Metabolomics

Prenatal alcohol exposure (PAE) is a major cause of neurodevelopmental disorders, yet most children are diagnosed late or misdiagnosed. Neuroplacentology suggest that placental factors released into maternal and/or umbilical cord blood contribute to fetal brain development. Consistently, a preclinical inter-organ transcriptomic database revealed that PAE disrupts the expression ratio of angiogenic and inflammatory factors suggesting an angio-inflammatory response. This study aimed i) to assay, by multiplex immunoassay, angiogenic and inflammatory factors in maternal and umbilical cord blood from alcohol-consuming women and ii) to perform a maternofetal analysis according to neonatal sex. Afterwards, dysregulated factors from mothers who gave birth to females or males were submitted to STRING and ShinyGO analyses. Results showed that PAE differently altered the distribution profiles of dysregulated angiogenic and inflammatory factors in maternal and umbilical cord blood. Moreover, sex-specific differences were observed, with 36% of dysregulated proteins specific to males, 48% to females, and 16% common to both. STRING analysis revealed robust functional protein-protein interactions linking together inflammatory and angiogenic clusters while the ShinyGO analysis identified enriched pathways related to vascular shear stress. These findings provide the first maternofetal analysis of combined angiogenic and inflammatory factors from alcohol-consuming mothers.

Background: Early detection of pancreatic ductal adenocarcinoma (PDAC) is critical. While faecal elastase-1 (FE-1) is a standard clinical marker for pancreatic function, its diagnostic accuracy for malignancy is limited. We sought to identify plasma metabolites that enhance FE-1 performance in symptomatic "at-risk" patients. Methods: Using the DEPEND cohort (CRUK C45617/A29908), plasma metabolomics was performed on patients with resectable PDAC (n=23) and healthy volunteers (n=24). Predictive modelling included feature selection and cross-validation, with further validation in an independent external cohort. Results: Citrulline was identified as significantly depleted in PDAC patients across discovery and validation cohorts. In isolation, Citrulline achieved an AUC of 0.86 (internal) and 0.88 (external validation). Standalone FE-1 demonstrated an AUC of 0.67. However, combining Citrulline and FE-1 significantly improved diagnostic performance, achieving a combined AUC of 0.96. Stratification revealed distinct metabolomic signatures associated with poorly differentiated tumours, suggesting a link to histological grade. Conclusions: Integrating Citrulline with FE-1 testing substantially improves PDAC detection in symptomatic patients. This non-invasive panel offers high diagnostic potential, though prospective validation is required to establish clinical cut-offs for routine practice.

N-glycolylneuraminic acid (Neu5Gc) has been repeatedly associated with human cancer, but reliable detection has remained elusive, generating controversy regarding its presence in human samples. To address this, matrix-matched calibration curves, which have been pioneered in proteomics and metabolomics for assessing changes in complex mixtures, were measured of released N-glycans at four orders of magnitude dynamic range in defined mixtures, systematically benchmarking Neu5Gc-containing N-glycan detection across multiple LC-MS platforms and sites. Orthogonally, the gold-standard analytical method, consisting of fluorescence detection of labelled monosaccharides separated by LC, was applied to the same samples, yielding absolute concentrations of Neu5Gc. LC-MS demonstrated an extended detection range of three or more orders of magnitude while retaining intact N-glycan measurement, improving assay specificity and enabling detection of the variety of Neu5Gc-bearing N-glycans. By combining orthogonal dimensions of evidence, including chromatographic separation, isotopic distribution matching, and composition-confirming MS/MS, LC-MS confidently resolved Neu5Gc signals from noise, even at low abundance. In comparison, DMB-LC-FLR was limited to two orders of magnitude dynamic range, insufficient for detection of Neu5Gc in commercially available pooled human sera. These findings strongly support that DMB-LC-FLR assay specificity and sensitivity are insufficient for Neu5Gc detection in human samples due to noise overwhelming the Neu5Gc signal. By establishing a reusable benchmarking framework for future glycomic studies, we aim to use LC-MS to improve the measurement of Neu5Gc in clinical samples.

Abstract Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is linked to obesity and cardiometabolic dysfunction, yet local prevalence data from Mexican first-level care units are scarce. We estimated the prevalence of MASLD by B-mode ultrasonography and characterized the clinical profile of obese adults in a family medicine unit in Leon, Mexico. Methods: Cross-sectional study of 55 adults with obesity (body mass index [BMI] >=30 kg/m2) registered at one medical office of a Mexican Social Security Institute family medicine unit (April-October 2024). Hepatic steatosis was graded by B-mode ultrasonography; MASLD was defined as ultrasonographic steatosis plus at least one cardiometabolic criterion (all participants met the obesity criterion). Prevalence was reported with Wilson 95% confidence intervals (CIs); associations with obesity grade were assessed by Cochran-Armitage trend and Fisher exact tests. Results: MASLD was present in 37 of 55 participants (67.3%; 95% CI, 54.1-78.2). Mild (grade 1) steatosis predominated (45.5%). Prevalence increased across obesity grades: 59.0%, 75.0%, and 100% for grades 1, 2, and 3, respectively (trend P=0.022). Type 2 diabetes and hypertension each affected 50.9% of participants; the sample was predominantly female (74.5%). Conclusion: Two of every three obese adults screened in primary care had ultrasonographic MASLD, with a significant rise across obesity grades. Opportunistic ultrasonography in obese primary-care patients may enable earlier MASLD detection. Keywords: Non-alcoholic Fatty Liver Disease; Fatty Liver; Obesity; Ultrasonography; Primary Health Care; Prevalence

BACKGROUND In line with International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommendations for high-sensitivity cardiac troponin assays, analytical validation and reference limit assessments are required to confirm that an assay meets performance criteria. This study evaluated the analytical performance and established the 99th percentile upper reference limit (URL) for the SPINCHIP High-Sensitivity Cardiac Troponin I (SPINCHIP hs-cTnI) point-of-care assay. METHODS Analytical performance characteristics, including the limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ), were assessed. Additionally, 1,053 plasma samples and 1,055 whole-blood samples were used to determine the URL. Imprecision around the 99th percentile URL was evaluated as part of the analytical validation. High-sensitivity criteria were assessed by confirming measurable cTnI in [&ge;]50% of healthy individuals (n=432 plasma; n=431 whole blood) and achieving imprecision <10% at the 99th percentile (plasma, n=960; whole blood, n=480). RESULTS SPINCHIP hs-cTnI demonstrated a LoB of 0.3 ng/L; LoDs of 0.8 ng/L (plasma) and 0.9 ng/L (whole blood); and LoQs of 1.1 ng/L (plasma) and 1.4 ng/L (whole blood). The analytical measuring range was 1.1-9,000 ng/L. Imprecision at the common 99th percentile URL (14 ng/L) was 5.8%; for men (URL=16 ng/L) 5.6% and for women (URL=10 ng/L) 6.3%. Greater than 85.2% (94.0% and 76.1% in men and women, respectively) of healthy individuals showed measurable cTnI above the LoD. CONCLUSIONS The SPINCHIP hs-cTnI assay meets the IFCC high-sensitivity requirements, demonstrating <10% imprecision at the 99th percentile, reliable low-concentration precision and cTnI detection in more than half of healthy individuals.

Background: Plasma phosphorylated tau-217 (pTau-217) measurements for use in Alzheimer disease (AD) identification require thresholds to define positivity and there exist different approaches to operationally defining the boundary. We compared amyloid {beta} (AB) PET-anchored and distribution-based positivity cut-off values and explored how these mapped onto latent biomarker states. Methods: We analysed plasma pTau-217 measured in the Bio-Hermes-001 cohort (N = 990) using an immunoassay (Lilly) and mass spectrometry assay (University of Gothenburg). Gaussian mixture models were used to identify latent classes and thresholds were derived in two ways: achieving 90% specificity for AB PET positivity and exceeding the mean + 2SDs of the lowest latent class. We explore classes in reference to AB PET status and clinical diagnosis, as well as agreement between approaches using Cohen kappa for both assays. Results: In both assays, three latent biomarker classes were identified with monotonic increases in AD clinical diagnosis and AB PET positivity. PET-anchored thresholds showed lower specificity but higher sensitivity to amyloid positivity than distribution-based thresholds. Overall agreement between the approaches was acceptable (k = 0.678 for Lilly and 0.575 for University of Gothenburg), with disagreement concentrated in the intermediate latent class. Classes with the lowest and highest pTau-217 concentrations were classified consistently using both thresholds Discussion: The two thresholding approaches yielded similar classifications at both the negative and positive tail of the observed biomarker distribution, but classify intermediate concentrations differently. The boundary definition influenced pTau-217 positivity more than the analytical platform itself. Thresholding approaches may capture different pTau-217 biomarker states, therefore such methodological decisions should be grounded in the context of the intended application.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

Background: Childhood obesity is a prevalent public health concern, particularly among children from marginalized backgrounds. Epigenomic mechanisms, including DNA methylation (DNAm), offer insight into the biological embedding of metabolic risk, yet protective family-level factors remain understudied. This study examined whether participation in a positive parenting intervention was associated with reduced epigenetic cardiometabolic risk among preschool-aged children. Methods: Participants were 74 children (n = 35 intervention; n = 39 services-as-usual; mean age = 36 months). Using a secondary analysis of a randomized controlled trial, DNAm-derived body mass index (BMI) and anthropometric BMI were assessed at baseline and 12-month follow-up, and parenting practices were observed post-treatment. Results: Children in the intervention group demonstrated significantly lower DNAm BMI at 12 months relative to controls, adjusting for child sex, race and/or ethnicity, and anthropometric BMI. No treatment effect was observed for anthropometric BMI, and DNAm BMI was not associated with anthropometric BMI at 12 months. Although parenting practices improved, they did not mediate intervention effects on DNAm-derived BMI. Conclusions: Parenting interventions may influence biological pathways related to cardiometabolic risk, even before changes in anthropometric outcomes, underscoring the potential of family-centered approaches to promote early metabolic health.

Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) cells to rapid cycling between castrate and supraphysiologic androgen concentrations, but clinical BAT uses testosterone, which can also activate wild-type androgen receptor (AR) in androgen-responsive tissues, causing systemic side effects. 5{beta}-dihydrotestosterone (5{beta}-DHT) is a naturally occurring testosterone metabolite generally considered androgenically inactive because it binds wild-type AR weakly, yet its activity against clinically relevant AR mutants has not been systematically evaluated. Here, we tested whether 5{beta}-DHT and related 5{beta}-reduced testosterone metabolites activate AR signaling and growth programs in prostate cancer models that carry AR mutations. In C4-2 cells, 5{beta}-DHT and 3{beta}-etiocholanediol (3{beta}-ecdiol) increased canonical AR target genes, including KLK3 and TMPRSS2, with weaker activity than testosterone, whereas other 5{beta} metabolites showed limited activity. In androgen-responsive LNCaP and C4-2 models, 5{beta}-DHT and 3{beta}-ecdiol promoted cell growth under androgen-depleted conditions, and this effect was suppressed by enzalutamide, supporting AR dependence. RNA-seq confirmed that 5{beta}-DHT and 3{beta}-ecdiol induced androgen-response gene sets substantially overlapping with testosterone, albeit at lower transcriptional magnitude. Further, we found that 5{beta}-DHT, but not 3{beta}-ecdiol, suppresses cell proliferation of LNCaP, C4-2, and PC-3 cells stably expressing the clinically relevant AR gain-of-function mutants W742C and H875Y through activating AR-induced senescence-like features after high-dose exposure, consistent with the therapeutic logic of BAT. These findings identify 5{beta}-DHT as an overlooked mutant-AR agonist capable of BAT-like tumor suppression and propose it as a testosterone surrogate in BAT with potentially reduced systemic androgenic side effects. HighlightsO_LI5{beta}-DHT and 3{beta}-ecdiol promote AR-dependent prostate cancer cell growth C_LIO_LIBoth are weaker AR agonists than testosterone by RNA-seq and qPCR C_LIO_LISupraphysiologic 5{beta}-DHT suppresses growth via AR-mediated senescence C_LIO_LIGrowth suppression extends to AR mutants W742C and H875Y C_LIO_LI5{beta}-DHT may be a lower-androgenicity testosterone surrogate for BAT C_LI

Background & Aims: The association between upper gastrointestinal (GI) symptoms and delayed gastric emptying time (GET) is debated. This study utilized Body Surface Gastric Mapping (BSGM) and the 'Auckland Classification' BSGM phenotyping scheme to investigate whether symptom associations with GET vary across different mechanistic phenotypes. Methods: A pooled analysis was performed on two prospective datasets of 194 patients with chronic upper GI symptoms. Participants underwent simultaneous BSGM (Gastric Alimetry) and gastric emptying testing (breath test or scintigraphy). Validated time-of-test symptom profiling was recorded at 15-minute intervals using 0-10 Likert-type scales. Patients were classified: 'Spectral Abnormal' (abnormal electrophysiology), 'Sensorimotor' (symptoms correlate with gastric electrical amplitude), 'Continuous' (symptoms uncorrelated), and 'NA' (no phenotype). Results: In the analyzed cohort, 24% had delayed GET. Overall, delayed GET was associated with a higher total symptom burden (15.6 delayed vs. 8.8 normal, p=0.006; r=0.30, p<0.001), specifically postprandial fullness (p=0.004) and early satiation (p=0.015). However, associations varied significantly by phenotype. In the 'Sensorimotor' phenotype, nausea was associated with delayed GET (4.9 vs. 1.0, p=0.027; r=0.59, p=0.009), while total symptom burden was 23.3 vs. 8.4 (p=0.135). Conversely, patients with 'Continuous' or 'Spectral Abnormal' phenotypes, or who were unclassified, showed no symptom associations. Conclusions: Delayed GET is weakly associated with an increased burden of upper GI symptoms. However, gastroduodenal disorders are heterogeneous, such that phenotyping reveals this association to be exclusive to patients with a 'Sensorimotor' phenotype. These results could improve targeting of therapies that address gastric emptying.

Background. Maximal oxygen uptake (VO2max) is a leading marker of cardiorespiratory fitness and a strong predictor of all-cause mortality. Cardiopulmonary exercise testing (CPET) is the reference method but is resource-intensive, so consumer wearables estimate VO2max from passively collected signals; these estimates compress the fitness range, returning near-correct group averages while ranking individuals poorly. No peer-reviewed validation of a smart-ring VO2max estimate against CPET has been reported, and none in a South Asian cohort. Objective. To validate the Ultrahuman Ring AIR VO2max estimate against laboratory CPET, benchmark it against published prediction equations, and assess its generalization and construct validity. Methods. In a single-site paired ring-CPET cohort (N = 101; mean CPET peak VO2 43.3 mL{middle dot}kg-{superscript 1}{middle dot}min-{superscript 1}, SD 9.9), peak oxygen uptake was measured by treadmill or cycle-ergometer CPET, and the Ultrahuman Ring AIR estimate was computed from passively collected signals using a transparent ensemble based on published equations. Ensemble weights and calibration were selected on an 85-subject development set by an automated search minimizing a composite 5-fold cross-validated error criterion; the locked estimate was evaluated on a 16-subject held-out test set. The calibrated coefficients are proprietary. Agreement was quantified with mean absolute error (MAE), bias, Pearson r, regression slope and Lin's concordance correlation coefficient (CCC; bootstrap 95% CIs), and Bland-Altman limits of agreement. Separately, in 181,133 de-identified Ring users (no CPET reference), construct validity was assessed against ring-measured sleep, continuous glucose monitoring (n = 2,597), and a venous blood panel (n up to 15,203), adjusted for age, sex, and BMI, with lipoprotein(a) as a pre-specified negative control. Reporting followed TRIPOD and STARD. Results. With a self-reported fitness level provided, the estimate agreed with CPET peak VO2 at MAE 4.68 mL{middle dot}kg-{superscript 1}{middle dot}min-{superscript 1} (95% CI 3.93 to 5.49), Pearson r 0.79, CCC 0.79, and slope 0.71. The five published equations were worse on every metric (MAE 6.2 to 10.6, CCC 0.28 to 0.56, slope 0.32 to 0.42), each compressing the fitness range. On the held-out test set (n = 16), agreement held (r 0.84, slope 0.81, MAE essentially unchanged). Without the fitness input, full-cohort MAE was 5.16, still ahead of every published equation. At population scale, higher estimated fitness tracked a healthier profile on measurements the estimate does not use: better ring-measured sleep; higher continuous-glucose time in target range (79.6% versus 61.5%, top versus bottom decile; n = 222 and 399 of 2,597 users); and lower triglycerides, fasting glucose, and HOMA-IR (n up to 15,203 assayed per marker). These associations held after adjustment for age, sex, and BMI, whereas the pre-specified negative control lipoprotein(a) did not separate the deciles. Conclusions. The Ultrahuman Ring AIR VO2max estimate agreed with laboratory CPET substantially better than published prediction equations, held its agreement on held-out subjects, and ordered a large population along independent cardiometabolic gradients consistent with true fitness.

Per- and polyfluoroalkyl substances (PFAS) are persistent toxicants with immunological, metabolic and epithelial effects, but their relevance to inflammatory skin disease remains unclear. We developed a computational toxicology framework to test whether perfluoroalkyl sulfonate programs, especially perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS), converge with psoriasis-associated keratinocyte inflammation. Exposure transcriptomes were derived from GSE236956, in which human embryonic stem cell-derived epithelial-lineage models were exposed to 10 M PFAS for 8-16 days. Six PFAS were prioritized using descriptors, Tanimoto similarity, toxicology evidence, adverse outcome pathway (AOP)-like key events, exposure differentially expressed gene burden and read-across support. PFAS signatures were integrated with psoriasis bulk transcriptomes, single-cell RNA sequencing, keratinocyte-state mapping, regulator and communication inference, spatial transcriptomics and token-aware Geneformer-compatible virtual perturbation. PFOS ranked highest in integrated prioritization, followed by PFHxS and perfluorooctanoic acid. PFHxS produced a smaller but directionally informative signature within a PFOS-dominant perfluoroalkyl sulfonate footprint. The shared PFOS and PFHxS program converged with psoriasis through inflammatory keratinocyte, epidermal-stress, cytoskeletal and lipid-related modules. Single-cell and spatial analyses localized the program to activated keratinocytes and inflammatory epidermal niches, with strong spatial co-localization with inflammatory keratinocyte and epidermal stress scores. Virtual perturbation prioritized S100A9, S100A8, KRT16, IL36G, CCL20, CXCL8, FABP5, KRT17, FOS, JUN and NFKBIZ as candidate effectors. These findings support an exposure-informed, experimentally testable hypothesis linking persistent perfluoroalkyl sulfonate programs to keratinocyte inflammatory niches in psoriasis.

Importance Sleep-disordered breathing (SDB) is common in childhood and is associated with attentional and behavioral impairments despite largely preserved sleep macrostructure and minimal abnormalities in conventional electroencephalographic measures. This discrepancy has contributed to the perception that sleep is relatively preserved in pediatric SDB and has limited understanding of the physiological mechanisms underlying morbidity. Objective To determine whether pediatric SDB is associated with disruption of the regional organization and homeostatic dynamics of slow-wave activity (SWA), a key physiological marker of sleep-dependent neural recovery and development. Design, Setting, and Participants Cross-sectional study of 62 children aged 4 to 12 years who underwent overnight polysomnography with high-density electroencephalography in a laboratory setting. Participants were recruited from clinical referrals and the community, spanning the full spectrum of SDB severity. Exposures SDB severity indexed by hypopnea index (HI), apnea-hypopnea index (AHI), and obstructive apnea index (OAI). Main Outcomes and Measures Regional electroencephalogram-derived SWA (0.5 to 4 Hz) topography and exponential decay parameters derived from frontal and posterior cortical regions. The frontal-to-posterior decay-rate ratio was evaluated as a summary measure of regional sleep homeostasis. Results In children with lower hypopnea index, SWA demonstrated the expected developmental pattern, with posterior predominance in younger children and a progressive shift toward a more balanced anterior-posterior distribution with age. Increasing HI was associated with attenuation or reversal of this spatial organization. Global SWA showed no meaningful association with SDB severity. In contrast, regional frontal and posterior decay parameters were strongly associated with HI (adjusted R2 = 0.53; p < 1e-6) but not OAI (adjusted R2 = 0.05; p = .95). The frontal-to-posterior decay-rate ratio showed the strongest association with HI {beta} = 4.15; 95% CI, 3.17-5.13; p < 1e-10; adjusted R2 = 0.55. Conclusions and Relevance Pediatric SDB was associated with regional disruption of slow-wave sleep homeostasis rather than global loss of deep sleep. These alterations affected both the spatial organization and temporal dynamics of SWA during a period of active cortical maturation and were not captured by conventional sleep metrics. Regional SWA dynamics may provide a developmentally sensitive marker of physiological disease burden in children with SDB.

Applications of tissue culture are critical for Cannabis sativa L. (cannabis), supporting clonal propagation, germplasm preservation, pathogen elimination, among other biotechnological applications. However, extensive genetic diversity associated with cannabis results in highly variable responses to in vitro conditioning, and no consensus basal media formulation exists to support reproducible micropropagation across genotypes. To address these limitations, a hybridized ensemble-NSGA-II approach was employed for concurrent optimization of individual media components to create a species specific, cultivar inclusive basal salt formulation for cannabis micropropagation. The resulting PHJ media represents a unique formulation that overcomes recalcitrance across a wide array of cannabis cultivars, facilitating improved growth and uniformity for the nine cultivars used in its development and validation. These results remain consistent from explant initiation through multiple rounds of subculture. The ability of PHJ to overcome genotypic recalcitrance is telling of its potential applicability with an array of plant species beyond cannabis. Additionally, robust performance both with and without plant growth regulators underscores the plausible use of PHJ for diverse applications beyond standard micropropagation. Ultimately, this cultivar-inclusive basal medium demonstrates utility for both scientific research and industrial-scale operations.

Background: Lead exposure is estimated to cause approximately 3.5 million premature deaths a year, yet the key ongoing sources of lead exposure are unclear. Methods: We estimated the contribution of dietary lead intake to global blood lead levels (BLLs) for 7-year-old children and 22-year-old adults by applying the All-Ages Lead Model (AALM) to calculate blood lead levels (BLLs) based on 25 total diet studies (TDS) that quantify dietary lead intake across 46 countries. Results: For children, the population-weighted average dietary lead intake in low- and middle-income countries (LMICs) (32.0 g/day) was found to be more than three times higher than in high-income countries (HICs) (9.3 g/day), and more than 10 times higher than the FDA reference level for children (2.2 g/day). The average impact on BLLs for children is estimated to be near 29 g/L in LMICs and near 12 g/L in HICs. Averaged across the TDS data, vegetables (27%) and cereals (24%) were found to contribute the most to dietary lead. Conclusions: While there are limitations associated with biokinetic modelling and the TDS data from LMICs, these results suggest that the contribution of dietary lead intake to global lead exposure is in the region of 40 to 50%, suggesting, in turn, that dietary lead intake is likely a major global driver of lead poisoning. Lead absorbed from the environment into food crops is expected to be the key driver of dietary lead. Current regulatory levels for maximum lead concentrations in foods (0.05-0.3 mg/kg) are out-of-date and may imply a dietary lead intake of 200 g/day, far higher than the FDA reference level (2.2 g/day). Collecting representative TDS data in high lead burden countries should be a priority. Further research is also recommended on upstream lead sources and pathways of lead uptake in plants, driving global food contamination.

Autonomic dysfunction is a common sequela of mild traumatic brain injury (mTBI). Physical activity progression is an integral component of mTBI rehabilitation, particularly in addressing autonomic dysfunction. However, clinicians often rely on point-in-time evaluation of orthostatic and exercise intolerance to guide activity recommendations. Commercially available wearable devices (e.g., Fitbits) provide an opportunity to evaluate heart rate response to activity in a real-world setting. Previous work has used physiologic (heart rate) and activity (step count) data to identify subgroups of adults with stroke that may be used to guide activity recommendations. This method may be useful to subgroup youth post-mTBI to identify those who have abnormal physiologic responses to activity. We aimed to identify subgroups using heart rate and step count data in adolescents presenting for specialty care after diagnosed mTBI. Eighty participants aged 13-18 within six months of mTBI diagnosis were recruited to wear a Fitbit Sense 2. Data from seven days and two nights collected within fourteen days of enrollment were included. A group-based steps per minute (SPM) threshold (25th percentile; 10 SPM) and individualized heart rate threshold (20% heart rate reserve (HRR)) were used to classify each minute of active daytime data into one of four quadrants: SPM>10 & HRR>20% (QI), SPM<10 & HRR>20% (QII), SPM<10 & HRR<20% (QIII), and SPM>10 & HRR<20% (QIV). We used percentage of minutes in each quadrant, mean steps per day, percentage of minutes with zero steps, mean SPM in QI, and resting heart rate in a k-means clustering algorithm to identify subgroups. We evaluated subgroup differences by clustering variables using Kruskal-Wallis tests. Sixty-one participants were included. Three subgroups emerged: Sedentary (n=12), Active (n=23), and Atypically Elevated Heart Rate (AEHR; n=26). Subgroups varied significantly on all clustering variables (p<0.01). The Active subgroup took a high number of steps per day, had lower sedentary time, and had the highest activity intensity (mean SPM in QI). The Sedentary subgroup took fewer steps per day compared to the Active subgroup, had high sedentary time, and showed the highest resting heart rate. The AEHR subgroup took fewer steps per day compared to the Active subgroup and had high sedentary time. The AEHR subgroup also spent a higher percentage of time with an atypically high heart rate response to low levels of activity compared to the other subgroups. Our findings suggest that data from wearable devices can identify subgroups of adolescents with mTBI with distinct physiologic/physical activity profiles, which may ultimately be used to inform personalized activity prescriptions. Future work should aim to understand how the identified subgroups relate to longitudinal outcomes.

Plant-associated bacteria can improve plant performance under abiotic stress, but beneficial functions in plant microbiomes may depend on defined combinations of microorganisms rather than individual isolates alone. Here, we developed a cube-based screening strategy to identify functional synthetic microbial communities (SynComs) from 135 quinoa-associated bacterial isolates while preserving combinatorial diversity and traceability of isolate-level contributions. The isolates were divided into five 27-isolate sets, each arranged as a 3 x 3 x 3 cube in which each 3 x 3 layer was defined as a 9-isolate SynCom, generating 45 SynComs in total. Screening under 100 mM NaCl identified SynCom DY1 (SCDY1) as a candidate salt stress-mitigating consortium. SCDY1 consisted of nine taxonomically diverse isolates and exhibited a multifunctional profile, including siderophore production, phosphate solubilization, carboxymethyl cellulose degradation, indole compound production, and growth under saline conditions. In Arabidopsis thaliana, SCDY1 promoted primary root elongation and biomass accumulation in a salinity-dependent manner, with the clearest effect under 120 mM NaCl, and at least a subset of constituent bacteria was recoverable from inoculated seedlings. RNA sequencing and targeted RT-qPCR indicated that SCDY1 modulated host gene expression under moderate salinity stress, with responsive genes associated with oxidative stress, water- and oxygen-related processes, phenylpropanoid biosynthesis, glutathione metabolism, and root epidermis-related processes. Root hair phenotyping further showed that SCDY1 enhanced root hair-related traits and shifted visible root hair formation closer to the root apex. These findings identify a quinoa-derived SynCom that improves plant performance under salinity stress and provide a practical, traceable framework for discovering beneficial microbial consortia from plant-associated bacterial collections. Scope statementThis manuscript fits the Research Topic "Harnessing Plant Microbiomes for Climate Resilience: From Ecological Insight to Synthetic Community Design" in Frontiers in Plant Science because it presents a traceable strategy for discovering functional synthetic microbial communities from a stress-adapted plant-associated bacterial collection. We developed a cube-based screening strategy using 135 quinoa-associated bacterial isolates and identified a nine-isolate synthetic microbial community, SCDY1, that promotes Arabidopsis growth under moderate salinity stress. The study integrates microbiological screening, characterization of plant growth-promoting traits, bacterial re-isolation, plant growth phenotyping, RNA-seq, RT-qPCR, and root hair phenotyping. These analyses link SCDY1 treatment to salinity-dependent growth promotion, recoverable bacterial members, stress- and redox-associated transcriptional changes, phenylpropanoid-related responses, and modulation of root epidermal phenotypes. By connecting a defined SynCom with host transcriptional and root epidermal responses, this work advances understanding of beneficial plant-microbe interactions under salt stress. The cube-based design also provides a practical and traceable framework for discovering functional SynComs from large plant-associated bacterial collections, which should be of interest to researchers studying plant symbiosis, microbiome engineering, abiotic stress tolerance, and sustainable crop improvement.

Background. Post acute sequelae of COVID 19 (PASC) is clinically heterogeneous and mechanistically unresolved, and single-analyte studies have struggled to explain it. Methods. We profiled matched plasma proteomics, metabolomics and whole-blood transcriptomics at acute infection and convalescence (mean 86 days later) in a Belgian cohort, using linear mixed models, multiomic gene-set enrichment, and a degree-matched differential-correlation approach to quantify how each node's interactions were rewired between patients who developed PASC and those who recovered; seven axis proteins were additionally quantified by multiplex immunoassay as orthogonal validation. Findings. Single omic testing yielded few FDR significant features, yet multi-omic enrichment showed sustained complement cascade involvement from acute illness to follow-up in PASC. Correlation networks re-organised topologically toward C3 and lost the immunoglobulin V gene coexpression seen in recovery. The most rewired nodes, heparin cofactor II (SERPIND1), alpha 1 antitrypsin (SERPINA1), complement factor H related 5 (CFHR5), prothrombin/thrombin (F2) and immunoglobulin V gene transcripts (notably IGLV3 21), changed in their co-expression structure rather than in abundance. In multiplex validation, acute CRP was elevated in patients who developed PASC (FDR = 0.012), whereas the directly measured abundances of the network-nominated proteins were unchanged. Interpretation. These trajectory aware, cross omic networks nominate a thrombo inflammatory axis in which complement and coagulation regulation remain dysregulated in PASC at the level of wiring rather than abundance, providing a systems framework for validation and for exploring interventions at the complement coagulation platelet interface.

Background: Short-chain fatty acids (SCFAs) are microbial metabolites with immunoregulatory properties. Human milk contains SCFAs which have been proposed as potential modulators of infant immune development. We aimed to examine associations between human milk SCFA concentrations and infant allergic disease outcomes in a high-risk cohort of infants of atopic mothers. Methods: SCFAs were measured by targeted liquid chromatography-mass spectrometry in human milk samples collected at 3 and 6 months postpartum from atopic mothers enrolled in the Infant Fish Oil Supplementation (IFOS) Study (n=147). Associations between milk SCFA concentrations and early childhood allergic disease outcomes (atopic dermatitis, food allergy, allergic rhinitis, and allergen sensitisation at 1 and 2-3 years) were examined using logistic regression. Results: Human milk SCFA concentrations were broadly stable between 3 and 6 months postpartum, except for acetate which was significantly elevated at 6 months. No significant associations were observed between human milk SCFA concentrations and any allergic disease outcome after correction for multiple comparisons (all p>0.05). Conclusions: Human milk SCFA concentrations are not associated with allergic disease outcomes up to 4 years of age. These findings suggest that oral SCFA exposure via human milk is insufficient to reduce infant allergy risk, and that gut SCFA production may be a more relevant target for future allergy prevention research.

Copper (Cu) is an essential metal involved in neurobiological processes including energy metabolism and neurotransmission, yet dysregulated Cu levels may adversely affect brain health and olfactory performance. Although olfactory dysfunction has primarily been studied in older adults and neurodegenerative disease, adolescence is a critical period of brain maturation during which the olfactory system may be particularly vulnerable. This cross-sectional study examined associations between Cu exposure, olfactory bulb (OB) volume, and olfactory performance in 200 adolescents and young adults (64% female; ages 13 - 25) from the Public Health Impact of Metals Exposure cohort. Cu concentrations in blood, urine, hair, and saliva were measured using inductively coupled plasma mass spectrometry. T2-weighted magnetic resonance imaging scans estimated left, right, and total OB volumes using a three-stage deep learning pipeline. Olfactory performance was assessed using the Sniffin Sticks test. Weighted quantile sum regression evaluated associations between a Cu mixture index and OB outcomes, while standard linear regression models assessed individual Cu biomarkers. Models were adjusted for age and sex. A higher Cu index was associated with reduced left (Beta= -0.72, 95% CI [-1.42, -0.02]), right (Beta = -0.79, 95% CI [-1.43, -0.15]), and total OB volume (Beta= -1.55, 95% CI [-2.85, -0.25]), as well as lower odor threshold scores (Beta = -0.23, 95% CI [-0.42, -0.03]). Individual biomarkers were not independently associated with outcomes. These findings suggest that Cu exposure may adversely affect olfactory neurodevelopment during adolescence and highlight the importance of studying environmental exposures relevant to long-term neurological health.