Racial Differences in Vaginal Fluid Metabolites and Association with Systemic Inflammation Markers among Ovarian Cancer Patients: A Pilot Study.

The vaginal microbiome differs by race and contributes to inflammation by directly producing or consuming metabolites or by indirectly inducing host immune response, but its potential contributions to ovarian cancer (OC) disparities remain unclear. In this exploratory cross-sectional study, we examine whether vaginal fluid metabolites differ by race among patients with OC, if they are associated with systemic inflammation, and if such associations differ by race. Study participants were recruited from the Ovarian Cancer Epidemiology, Healthcare Access, and Disparities Study between March 2021 and September 2022. Our study included 36 study participants with ovarian cancer who provided biospecimens; 20 randomly selected White patients and all 16 eligible Black patients, aged 50-70 years. Acylcarnitines (n = 45 species), sphingomyelins (n = 34), and ceramides (n = 21) were assayed on cervicovaginal fluid, while four cytokines (IL-1ß, IL-10, TNF-α, and IL-6) were assayed on saliva. Seven metabolites showed >2-fold differences, two showed significant differences using the Wilcoxon rank-sum test (p < 0.05; False Discovery Rate > 0.05), and 30 metabolites had coefficients > ±0.1 in a Penalized Discriminant Analysis that achieved two distinct clusters by race. Arachidonoylcarnitine, the carnitine adduct of arachidonic acid, appeared to be consistently different by race. Thirty-eight vaginal fluid metabolites were significantly correlated with systemic inflammation biomarkers, irrespective of race. These findings suggest that vaginal fluid metabolites may differ by race, are linked with systemic inflammation, and hint at a potential role for mitochondrial dysfunction and sphingolipid metabolism in OC disparities. Larger studies are needed to verify these findings and further establish specific biological mechanisms that may link the vaginal microbiome with OC racial disparities.

Peripheral nerve decompression for pain relief in the setting of previous obstetrical brachial plexus injury: a retrospective case series.

PURPOSE: In children with previous obstetrical brachial plexus injury (OBPI), upper extremity pain is present in 45 to 66% of patients. Recent literature reports this as musculoskeletal or neuropathic in nature. The purpose of the study is to demonstrate that peripheral nerve decompression, and neurolysis may be an effective treatment for patients with upper extremity pain in the context of previous OBPI. METHODS: A retrospective chart review was performed on patients undergoing peripheral nerve decompression and neurolysis after OBPI by senior author. The primary outcome assessed was pain, and secondary outcome measure was range of motion of the wrist and elbow. Outcome measures were assessed preoperatively as well as at their subsequent follow-up. RESULTS: Six patients were included, with a mean age of 14 years old at time of decompression. Three patients underwent median nerve, two patients underwent ulnar nerve, and one patient underwent posterior interosseous nerve decompression. There was a substantial improvement in pain post-operatively, demonstrated by reduction or resolution of subjective pain in all patients and resolution of Tinel's sign. There was a modest improvement in range of motion. CONCLUSION: This study demonstrates an improvement in subjective pain and range of motion after decompression and neurolysis in small subset of OBPI patients. It generates the hypothesis that peripheral nerve compression is a source of pain that can be addressed in this population. Future research should focus on confirming this hypothesis and assessing treatment options on a larger scale.

Utilization of the Rat Tibial Nerve Transection Model to Evaluate Cellular and Molecular Mechanisms Underpinning Denervation-Mediated Muscle Injury.

Peripheral nerve injury denervates muscle, resulting in muscle paralysis and atrophy. This is reversible if timely muscle reinnervation occurs. With delayed reinnervation, the muscle's reparative ability declines, and muscle-resident fibro-adipogenic progenitor cells (FAPs) proliferate and differentiate, inducing fibro-fatty muscle degradation and thereby physical disability. The mechanisms by which the peripheral nerve regulates FAPs expansion and differentiation are incompletely understood. Using the rat tibial neve transection model, we demonstrated an increased FAPs content and a changing FAPs phenotype, with an increased capacity for adipocyte and fibroblast differentiation, in gastrocnemius muscle post-denervation. The FAPs response was inhibited by immediate tibial nerve repair with muscle reinnervation via neuromuscular junctions (NMJs) and sensory organs (e.g., muscle spindles) or the sensory protection of muscle (where a pure sensory nerve is sutured to the distal tibial nerve stump) with reinnervation by muscle spindles alone. We found that both procedures reduced denervation-mediated increases in glial-cell-line-derived neurotrophic factor (GDNF) in muscle and that GDNF promoted FAPs adipogenic and fibrogenic differentiation in vitro. These results suggest that the peripheral nerve controls FAPs recruitment and differentiation via the modulation of muscle GDNF expression through NMJs and muscle spindles. GDNF can serve as a therapeutic target in the management of denervation-induced muscle injury.

Metabolomic and genetic architecture of gestational diabetes subtypes.

AIMS/HYPOTHESIS: Physiological gestational diabetes mellitus (GDM) subtypes that may confer different risks for adverse pregnancy outcomes have been defined. The aim of this study was to characterise the metabolome and genetic architecture of GDM subtypes to address the hypothesis that they differ between GDM subtypes. METHODS: This was a cross-sectional study of participants in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study who underwent an OGTT at approximately 28 weeks' gestation. GDM was defined retrospectively using International Association of Diabetes and Pregnancy Study Groups/WHO criteria, and classified as insulin-deficient GDM (insulin secretion <25th percentile with preserved insulin sensitivity) or insulin-resistant GDM (insulin sensitivity <25th percentile with preserved insulin secretion). Metabolomic analyses were performed on fasting and 1 h serum samples in 3463 individuals (576 with GDM). Genome-wide genotype data were obtained for 8067 individuals (1323 with GDM). RESULTS: Regression analyses demonstrated striking differences between the metabolomes for insulin-deficient or insulin-resistant GDM compared to those with normal glucose tolerance. After adjustment for covariates, 33 fasting metabolites, including 22 medium- and long-chain acylcarnitines, were uniquely associated with insulin-deficient GDM; 23 metabolites, including the branched-chain amino acids and their metabolites, were uniquely associated with insulin-resistant GDM; two metabolites (glycerol and 2-hydroxybutyrate) were associated with the same direction of association with both subtypes. Subtype differences were also observed 1 h after a glucose load. In genome-wide association studies, variants within MTNR1B (rs10830963, p=3.43×10-18, OR 1.55) and GCKR (rs1260326, p=5.17×10-13, OR 1.43) were associated with GDM. Variants in GCKR (rs1260326, p=1.36×10-13, OR 1.60) and MTNR1B (rs10830963, p=1.22×10-9, OR 1.49) demonstrated genome-wide significant association with insulin-resistant GDM; there were no significant associations with insulin-deficient GDM. The lead SNP in GCKR, rs1260326, was associated with the levels of eight of the 25 fasting metabolites that were associated with insulin-resistant GDM and ten of 41 1 h metabolites that were associated with insulin-resistant GDM. CONCLUSIONS/INTERPRETATION: This study demonstrates that physiological GDM subtypes differ in their metabolome and genetic architecture. These findings require replication in additional cohorts, but suggest that these differences may contribute to subtype-related adverse pregnancy outcomes.

APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease.

Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3-mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ-mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.

Sex Differences in Branched-chain Amino Acid and Tryptophan Metabolism and Pathogenesis of Youth-onset Type 2 Diabetes.

OBJECTIVES: Insulin resistance is associated with elevations in plasma branched-chain amino acids (BCAAs). BCAAs compete with aromatic amino acids including tryptophan for uptake into ß cells. To explore relationships between BCAAs and tryptophan metabolism, adiposity, and glucose tolerance, we compared urine metabolites in overweight/obese youth with type 2 diabetes (T2D) with those in nondiabetic overweight/obese and lean youth. METHODS: Metabolites were measured in 24-hour and first-morning urine samples of 56 nondiabetic adolescents with overweight/obesity, 42 adolescents with T2D, and 43 lean controls, aged 12 to 21 years. Group differences were assessed by Kruskal Wallis or ANOVA. RESULTS: Groups were comparable for age, pubertal status, and ethnicity. Youth with T2D were predominantly female and had highest percent body fat. BCAAs, branched-chain ketoacids (BCKAs), tryptophan, and kynurenine were higher in urine of subjects with T2D. There were no differences between lean controls and nondiabetic youth with overweight/obesity. T2D was associated with diversion of tryptophan from the serotonin to the kynurenine pathway, with higher urinary kynurenine/serotonin ratio and lower serotonin/tryptophan and 5-HIAA/kynurenine ratios. Urinary BCAAs, BCKAs, tryptophan, and ratios reflecting diversion to the kynurenine pathway correlated positively with metrics of body fat and hemoglobin A1c. Increases in these metabolites in the obese T2D group were more pronounced and statistically significant only in adolescent girls. CONCLUSION: Increases in urinary BCAAs and BCKAs in adolescent females with T2D are accompanied by diversion of tryptophan metabolism from the serotonin to the kynurenine pathway. These adaptations associate with higher risks of T2D in obese adolescent females than adolescent males.

Maternal and Cord Blood Serum Metabolite Associations with Childhood Adiposity and Body Composition Outcomes.

Maternal metabolites influence the size of newborns independently of maternal body mass index (BMI) and glycemia, highlighting the importance of maternal metabolism on offspring outcomes. This study examined associations of maternal metabolites during pregnancy with childhood adiposity, and cord blood metabolites with childhood adiposity using phenotype and metabolomic data from the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study and the HAPO Follow-Up Study. The maternal metabolites analyses included 2324 mother-offspring pairs, while the cord blood metabolites analyses included 937 offspring. Multiple logistic and linear regression were used to examine associations between primary predictors, maternal or cord blood metabolites, and childhood adiposity outcomes. Multiple maternal fasting and 1 hr metabolites were significantly associated with childhood adiposity outcomes in Model 1 but were no longer significant after adjusting for maternal BMI and/or maternal glycemia. In the fully adjusted model, fasting lactose levels were negatively associated with child BMI z-scores and waist circumference, while fasting urea levels were positively associated with waist circumference. One-hour methionine was positively associated with fat-free mass. There were no significant associations between cord blood metabolites and childhood adiposity outcomes. Few metabolites were associated with childhood adiposity outcomes after adjusting for maternal BMI and glucose, suggesting that maternal BMI accounts for the association between maternal metabolites and childhood adiposity.

Association of Maternal Metabolites and Metabolite Networks with Newborn Outcomes in a Multi-Ancestry Cohort.

The in utero environment is important for newborn size at birth, which is associated with childhood adiposity. We examined associations between maternal metabolite levels and newborn birthweight, sum of skinfolds (SSF), and cord C-peptide in a multinational and multi-ancestry cohort of 2337 mother-newborn dyads. Targeted and untargeted metabolomic assays were performed on fasting and 1 h maternal serum samples collected during an oral glucose tolerance test performed at 24-32 week gestation in women participating in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. Anthropometric measurements were obtained on newborns at birth. Following adjustment for maternal BMI and glucose, per-metabolite analyses demonstrated significant associations between maternal metabolite levels and birthweight, SSF, and cord C-peptide. In the fasting state, triglycerides were positively associated and several long-chain acylcarnitines were inversely associated with birthweight and SSF. At 1 h, additional metabolites including branched-chain amino acids, proline, and alanine were positively associated with newborn outcomes. Network analyses demonstrated distinct clusters of inter-connected metabolites significantly associated with newborn phenotypes. In conclusion, numerous maternal metabolites during pregnancy are significantly associated with newborn birthweight, SSF, and cord C-peptide independent of maternal BMI and glucose, suggesting that metabolites in addition to glucose contribute to newborn size at birth and adiposity.

Metabolic factors associated with incident fracture among older adults with type 2 diabetes mellitus: a nested case-control study.

Older adults with type 2 diabetes mellitus have an increased risk of fracture despite a paradoxically higher average bone mineral density. This study identified additional markers of fracture risk in this at-risk population. Non-esterified fatty acids and the amino acids glutamine/glutamate and asparagine/aspartate were associated with incident fractures. PURPOSE: Type 2 diabetes mellitus (T2D) is associated with an increased risk of fracture despite a paradoxically higher bone mineral density. Additional markers of fracture risk are needed to identify at-risk individuals. METHOD: The MURDOCK study is an ongoing study, initiated in 2007, of residents in central North Carolina. At enrollment, participants completed health questionnaires and provided biospecimen samples. In this nested case-control analysis, incident fractures among adults with T2D, age ≥ 50 years, were identified by self-report and electronic medical record query. Fracture cases were matched 1:2 by age, gender, race/ethnicity, and BMI to those without incident fracture. Stored sera were analyzed for conventional metabolites and targeted metabolomics (amino acids and acylcarnitines). The association between incident fracture and metabolic profile was assessed using conditional logistic regression, controlled for multiple confounders including tobacco and alcohol use, medical comorbidities, and medications. RESULTS: 107 incident fractures were identified with 210 matched controls. Targeted metabolomics analysis included 2 amino acid factors, consisting of: 1) the branched chain amino acids, phenylalanine and tyrosine; and 2) glutamine/glutamate, asparagine/aspartate, arginine, and serine [E/QD/NRS]. After controlling for multiple risk factors, E/QD/NRS was significantly associated with incident fracture (OR 2.50, 95% CI: 1.36-4.63). Non-esterified fatty acids were associated with lower odds of fracture (OR 0.17, 95% CI: 0.03-0.87). There were no associations with fracture among other conventional metabolites, acylcarnitine factors, nor the other amino acid factors. CONCLUSION: Our results indicate novel biomarkers, and suggest potential mechanisms, of fracture risk among older adults with T2D.

Type-2 Diabetes, Pancreatic Amylin, and Neuronal Metabolic Remodeling in Alzheimer's Disease.

Type-2 diabetes raises the risk for Alzheimer's disease (AD)-type dementia and the conversion from mild cognitive impairment to dementia, yet mechanisms connecting type-2 diabetes to AD remain largely unknown. Amylin, a pancreatic ß-cell hormone co-secreted with insulin, participates in the central regulation of satiation, but also forms pancreatic amyloid in persons with type-2 diabetes and synergistically interacts with brain amyloid ß (Aß) pathology, in both sporadic and familial Alzheimer's disease (AD). Growing evidence from studies of tumor growth, together with early observations in skeletal muscle, indicates amylin as a potential trigger of cellular metabolic reprogramming. Because the blood, cerebrospinal fluid, and brain parenchyma in humans with AD have increased concentrations of amylin, amylin-mediated pathological processes in the brain may involve neuronal metabolic remodeling. This review summarizes recent progress in understanding the link between prediabetic hypersecretion of amylin and risk of neuronal metabolic remodeling and AD and suggests nutritional and medical effects of food constituents that might prevent and/or ameliorate amylin-mediated neuronal metabolic remodeling.

Tryptophan Metabolism and Neurodegeneration: Longitudinal Associations of Kynurenine Pathway Metabolites with Cognitive Performance and Plasma Alzheimer's Disease and Related Dementias Biomarkers in the Duke Physical Performance Across the LifeSpan Study.

BACKGROUND: The kynurenine pathway (KP) comprises a family of tryptophan-derived metabolites that some studies have reported are associated with poorer cognitive performance and an increased risk of Alzheimer's disease and related dementias (ADRD). OBJECTIVE: The objective of this study was to determine the associations of plasma KP metabolites (kynurenine [KYN], kynurenic acid [KA], and tryptophan [TRP]) with a panel of plasma ADRD biomarkers (Aß42/ ß40 ratio, pTau-181, glial fibrillary acidic protein [GFAP], and neurofilament light [NfL]) and cognitive performance in a subset of older adults drawn from the Duke Physical Performance Across the LifeSpan (PALS) study. METHODS: The Montreal Cognitive Assessment (MoCA) was used to assess cognitive performance. We used multivariate multiple regression to evaluate associations of the KYN/TRP and KA/KYN ratios with MoCA score and plasma ADRD biomarkers at baseline and over two years (n = 301; Age = 74.8±8.7). RESULTS: Over two years, an increasing KYN/TRP ratio was associated with increasing plasma concentrations of plasma p-Tau181 (ß= 6.151; 95% CI [0.29, 12.01]; p = 0.040), GFAP (ß= 11.12; 95% CI [1.73, 20.51]; p = 0.020), and NfL (ß= 11.13; 95% CI [2.745, 19.52]; p = 0.009), but not MoCA score or the Aß42/Aß40 ratio. There were no significant associations of KA/KYN with MoCA score or plasma ADRD biomarkers. CONCLUSION: Our findings provide evidence that greater concentrations of KP metabolites are associated longitudinally over two years with greater biomarker evidence of neurofibrillary tau pathology (pTau-181), neuroinflammation (GFAP), and neurodegeneration (NfL), suggesting that dysregulated KP metabolism may play a role in ADRD pathogenesis.

Branched-chain α-keto acids and glutamate/glutamine: Biomarkers of insulin resistance in childhood obesity.

OBJECTIVES: Insulin resistance (IR) in adolescents with obesity is associated with a sex-dependent metabolic 'signature' comprising the branched-chain amino acids (BCAAs), glutamate/glutamine, C3/C5 acylcarnitines and uric acid. Here, we compared the levels of branched-chain α-keto acids (BCKAs) and glutamate/glutamine, which are the byproducts of BCAA catabolism and uric acid among adolescents with obesity prior to and following a 6-month lifestyle-intervention program. METHODS: Fasting plasma samples from 33 adolescents with obesity (16 males, 17 females, aged 12-18 year) were analysed by flow-injection tandem MS and LC-MS/MS. Multiple linear regression models were used to correlate changes in BCKAs, glutamate/glutamine and uric acid with changes in weight and insulin sensitivity as assessed by HOMA-IR, adiponectin and the ratio of triglyceride (TG) to HDL. In predictive models, BCKAs, glutamate/glutamine and uric acid at baseline were used as explanatory variables. RESULTS: Baseline BCKAs, glutamate/glutamine and uric acid were higher in males than females despite comparable BMI-metrics. Following lifestyle-intervention, α-keto-ß-methylvalerate (α-KMV, a metabolic by product of isoleucine) decreased in males but not in females. The ratio of BCKA/BCAA trended lower in males. In the cohort as a whole, BCKAs correlated positively with the ratio of TG to HDL at baseline and HOMA-IR at 6-month-follow-up. Glutamate/glutamine was positively associated with HOMA-IR at baseline and 6-month-follow-up. A reduction in BCKAs was associated with an increase in adiponectin, and those with higher BCKAs at baseline had higher adiponectin levels at 6-month-follow-up. Interestingly those adolescents with higher uric acid levels at baseline had greater reduction in weight. CONCLUSIONS: BCKAs and glutamate/glutamine may serve as biomarkers of IR in adolescents with obesity, and uric acid might serve as a predictor of weight loss in response to lifestyle-intervention. Differential regulation of BCAA catabolism in adolescent males and females implicates critical roles for sex steroids in metabolic homeostasis.

Ecological memory of prior nutrient exposure in the human gut microbiome.

Many ecosystems have been shown to retain a memory of past conditions, which in turn affects how they respond to future stimuli. In microbial ecosystems, community disturbance has been associated with lasting impacts on microbiome structure. However, whether microbial communities alter their response to repeated stimulus remains incompletely understood. Using the human gut microbiome as a model, we show that bacterial communities retain an "ecological memory" of past carbohydrate exposures. Memory of the prebiotic inulin was encoded within a day of supplementation among a cohort of human study participants. Using in vitro gut microbial models, we demonstrated that the strength of ecological memory scales with nutrient dose and persists for days. We found evidence that memory is seeded by transcriptional changes among primary degraders of inulin within hours of nutrient exposure, and that subsequent changes in the activity and abundance of these taxa are sufficient to enhance overall community nutrient metabolism. We also observed that ecological memory of one carbohydrate species impacts microbiome response to other carbohydrates, and that an individual's habitual exposure to dietary fiber was associated with their gut microbiome's efficiency at digesting inulin. Together, these findings suggest that the human gut microbiome's metabolic potential reflects dietary exposures over preceding days and changes within hours of exposure to a novel nutrient. The dynamics of this ecological memory also highlight the potential for intra-individual microbiome variation to affect the design and interpretation of interventions involving the gut microbiome.

Evaluating immune response and metabolic related biomarkers pre-allogenic hematopoietic stem cell transplant in acute myeloid leukemia.

Although hematopoietic stem cell transplantation (HCT) is the only curative treatment for acute myeloid leukemia (AML), it is associated with significant treatment related morbidity and mortality. There is great need for predictive biomarkers associated with overall survival (OS) and clinical outcomes. We hypothesized that circulating metabolic, inflammatory, and immune molecules have potential as predictive biomarkers for AML patients who receive HCT treatment. This retrospective study was designed with an exploratory approach to comprehensively characterize immune, inflammatory, and metabolomic biomarkers. We identified patients with AML who underwent HCT and had existing baseline plasma samples. Using those samples (n = 34), we studied 65 blood based metabolomic and 61 immune/inflammatory related biomarkers, comparing patients with either long-term OS (≥ 3 years) or short-term OS (OS ≤ 1 years). We also compared the immune/inflammatory response and metabolomic biomarkers in younger vs. older AML patients (≤30 years vs. ≥ 55 years old). In addition, the biomarker profiles were analyzed for their association with clinical outcomes, namely OS, chronic graft versus host disease (cGVHD), acute graft versus host disease (aGVHD), infection and relapse. Several baseline biomarkers were elevated in older versus younger patients, and baseline levels were lower for three markers (IL13, SAA, CRP) in patients with OS ≥ 3 years. We also identified immune/inflammatory response markers associated with aGVHD (IL-9, Eotaxin-3), cGVHD (Flt-1), infection (D-dimer), or relapse (IL-17D, bFGF, Eotaxin-3). Evaluation of metabolic markers demonstrated higher baseline levels of medium- and long-chain acylcarnitines (AC) in older patients, association with aGVHD (lactate, long-chain AC), and cGVHD (medium-chain AC). These differentially expressed profiles merit further evaluation as predictive biomarkers.

A Multidimensional Bioinformatic Platform for the Study of Human Response to Surgery.

OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

Path-level interpretation of Gaussian graphical models using the pair-path subscore.

BACKGROUND : Construction of networks from cross-sectional biological data is increasingly common. Many recent methods have been based on Gaussian graphical modeling, and prioritize estimation of conditional pairwise dependencies among nodes in the network. However, challenges remain on how specific paths through the resultant network contribute to overall 'network-level' correlations. For biological applications, understanding these relationships is particularly relevant for parsing structural information contained in complex subnetworks. RESULTS: We propose the pair-path subscore (PPS), a method for interpreting Gaussian graphical models at the level of individual network paths. The scoring is based on the relative importance of such paths in determining the Pearson correlation between their terminal nodes. PPS is validated using human metabolomics data from the Hyperglycemia and adverse pregnancy outcome (HAPO) study, with observations confirming well-documented biological relationships among the metabolites. We also highlight how the PPS can be used in an exploratory fashion to generate new biological hypotheses. Our method is implemented in the R package pps, available at https://github.com/nathan-gill/pps . CONCLUSIONS: The PPS can be used to probe network structure on a finer scale by investigating which paths in a potentially intricate topology contribute most substantially to marginal behavior. Adding PPS to the network analysis toolkit may enable researchers to ask new questions about the relationships among nodes in network data.

Accuracy of the Vesical Imaging-Reporting and Data System (VIRADS) for pre-treatment staging of bladder cancer in an Australian cohort.

INTRODUCTION: To evaluate the performance of the Vesical Imaging-Reporting and Data System (VIRADS) in differentiating muscle-invasive and non-muscle-invasive bladder cancer and whether this reporting system improves inter-reader agreement. METHODS: Sixty-four cases of multiparametric 3 tesla bladder MRI from January 2014 to May 2020 were reviewed retrospectively. T2-weighted, diffusion and post-contrast images were reviewed. All magnetic resonance images were reported by a radiologist with 15 years' experience (Reader 1) and a final year radiology trainee with a special interest in urogenital imaging with 3 years of experience (Reader 2). Both readers were blinded to clinical history and histopathology results when scoring each lesion. RESULTS: The sensitivity and specificity for differentiating MIBC and NMIBC were 91% and 68%, respectively, for Reader 1 and 91% and 63%, respectively, for Reader 2. The inter-reader agreement for assigning VIRADS scores was 0.79. The area under the receiver operator curve for Reader 1 and 2 were not significantly different (Reader 1 = 0.79, Reader 2 = 0.77, P = 0.83). CONCLUSIONS: Staging of bladder cancer prior to treatment can be accurately and reliably diagnosed using VIRADS, a novel, standardised reporting system for bladder MRI.

Disrupted Circadian Rhythm of Epinephrine in Males With Youth-Onset Type 2 Diabetes.

Context: Blood pressure and plasma catecholamines normally decline during sleep and rapidly increase in early morning. This is blunted in adults with type 2 diabetes (T2D). Objective: We hypothesize that increased sympatho-adrenal activity during sleep differentiates youth with T2D from nondiabetic obese youth and lean youth. Methods: Fasting spot morning and 24-hour urines were collected in obese adolescents with and without T2D, and normal-weight controls. Fractionated free urine catecholamines (epinephrine, norepinephrine, and dopamine) were measured, and the ratio of fasting spot morning to 24-hour catecholamines was calculated. Results: Urinary 24-hour catecholamine levels were comparable across the 3 groups. Fasting morning epinephrine and the ratio of fasting morning/24-hour epinephrine were higher in youth with T2D (P = 0.004 and P = 0.035, respectively). In males, the ratio of fasting morning/24-hour epinephrine was also higher in youth with T2D (P = 0.005). In females, fasting morning norepinephrine and the ratio of fasting morning/24-hour dopamine were lower in obese youth with and without T2D (P = 0.013 and P = 0.005, respectively) compared with lean youth. Systolic blood pressure was higher in diabetic participants than other groups; males trended higher than females. Conclusion: Circadian rhythm in catecholamines is disrupted in youth-onset T2D, with a blunted overnight fall in urinary epinephrine in males. Conversely, fasting morning norepinephrine and dopamine levels were lower in obese females with or without T2D. Higher nocturnal catecholamines in males with T2D might associate with, or predispose to, hypertension and cardiovascular complications. Lower catecholamine excretion in females with obesity might serve an adaptive, protective role.

Urine tricarboxylic acid cycle signatures of early-stage diabetic kidney disease.

INTRODUCTION: Urine tricarboxylic acid (TCA) cycle organic anions (OAs) are elevated in diabetes and may be biomarkers for diabetic kidney disease (DKD) progression. OBJECTIVES: We assessed associations of 10 urine TCA cycle OAs with estimated glomerular filtration rate (eGFR) and eGFR slope. METHODS: This study is ancillary to the Simultaneous Risk Factor Control Using Telehealth to SlOw Progression of Diabetic Kidney Disease (STOP-DKD) Trial-a randomized trial of pharmacist-led medication and behavior management in 281 patients with early to moderate DKD at Duke from 2014 to 2015. We used linear mixed models to assess associations of urine TCA cycle OAs with outcomes and modelled TCA cycle OAs as: (1) the average of z-scores for each OA; and (2) principal component (PC) scores derived by principal component analysis (PCA). Untargeted urine metabolomics were added for additional discovery. RESULTS: Among 132 participants with 24 h urine samples (50% men; 58% Black; mean age 64 years [SD 9]; mean eGFR 74 ml/min/1.73m2 [SD 21] and median urine albumin-to-creatinine [UACR] 20 mg/g [IQR 8-95]), PCA identified 3 OA metabolite PCs. Malate, fumarate, pyruvate, α-ketoglutarate, lactate, succinate and citrate/isocitrate loaded positively on PC1; methylsuccinate, ethylmalonate and succinate loaded positively on PC2; and methylmalonate, ethylmalonate and citrate/isocitrate loaded negatively on PC3. Over a median follow-up of 1.8 years (IQR, 1.2 to 2.2), higher average OA z-score was strongly associated with higher eGFR after covariate adjustment (p = 0.01), but not with eGFR slope (p = 0.9). Higher PC3, but not other PCs, was associated with lower eGFR (p < 0.001). Conditional random forests and smooth clipped absolute deviation models confirmed methylmalonate, citrate/isocitrate, and ethylmalonate, and added lactate as top ranked metabolites in models of baseline eGFR (R-squared 0.32 and 0.33, respectively). Untargeted urine metabolites confirmed association of urine TCA cycle OAs with kidney function. CONCLUSION: Thus, lower urine TCA cycle OAs, most notably lower methylmalonate, ethylmalonate and citrate/isocitrate, are potential indicators of kidney impairment in early stage DKD.