Journal of the Endocrine Society

Background: Recent primary aldosteronism (PA) guidelines proposed probability-based stratifications, and use of aldosterone suppression testing, to predict lateralizing PA subtype. This guideline framework was based on very low-quality evidence. Methods: The discriminatory capacity of guideline-endorsed probability frameworks for PA subtyping were evaluated in this retrospective study of 319 PA patients, from two large tertiary centers in Bangkok, Thailand, who underwent subtyping assessments regardless of probability status. PA subtypes were determined by adrenal venous sampling (AVS) and/or post-adrenalectomy outcomes using PASO criteria. The main objectives were to evaluate the accuracy of predicting PA subtype using: 1) guideline-endorsed classification to high, intermediate, and low probabilities of lateralization; and 2) the seated saline suppression test (SST). Results: The majority of PA patients were characterized as having intermediate probability for lateralizing PA (75%); however, lateralizing PA was ultimately confirmed in 61-78% of all patients, regardless of guideline-based probability classification. The vast majority of SST results were positive using guideline-derived criteria, regardless of probability stratification or ultimate subtype: 89.3% of patients with lateralizing PA and 80.6% of those with bilateral PA had a positive SST. Among patients with intermediate probability of lateralizing PA, where guidelines specifically endorse the value of SST, the SST had a sensitivity of 89.4% and specificity of 22.0% for detecting lateralizing PA, with 78.0% false-positive and 10.6% false-negative rates. Consistently, post-SST aldosterone concentrations exhibited near-complete overlap between those with and without lateralizing PA. Conclusion: Guideline-endorsed probability frameworks, and the use of SST, lacked discriminatory capacity to predict PA subtype.

Zinc is an essential trace element involved in numerous biological processes, including cellular signalling, development, and reproduction. Zinc homeostasis is regulated by zinc transporters, yet the physiological roles of many transporters remain poorly understood in vivo. Here, we investigated the function of the zinc transporter ZIP9 (SLC39A9) using a zebrafish (Danio rerio) knockout model. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed altered zinc distribution in zip9-deficient larvae. Synchrotron-based X-ray fluorescence (XRF) imaging further showed reduced zinc levels in the brain region of mutant zebrafish. Consistent with these observations, loss of zip9 was associated with altered expression of key neuroendocrine genes within the hypothalamic-pituitary-gonadal (HPG) axis. Zip9 mutant females exhibited disrupted ovarian follicle development, reduced spawning rates, and decreased egg production. In addition, embryos derived from zip9 mutant parents displayed reduced size, impaired early development, and decreased survival. Together, these findings identify ZIP9 as a regulator of zinc distribution in vivo and suggest that ZIP9-mediated zinc signalling contributes to reproductive regulation in zebrafish.

Background: C-terminal binding protein 2 (CtBP2) has been implicated in metabolic regulation, but its association with specific measures of adiposity and lipid profiles in humans remains unclear. This study examined the relationship between circulating CtBP2 levels and key components of metabolic syndrome, focusing on body fat distribution and lipid markers. Methods: Data from 508 participants (259 men, 249 women) from a publicly available dataset were analyzed. Serum CtBP2 concentrations were measured using ELISA. Associations with obesity markers (BMI, waist circumference, waist-to-hip ratio) and lipid profiles (triglycerides, HDL cholesterol) were assessed using Spearman correlation and linear regression, adjusting for age and sex. Results: CtBP2 levels showed weak but statistically significant positive correlations with all measures of adiposity, with the strongest association observed for waist circumference ({rho} = 0.150, p < 0.001), followed by BMI ({rho} = 0.120, p = 0.007) and waist-to-hip ratio ({rho} = 0.098, p = 0.027). No significant correlations were found with triglycerides or HDL cholesterol. In the regression model predicting BMI, age, and sex were significant predictors, while CtBP2 demonstrated a trend toward association ({beta} = 0.080, p = 0.052). Conclusion: Circulating CtBP2 appears to be modestly associated with measures of adiposity, particularly abdominal fat, but not with lipid abnormalities. These findings suggest a potential role for CtBP2 in obesity-related metabolic dysregulation and underscore the need for further mechanistic studies to clarify its clinical relevance.

Introduction Denosumab increases bone mineral density and reduces fracture risk in patients with osteoporosis. However, whether BMD response to denosumab differs by age, particularly during longer term treatment, remains unclear. This study investigated the association between baseline age and BMD gain during 3 years of denosumab treatment in patients with osteoporosis. Methods This retrospective study included patients with osteoporosis who were treated with denosumab. DXA-based BMD and bone turnover markers were followed for up to 3 years. Percent BMD gain from baseline, defined as %BMD gain, was evaluated. The longitudinal association between baseline age and %BMD gain was assessed using multivariable linear mixed-effects models for the lumbar spine and total hip. Analyses were performed in the treatment naive cohort and the overall cohort according to prior osteoporosis treatment status. Results A total of 255 patients were included in the analysis, of whom 110 had not received prior osteoporosis treatment. In multivariable linear mixed-effects models, older baseline age was associated with smaller lumbar spine %BMD gain in the treatment naive cohort at both 1 and 3 years. Each 1-year increase in age was associated with a 0.187 percentage-point lower lumbar spine %BMD gain at 1 year and a 0.293 percentage-point lower gain at 3 years (1 year: {beta} = -0.187, p = 0.006, 3 years: {beta} = -0.293, p = 0.031). In contrast, baseline age was not significantly associated with total hip %BMD gain in the treatment naive cohort (1 year: {beta} = -0.011, p = 0.826; 3 years: {beta} = 0.028, p = 0.727). In the overall cohort, baseline age was not significantly associated with %BMD gain at either the lumbar spine or total hip at 1 or 3 years (all p > 0.05). Conclusion Older baseline age was associated with a modestly smaller lumbar spine BMD gain in treatment naive patients, whereas no significant age-related association was observed at the total hip. In the overall cohort, age was not significantly associated with BMD gain at either site. These findings suggest that age may have a limited, site specific influence on BMD response to denosumab, particularly in treatment naive patients, and may support more individualized treatment planning in patients with osteoporosis.

Introduction Air pollution is a leading driver of cardiovascular disease with a growing body of literature implicating this in worse glucose homeostasis. Increases in fine particulate matter air pollution (PM2.5) are associated with increased blood glucose and hemoglobin A1c across the glycemic spectrum from normoglycemia to prediabetes to all forms of diabetes. Despite strong evidence for positive associations of PM2.5 with dysglycemia, it remains unknown if reducing air pollution exposure through air filtration can effect improvements in glucose. This study aims to test the hypothesis that short-term, in-home air pollution reduction using high efficiency particulate air (HEPA) filtration will improve blood sugar in adults with prediabetes. Methods and analysis This trial is a randomized, double-blind, sham-controlled trial of the effects of lowering air pollution exposure using HEPA filtration on cardiometabolic health in adults with prediabetes living in the New York City area. Participants will be randomly assigned to use bedroom air cleaners, or sham air cleaners, while measuring PM2.5 continuously for 1 month. The primary outcomes will be continuous glucose monitoring metrics measured before and after HEPA air filtration. Exploratory outcomes will include insulin resistance measures, serum biomarkers and transcriptomics measured before and after HEPA intervention. We will quantify effects of HEPA filtration with models using treatment arm (true versus sham filtration) as the independent variable. Secondary analyses will model continuous measures of PM2.5 as the independent variable. Ethics and Dissemination This study has undergone peer review; and the work was supported by Grant 2023-0214 from the Doris Duke Foundation, who had no other role in study design or implementation. The study was registered in ClinicalTrials.gov (NCT05994937) prior to recruitment. Clinical Trials Clinical Trials NCT05994937; https://clinicaltrials.gov/study/NCT05994937

Gestational diabetes mellitus (GDM) is a major metabolic complication of pregnancy with significant maternal and fetal adverse consequences. Beyond classical mechanisms, emerging evidence suggests that gut-derived metabolic endotoxemia may contribute to dysglycemia. Intestinal alkaline phosphatase (IAP), a key enzyme involved in maintaining gut barrier integrity and detoxifying lipopolysaccharides, has been linked to type 2 diabetes mellitus; however, its role in GDM remains largely unexplored. This hospital-based cross-sectional analytical study evaluated fecal IAP levels and their association with GDM among 198 pregnant women recruited from three antenatal care clinics representing three tiers of ANC services. Participants were screened for GDM using a 75-g oral glucose tolerance test and classified as having GDM (n=55) or normal glucose tolerance (NGT; n=143) according to WHO 2013 criteria. Stool samples were collected, and fecal IAP levels were measured using an enzymatic colorimetric assay. Fecal IAP level was significantly lower in women with GDM than in those with NGT (median 23.59 vs 46.48 U/g stool; p<0.001). Lower IAP level remained independently associated with GDM after adjustment for body mass index and previous GDM (adjusted OR 0.98 per unit increase; 95% CI 0.97-0.99; p<0.001). A graded relationship was observed between declining IAP level and GDM. Receiver operating characteristic analysis demonstrated modest discrimination (AUC 0.676), while a threshold of approximately 65 U/g stool yielded high sensitivity (89.1%) but lower specificity. Reduced fecal IAP is independently associated with GDM, supporting a potential role of gut-derived metabolic dysregulation in gestational glucose intolerance. While not suitable as a standalone diagnostic tool, fecal IAP may serve as a complementary biomarker for risk stratification during pregnancy. Prospective studies are warranted to determine its predictive value and explore its potential as a therapeutic target.

Pregnancy leads to many adaptations in the maternal body, most of which are reversible. However, reproductive experience can also result in permanent effects. Here, we investigated how pregnancy influences the somatotrophic system and the lasting effects of reproductive experience on the maternal organism. Reproductive experience induced a pronounced increase in lean body mass and longitudinal growth in both wild-type and growth hormone (GH)-deficient mice compared with age-matched virgins. Body growth was primarily observed during the first pregnancy, whereas a second gestation was mostly associated with increased adiposity. Data from a cohort of women with isolated GH deficiency (IGHD) caused by a loss-of-function mutation in the GHRHR gene revealed that nulliparous women were 7 cm shorter than those with one or more pregnancies. Increased GH secretion was observed in pregnant wild-type mice but not in pregnant GHRHR-deficient mice. Pregnancy-induced body growth is preserved despite disruption of GH-, ghrelin-, and estrogen-related signaling pathways. In conclusion, reproductive experience induces permanent changes in the maternal organism, promoting body growth in models that allow this response. Pregnancy-induced body growth appears to be independent of GH action. These findings underscore the need for further studies to investigate the long-lasting consequences of reproductive experience in females.

Estrogen is an essential hormone that critically impacts bodily and brain functions, supporting learning, memory, and motor activities. A decrease in estrogen levels is associated with cognitive decline and motor dysfunction, such as muscle weakness. While conventional hormone replacement treatments (HRT) exist, those have limitations and potentially severe side effects. NAP (davunetide) is the smallest neuroprotective peptide site of activity-dependent neuroprotective protein (ADNP), a master regulator of cognition, essential for brain formation. It is known that NAP restores ADNP activity in cases of deficiency and it has already shown potential in preventing cognitive impairment, protecting against tauopathy, and improving motor function in various animal models and in clinical trials. Based on the dynamic regulation of ADNP by the estrous cycle and its involvement in steroidogenic pathways, we hypothesize that NAP may restore ADNP activity and thus serve as an alternative to conventional hormonal treatments. To test this, 3-month-old female ICR mice underwent bilateral ovariectomy (OVX) or Sham surgery and received daily intranasal administration of NAP, estrogen, or vehicle. Results showed a significant reduction in weight-normalized forelimb grip strength in the OVX model. Daily administration of NAP or estrogen resulted in intermediate grip strength levels that did not statistically differ from either the Sham control or untreated OVX groups. Interestingly, grip strength was the only test that yielded significant results, and no significant differences were observed in the Novel Object Recognition (NOR) test or computed tomography (CT) scans. These findings suggest that NAP may effectively prevent the loss of physical force production typically seen following ovarian hormone depletion, presenting a viable, non-hormonal candidate strategy for managing musculoskeletal symptoms. We hypothesize that the lack of significance in other parameters was due to soy-derived phytoestrogens in the diet, which may have exerted a systemic estrogenic effect that masked the expected physiological phenotypes typically observed in OVX models. Future replication using phytoestrogen-deficient food is required to isolate the specific neuroprotective and musculoskeletal effects of NAP from dietary influence and clarify the broader therapeutic benefits of NAP.

BackgroundPolycystic kidney disease (PKD) is the leading genetic cause of renal failure, resulting in the accumulation of fluid filled cysts and gross enlargement of the kidney. Mutations in PKD1 or PKD2, which encode ciliary polycystin proteins, are the most common cause of PKD. These proteins function in a cilia-dependent cyst activation (CDCA) pathway-one that requires cilia for its pro-cystic function-yet the molecular driver(s) of this pathway are unknown. ARL13B is a regulatory GTPase enriched in cilia and links to renal cystogenesis. ARL13B possesses guanine nucleotide exchange factor (GEF) activity for ARL3, another ARL with links to cilia. MethodsWe used two distinct Arl13b mouse alleles to investigate whether ARL13B is a component of the CDCA pathway: Arl13bV358Aencodes for enzymatically normal ARL13B that is undetectable in cilia, and Arl13bR79Qencodes for cilia-localized ARL13B lacking a residue critical for its ARL3 GEF activity. We used these alleles in a Pkd1-deficient adult mouse model and investigated renal morphology (H&E and cystic index analysis), physiology (blood urea nitrogen measurements), renal fibrosis (picrosirius staining and -smooth muscle actin levels), renal injury (SOX9 immunofluorescent staining and quantification), and Wnt signaling ({beta}-catenin and cyclin D1 protein levels). ResultsWe found that loss of ciliary ARL13B or mutation of a single residue critical for its ARL3 GEF activity suppressed Pkd1-dependent cysts. We observed reductions in kidney size, cystic index, and blood urea nitrogen. We also observed suppression of renal fibrosis, renal injury, and {beta}-catenin and cyclin D1 protein levels. ConclusionsOur results identified a subcellular location and mechanism driving Pkd1-dependent renal cystogenesis. We demonstrated that expression of a critical residue for ARL13Bs GEF activity specifically in cilia is a key mechanism of the CDCA pathway driving renal cystogenesis. Key PointsO_LILoss of ciliary ARL13B suppressed renal cystogenesis in an adult mouse model of polycystic kidney disease (PKD) without ablating cilia C_LIO_LILoss of ciliary ARL13B or mutation of the residue critical for its GEF activity did not affect renal morphology or physiology in a PKD mouse model C_LIO_LIMutation of a residue critical for ARL13B activation of ARL3 suppressed cystic phenotypes in Pkd1-dependent cysts C_LI

Lead is a toxic metal ubiquitous in our environment. While dramatic reductions in lead sources have paralleled equivalent decreases in lead-poisoning rates, chronic lead exposure remains a critical public health concern. Childhood lead exposure (at its lowest levels) is liked to changes in cognitive development but less is known about lead's effects on children's brain structure, especially as a result of in utero exposure. We measured prenatal and early-postnatal lead exposure in shed deciduous teeth of 448 9- and 10-year-old children (from 20 United States cities) and linked those lead levels to childhood brain structure, cognition/behavior, and neighborhood- and family-level socioeconomic characteristics. Here we show negative associations between tooth-lead levels and the thickness of the brain's cortex, particularly in regions linked to language processing. With increasing tooth-lead levels, children of lower-income (versus higher-income) families showed steeper declines in receptive vocabulary. Caregiver-reported behavioral problems exhibited similar associations. With in utero exposure linked to adverse neurodevelopmental outcomes (well before lead exposure and its risks are evaluated by healthcare professionals), prenatal screening of maternal lead levels/exposure, coupled with recommended strategies to reduce its placental transmission, may help reduce lead's effects on future generations.

Objective Fatty Acid esters of Hydroxy-Fatty Acids (FAHFAs) are anti-diabetic and anti-inflammatory lipokines produced mainly by adipose tissue (AT). As exercise training enhances FAHFA levels, we investigated the impact of acute exercise (AE) and exercise-mimicking conditions on circulating and adipocyte FAHFA levels. Methods Clinical trial (NCT05572905) in 60 women, grouped by BMI (lean vs. obese) and age (young vs. older), was combined with in vitro experiments on human adipocytes. Following baseline characterization (body composition, VO2max, insulin sensitivity, AT/plasma FAHFAs), women underwent a cross-over AE and control interventions with repeated blood sampling for FAHFA analysis. Results In AT, lean and older women exhibited higher FAHFA levels than obese and young women, respectively; older women also showed a shift toward higher levels of 13/12-carbon-branched FAHFAs. Circulating FAHFA levels were similar across all groups and were not positively associated with insulin sensitivity, VO2max or FAHFA levels in AT. Although AE increased circulating free fatty acids (FFA), plasma FAHFAs dropped in response to both AE and control interventions. In adipocytes, FAHFAs were unaffected by glucocorticoids but increased in response to lipolysis together with gene expression related to FFA oxidation (FAO). Nevertheless, blocking mitochondrial FAO partially mimicked the lipolytic effect, while peroxisomal inhibition synergistically boosted FAHFA lipolysis-driven production despite having no effect alone. Conclusions While adiposity and aging modulate FAHFA levels in AT, circulating levels remain stable and unaffected by AE, challenging subcutaneous AT as their primary systemic source. In vitro, FAHFA synthesis is driven by high FFA availability but limited by competing peroxisomal FAO.

Circadian rhythms regulate diverse physiological processes, including metabolism, and their disruption has been implicated in metabolic disorders such as obesity. However, the tissue-specific effects of obesity on peripheral circadian clocks remain incompletely understood. Here, we investigated the impact of high-fat diet (HFD)-induced obesity on circadian gene expression in skeletal muscle, liver, and white adipose tissue (WAT). Mice were fed either a regular diet (RD) or HFD for 6 weeks, followed by tissue collection at 4-hour intervals over a 24-hour period. Under RD conditions, key circadian regulators and their downstream targets exhibited robust 24-hour oscillations across all tissues. In contrast, HFD feeding induced distinct, tissue-specific alterations. In the liver, Per2, Dbp, and Rev-erb showed phase-advanced expression patterns, whereas in WAT, rhythmic expression was markedly attenuated. Notably, skeletal muscle largely preserved circadian gene expression patterns, indicating relative resistance to HFD-induced circadian disruption. In addition, HFD feeding altered metabolic gene expression in adipose tissue, characterized by reduced Pgc1 expression and increased Leptin expression. Together, these findings demonstrate that HFD-induced obesity differentially disrupts peripheral circadian clocks in a tissue-specific manner and highlight skeletal muscle as a relatively resilient tissue. These results provide insight into how circadian dysregulation contributes to metabolic abnormalities in obesity.

Polycystic ovary syndrome (PCOS) is linked to adverse pregnancy outcomes and increased cardiometabolic risk in offspring, yet the placental mechanisms underlying these risks remain poorly understood. Metformin is prescribed during PCOS pregnancies despite limited mechanistic justification. Using multi-modal molecular analyses of placentas from healthy controls and women with PCOS randomized to placebo or metformin (PregMet trial), restricted to uncomplicated pregnancies, we characterized direct PCOS associated placental alterations independent of confounding complications. PCOS placentas showed transcriptional downregulation across multiple cell types and shifts in cell type proportions. Specifically, syncytiotrophoblasts exhibited reduced expression activity of growth hormone receptor signaling and glycosaminoglycan biosynthesis. Endothelial cells displayed diminished receptor tyrosine kinase pathway activity, including VEGFC, despite increased cell proportion and hypervascularity. Intercellular communication networks were globally suppressed, including reductions in PDGF signaling from Hofbauer cells to fibroblasts. Notably, metformin did not reverse most PCOS-associated molecular alterations and induced transcriptional changes correlated to birth weight and childhood BMI. These findings indicate that PCOS-associated placental features are driven by cell type specific dysregulation of growth factor, angiogenic signaling pathways that are largely unresponsive to metformin. This underscores the need to develop mechanism based, placenta targeted therapeutic alternatives for future pregnancy management.

Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na+, Cl-, and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity brackish water (BW), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na+ and Cl- but not water. To identify aquaporins (Aqps) involved in regulating water permeability in the collecting ducts of teleosts, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish (Takifugu rubripes), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to BW. Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4 in collecting duct cells, with apical Aqp1aa downregulated in BW. These results suggest that Aqp1aa and Aqp4 mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in BW. NEW & NOTEWORTHYRegulation of water permeability in the collecting duct is important for osmoregulation in teleost fish. Expression analyses of aquaporins (Aqps) in the marine pufferfish Takifugu rubripes showed that Aqp1aa and Aqp4a are highly expressed in the collecting duct and localized to the apical and basolateral membranes, respectively. Renal Aqp1aa expression was markedly reduced in fish acclimated to hypoosmotic brackish water. These results indicate that collecting duct water permeability is regulated by Aqp1aa expression.

Perinatal opioid exposure is a prevalent clinical concern linked to respiratory instability and adverse infant outcomes. The opioid buprenorphine is prescribed as a medication for opioid use disorder during pregnancy and used to treat neonatal opioid withdrawal syndrome, yet its direct effects on neonatal control of breathing have not been examined. Here, we asked how acute buprenorphine exposure affects breathing at rest, and during chemoreceptor stimulation. Using dual-chamber head-out plethysmography, we measured pulmonary ventilation rate ([V]I) and metabolic rate in awake male and female Sprague-Dawley neonatal rats on postnatal days 4-5 (P4-5) during eupnea and a hypoxic-hypercapnic (HH) challenge. The effects of buprenorphine and two opioid receptor antagonists, naloxone hydrochloride, or peripherally restricted naloxone methiodide, were assessed using a repeated measures design. [V]I during eupnea and HH were markedly depressed following buprenorphine administration. Buprenorphine reduced [V]O2 and [V]CO2 and produced ventilatory equivalents for O2 and CO2 consistent with frank hypoventilation, driven by reduced breathing frequency and tidal volume (VT). When administered after buprenorphine, neither naloxone hydrochloride nor naloxone methiodide could rescue the buprenorphine-mediated hypoventilation in eupnea or during HH. In contrast, pre-treatment with either naloxone hydrochloride or naloxone methiodide attenuated buprenorphine-induced hypoventilation by preserving VT. These findings demonstrate that neonatal protective chemoreceptor reflexes are depressed by buprenorphine and suggest that pre-treatment with a peripheral opioid receptor antagonist could mitigate buprenorphine-induced hypoventilation without inducing opioid withdrawal. Key PointsO_LIAcute buprenorphine exposure significantly depressed pulmonary ventilation rate ([V]I) during eupnea and hypoxic hypercapnia (HH) in awake neonatal rats. C_LIO_LIBuprenorphine-induced hypoventilation was driven by reduced tidal volume (VT) and breathing frequency. C_LIO_LIBuprenorphine also reduced oxygen consumption ([V]O2) and carbon dioxide production ([V]CO2). C_LIO_LINaloxone given after buprenorphine failed to reverse hypoventilation. C_LIO_LIIn contrast, pre-treatment with either naloxone hydrochloride or peripherally restricted naloxone methiodide mitigated buprenorphine-induced hypoventilation by preserving VT. C_LI

Background Bacteriological contamination of drinking water remains a major public health burden in sub-Saharan Africa, yet the full contamination chain from source to household has rarely been quantified at national scale. This study analyses water quality at both levels using the 2021 Cote d'Ivoire Demographic and Health Survey (DHS-CI 2021). Methods Cross-sectional secondary analysis of DHS-CI 2021 data. Households with paired bacteriological tests at the source (SH3227) and at the household (SH3225) were included (n = 2,541 for determinants; n = 2,528 for chain analysis). Contamination was defined as >0 CFU/100 ml. Determinants of source contamination were assessed by weighted logistic regression accounting for complex survey design. The contamination chain was described across four categories: safe throughout, recontaminated during transport/storage, decontaminated at home, and contaminated throughout. Results Weighted prevalence of source contamination was 63.6% [95% CI: 60.7-66.5%] and 77.0% [74.1-79.9%] at the household. Only 15.0% of households had safe water throughout the chain; 21.2% showed domestic recontamination and 60.8% consumed water contaminated at both levels. Key determinants of source contamination were use of an unimproved source (aOR = 8.15; 95% CI: 4.54-14.66), administrative region, travel time [&le;]30 minutes (aOR = 1.92; 95% CI: 1.41-2.62), and higher wealth quintiles (protective; aOR = 0.25 for richest). Model discrimination was good (AUC = 0.809). Conclusions The vast majority of Ivorian households consume bacteriologically unsafe water, with domestic recontamination representing a distinct and significant degradation pathway even among users of improved sources. Dual interventions targeting source protection and safe household water storage are urgently needed to advance progress toward SDG 6 in Cote d'Ivoire.

Studies of protein quantitative trait loci (pQTLs) provide opportunities to interpret complex trait genetics and identify potential biomarkers and therapeutic targets. Circulating proteins are commonly used in pQTL studies due to the accessibility of blood-based measurements, but their levels may not always reflect regulation in disease-relevant tissues. We assessed colocalization and discordance between plasma and dorsal prefrontal cortex cis-pQTLs using data from four large-scale studies and investigated their implications for downstream analyses. Across the proteins examined, at most 80% of the cis-pQTLs showed evidence of colocalization. Among the colocalized loci, approximately 20% exhibited opposite directions of genetic effects. We characterized tissue-specific gene expression profiles based on data from the Genotype-Tissue Expression project. Proteins with colocalized cis-pQTLs were more likely to have high gene expression levels in systemic tissues and immune cells, whereas the remaining proteins were more likely to have high expression in brain tissues. We conducted Mendelian randomization (MR) analyses using neuroticism as an illustrative outcome to compare effect estimates derived using instruments from different pQTL studies. MR analyses identified 13 proteins significantly associated with neuroticism, including six with opposite effect directions between plasma and dorsal prefrontal cortex, highlighting the importance of tissue context. Overall, circulating pQTLs remain informative for proteins from systemic and immune pathways, while incorporating tissue-specific data may provide additional insight for proteins with more localized expression. Considering multiple tissue contexts may refine the interpretation of protein-trait associations and may improve the prioritization of candidate targets.

Aminoglycoside (AG) antibiotic safety is limited by ototoxicity, the mitigation of which is vital considering bacterial resistance mediated erosion of our antibiotic arsenal. Previously, we observed tectorial membrane (TM) sequestration of Ca2+. We hypothesized that the TM sequesters other cations, including the AG gentamicin. We proposed to test the effect of TM genetic ablation on ototoxicity and TM-AG sequestration. After intraperitoneal AG-furosemide, TM-lacking Tecta{Delta}ENT/{Delta}ENT mice showed limited outer hair cell loss, unlike wildtype littermates. Spectroscopy measurements of gentamicin-Texas red (GTTR) were made in isolated wildtype and TectaY1870C TMs and guinea pig cochleae following direct or intraperitoneal GTTR administration. TM-GTTR sequestration was observed in all cases, while negatively correlated with TectaY1870C zygosity. In summary, we discovered a novel TM component in the AG ototoxicity pathway. Intact TM structure is necessary for sequestration, and the TM modulates AG ototoxicity. TM-GTTR sequestration following systemic injection indicates that this phenomenon occurs during AG therapy. Single sentence summaryOtotoxic aminoglycosides collect inside the acellular tectorial membrane of the inner ear, likely due to electrostatic interactions, and the structural status of that membrane modulates the toxic effect of those aminoglycosides on sensory hair cells.

Brain metastases from renal cell carcinoma (RCC) remain a major cause of morbidity and mortality, yet the genomic features associated with metastatic dissemination remain poorly understood. Whole-exome sequencing was performed on 72 RCC brain metastasis samples with matched normal. To identify candidate genomic alterations associated with brain metastasis, the genomic alterations detected in the brain metastases were compared against alterations in extracranial metastases from the MSK-ECM cohort (n=137) and primary RCC tumors from TCGA (n=432) by case-control analyses. Candidate alterations were also identified through matched-pair analyses comparing brain metastases with matched primary tumors or extracranial metastases from the same patient (n=25). A random survival forest model incorporating the candidate CNA events was developed to predict overall survival. The candidate CNAs were further evaluated using functional experimental data from MetMap and DepMap. Survival analyses were conducted to assess the prognostic relevance of these alterations. We identified recurrent CNAs enriched in RCC brain metastases, including 4q loss, 7p gain, 7q gain, 8p loss, 8q gain, 9p21.3 deletion, 12q15 amplification, and 14q loss. These alterations were associated with significantly poorer patient survival among RCC patients. A random survival forest model based on these CNA events stratified TCGA-KIRC patients into prognostically distinct risk groups (C-index = 0.64). Among the recurrent CNAs, 8p loss, 8q gain, 9p21.3 deletion were associated with increased incidence of brain metastases across multiple primary cancer types in xenograft mouse models. These alterations were also strongly associated with metastatic progression and poor prognosis across RCC, lung adenocarcinoma, breast cancer, and melanoma. These findings indicate a shared genomic basis for brain tropism and highlight the potential utility of copy-number alterations as biomarkers for risk stratification and clinical decision-making.

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.