.99], respectively. The full model using all biomarkers and age presented an AUC of 0.73 [0.70-0.77]. Conclusions: Changed serum levels of nine novel biomarkers seem to be potential predictors for early detection of CRC, especially hepsin, IL-8 and OPG. [Table: see text]

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that often persists into adulthood and is accompanied by comorbid mental health problems. This cross-sectional cohort study analyzed 411 18-year-olds from the Danish COPSAC2000 birth cohort to investigate the relationship between the gut microbiome, fasting and postprandial systemic inflammation, ADHD symptoms, and symptoms of anxiety, stress, and depression. ADHD was assessed using the Adult ADHD Self-Report Scale (ASRS), while depression, stress, and anxiety were evaluated with the Depression, Anxiety, and Stress Scale 21 (DASS-21). Fecal metagenomic data and inflammation levels, measured as glycosylated protein A (GlycA), were analyzed following a standardized meal challenge. In males, higher ADHD symptom scores correlated significantly with increased abundance of a tryptophan biosynthesis pathway (MetaCyc Metabolic Pathways Database) and elevated fasting and postprandial GlycA levels (p < 0.05). While the severity of depression, anxiety, and stress symptoms showed weak associations with GlycA and the gut microbiome, our findings indicate a significant link between ADHD symptoms and postprandial inflammation, warranting further investigation into underlying mechanisms.

Background Early detection is paramount when reducing incidence and mortality of colorectal cancer (CRC). Current population-based screening programs primarily use fecal immunochemical test (FIT) to allocate individuals for colonoscopy although low specificity challenges colonoscopy capacities. We aimed to assess the potential of circulating tumor (ct)DNA markers for early CRC detection in a dual-test CRC screening approach among FIT positive individuals. Methods Plasma samples from 774 FIT positive (≥100 ng Hemoglobin/mL) individuals from the Danish CRC screening program were analyzed for hypermethylated DNA in the genes Branched Chain Amino-acid Transaminase 1 (BCAT1), Ikaros-Family Zinc Finger transcription 1 (IKZF1), and Interferon Regulator Factor 4 (IRF4). Multivariate logistic regression models were generated adding the ctDNA markers and age to the FIT value. The dual-test approach was benchmarked to FIT at specific thresholds. Results The dual-test approach improved CRC detection compared to the FIT alone (AUC of 87.2 [95% CI, 82.9-91.4] vs AUC of 72.5 [95% CI, 67.0-77.9]). This was also seen when adding advanced adenomas to the outcome resulting in AUCs of 71.8 [95% CI, 67.8-75.8] for the dual-test approach compared to 65.5 [95% CI, 61.3-69.7] for the FIT model alone. Benchmarking the dual-test approach at FIT cut-offs between 100 and 600 ng Hb/mL showed a potential for either reducing the colonoscopy requirement by up to 56% or increasing CRC detection by up to 28%. Conclusions As increasing FIT cutoff will decrease CRC detection rate, application of the ctDNA panel can increase the sensitivity and specificity in a dual-test approach among asymptomatic individuals.