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Hepatocellular carcinoma (HCC) is the third deadliest and sixth most common cancer in the world. Histone‐lysine N‐methyltransferase EHMT2 (also known as G9a) is a histone methyltransferase frequently overexpressed in many cancer types, including HCC. We showed that Myc‐driven liver tumours have a unique H3K9 methylation pattern with corresponding G9a overexpression. This phenomenon of increased G9a was further observed in our c‐Myc‐positive HCC patient‐derived xenografts. More importantly, we showed that HCC patients with higher c‐Myc and G9a expression levels portend a poorer survival with lower median survival months. We demonstrated that c‐Myc interacts with G9a in HCC and cooperates to regulate c‐Myc‐dependent gene repression. In addition, G9a stabilises c‐Myc to promote cancer development, contributing to the growth and invasive capacity in HCC. Furthermore, combination therapy between G9a and synthetic‐lethal target of c‐Myc, CDK9, demonstrates strong efficacy in patient‐derived avatars of Myc‐driven HCC. Our work suggests that targeting G9a could prove to be a potential therapeutic avenue for Myc‐driven liver cancer. This will increase our understanding of the underlying epigenetic mechanisms of aggressive tumour initiation and lead to improved therapeutic and diagnostic options for Myc‐driven hepatic tumours.

Even though the combined use of ultrasound (US) and alpha-fetoprotein (AFP) is recommended for the surveillance of hepatocellular carcinoma (HCC), the utilization of AFP has its challenges, including accuracy dependent on its cut-off levels, degree of liver necroinflammation, and etiology of liver disease. Though various studies have demonstrated the utility of protein induced by vitamin K absence II (PIVKA-II) in surveillance, treatment monitoring, and predicting recurrence, it is still not recommended as a routine biomarker test. A panel of 17 experts from Asia-Pacific, gathered to discuss and reach a consensus on the clinical usefulness and value of PIVKA-II for the surveillance and treatment monitoring of HCC, based on six predetermined statements. The experts agreed that PIVKA-II was valuable in the detection of HCC in AFP-negative patients, and could potentially benefit detection of early HCC in combination with AFP. PIVKA-II is clinically useful for monitoring curative and intra-arterial locoregional treatments, outcomes, and recurrence, and could potentially predict microvascular invasion risk and facilitate patient selection for liver transplant. However, combining PIVKA-II with US and AFP for HCC surveillance, including small HCC, still requires more evidence, whilst its role in detecting AFP-negative HCC will potentially increase as more patients are treated for hepatitis-related HCC. PIVKA-II in combination with AFP and US has a clinical role in the Asia-Pacific region for surveillance. However, implementation of PIVKA-II in the region will have some challenges, such as requiring standardization of cut-off values, its cost-effectiveness and improving awareness among healthcare providers.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for ~90% of liver neoplasms. It is the second leading cause of cancer-related deaths and the seventh most common cancer worldwide. Although there have been rapid developments in the treatment of HCC over the past decade, the incidence and mortality rates of HCC remain a challenge. With the widespread use of the hepatitis B vaccine and antiviral therapy, the etiology of HCC is shifting more toward metabolic-associated steatohepatitis (MASH). Early-stage HCC can be treated with potentially curative strategies such as surgical resection, liver transplantation, and radiofrequency ablation, improving long-term survival. However, most HCC patients, when diagnosed, are already in the intermediate or advanced stages. Molecular targeted therapy, followed by immune checkpoint inhibitor immunotherapy, has been a revolution in HCC systemic treatment. Systemic treatment of HCC especially for patients with compromised liver function is still a challenge due to a significant resistance to immune checkpoint blockade, tumor heterogeneity, lack of oncogenic addiction, and lack of effective predictive and therapeutic biomarkers.

Background & Aims: Lifestyle and environmental-related exposures are important risk factors for hepatocellular carcinoma (HCC), suggesting that epigenetic dysregulation significantly underpins HCC. We profiled 30 surgically resected tumours and the matched adjacent normal tissues to understand the aberrant epigenetic events associated with HCC. Methods: We identified tumour differential enhancers and the associated genes by analysing H3K27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) and Hi-C/HiChIP data from the resected tumour samples of 30 patients with early-stage HCC. This epigenome dataset was analysed with previously reported genome and transcriptome data of the overlapping group of patients from the same cohort. We performed patient-specific differential expression testing using multiregion sequencing data to identify genes that undergo both enhancer and gene expression changes. Based on the genes selected, we identified two patient groups and performed a recurrence-free survival analysis. Results: We observed large-scale changes in the enhancer distribution between HCC tumours and the adjacent normal samples. Many of the gain-in-tumour enhancers showed corresponding upregulation of the associated genes and vice versa, but much of the enhancer and gene expression changes were patient-specific. A subset of the upregulated genes was activated in a subgroup of patients’ tumours. Recurrence-free survival analysis revealed that the patients with a more robust upregulation of those genes showed a worse prognosis. Conclusions: We report the genomic enhancer signature associated with differential prognosis in HCC. Findings that cohere with oncofoetal reprogramming in HCC were underpinned by genome-wide enhancer rewiring. Our results present the epigenetic changes in HCC that offer the rational selection of epigenetic-driven gene targets for therapeutic intervention or disease prognostication in HCC. Impact and Implications: Lifestyle and environmental-related exposures are the important risk factors of hepatocellular carcinoma (HCC), suggesting that tumour-associated epigenetic dysregulations may significantly underpin HCC. We profiled tumour tissues and their matched normal from 30 patients with early-stage HCC to study the dysregulated epigenetic changes associated with HCC. By also analysing the patients’ RNA-seq and clinical data, we found the signature genes – with epigenetic and transcriptomic dysregulation – associated with worse prognosis. Our findings suggest that systemic approaches are needed to consider the surrounding cellular environmental and epigenetic changes in HCC tumours.

[This corrects the article DOI: 10.1371/journal.pone.0251998.].

Abstract Deregulation of MYC is among the most frequent oncogenic drivers in hepatocellular carcinoma (HCC). Unfortunately, the clinical success of MYC‐targeted therapies is limited. Synthetic lethality offers an alternative therapeutic strategy by leveraging on vulnerabilities in tumors with MYC deregulation. While several synthetic lethal targets of MYC have been identified in HCC, the need to prioritize targets with the greatest therapeutic potential has been unmet. Here, we demonstrate that by pairing splice‐switch oligonucleotide (SSO) technologies with our phenotypic‐analytical hybrid multidrug interrogation platform, quadratic phenotypic optimization platform (QPOP), we can disrupt the functional expression of these targets in specific combinatorial tests to rapidly determine target–target interactions and rank synthetic lethality targets. Our SSO‐QPOP analyses revealed that simultaneous attenuation of CHK1 and BRD4 function is an effective combination specific in MYC‐deregulated HCC, successfully suppressing HCC progression in vitro. Pharmacological inhibitors of CHK1 and BRD4 further demonstrated its translational value by exhibiting synergistic interactions in patient‐derived xenograft organoid models of HCC harboring high levels of MYC deregulation. Collectively, our work demonstrates the capacity of SSO‐QPOP as a target prioritization tool in the drug development pipeline, as well as the therapeutic potential of CHK1 and BRD4 in MYC‐driven HCC.

Abstract Background Burnout is a serious issue plaguing the medical profession with potential negative consequences on patient care. Burnout symptoms are observed as early as medical school. Based on a Job Demands-Resources model, this study aims to assess associations between specific job resources measured at the beginning of the first year of medical school with burnout symptoms occurring later in the first year. Methods The specific job resources of grit, tolerance for ambiguity, social support and gender were measured in Duke-NUS Medical School students at the start of Year 1. Students were then surveyed for burnout symptoms at approximately quarterly intervals throughout the year. Using high ratings of cynicism and exhaustion as the definition of burnout, we investigated the associations of the occurrence of burnout with student job resources using multivariable logistic regression analysis. Results Out of 59 students, 19 (32.2%) indicated evidence of burnout at some point across the first year of medical school. Stepwise multivariable logistic regression analysis identified grit as having a significant protective effect against experiencing burnout (Odds Ratio, 0.84; 95%CI 0.74 to 0.96). Using grit as a single predictor of burnout, area under the ROC curve was 0.76 (95%CI: 0.62 to 0.89). Conclusions Grit was identified as a protective factor against later burnout, suggesting that less gritty students are more susceptible to burnout. The results indicate that grit is a robust character trait which can prognosticate burnout in medical students. These students would potentially benefit from enhanced efforts to develop grit as a personal job resource.

Abstract Background Pancreatic adenocarcinoma (PDAC) is highly lethal. Surgery offers the only chance of cure, but 5‐year overall survival (OS) after surgical resection and adjuvant therapy remains dismal. Adjuvant trials were mostly conducted in the West enrolling fit patients. Applicability to a general population, especially Asia has not been described adequately. Aim We aimed to evaluate the clinical outcomes, prognostic factors of survival, pattern, and timing of recurrence after curative resection in an Asian institution. Methods and Results The clinicopathologic and survival outcomes of 165 PDAC patients who underwent curative resection between 1998 and 2013 were reviewed retrospectively. Median age at surgery was 62.0 years. 55.2% were male, and 73.3% had tumors involving the head of pancreas. The median OS of the entire cohort was 19.7 months. Median OS of patients who received adjuvant chemotherapy was 23.8 months. Negative predictors of survival include lymph node ratio (LNR) of >0.3 (HR = 3.36, P = .001), tumor site involving the body or tail of pancreas (HR = 1.59, P = .046), presence of perineural invasion (PNI) (HR = 2.36, P = .018) and poorly differentiated/undifferentiated tumor grade (HR = 1.86, P = .058). The median time to recurrence was 8.87 months, with 66.1% and 81.2% of patients developing recurrence at 12 months and 24 months respectively. The most common site of recurrence was the liver. Conclusion The survival of Asian patients with resected PDAC who received adjuvant chemotherapy is comparable to reported randomized trials. Clinical characteristics seem similar to Western patients. Hence, geographical locations may not be a necessary stratification factor in RCTs. Conversely, lymph node ratio and status of PNI ought to be incorporated.

Abstract Background Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC. Methods Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed. Results Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis. Conclusions Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In the chip, patient-derived cells were encapsulated in alginate and grown to maturity for more than 7 days to form cancer multi-spheroids. Multi-spheroids grown in conventional well plates require many cells and are easily damaged as a result of multiple pipetting during maintenance culture or experimental procedures. To address these issues, we applied a micropillar and microwell structure to the multi-spheroids array. Patient-derived cells from patients with Glioblastoma (GBM), the most common and lethal form of central nervous system cancer, were used to validate the array chip performance. After forming multi-spheroids with a diameter greater than 100μm in a 12×36 pillar array chip (25mm × 75mm), we tested 70 drug compounds (6 replicates) using a high-dose to determine safety and efficacy for drug candidates. Comparing the drug response of multi-spheroids derived from normal cells and cancer cells, we found that four compounds (Dacomitinib, Cediranib, LY2835219, BGJ398) did not show toxicity to astrocyte cell and were efficacious to patient-derived GBM cells.

BackgroundVenous reconstruction has been recently demonstrated to be safe for tumours with invasion into portal vein and/or superior mesenteric vein. This study aims to compare the patency between various venous reconstructions.MethodsThis is retrospective study of 76 patients who underwent pancreaticoduodenectomy or total pancreatectomy with venous reconstruction from 2006 to 2018. Patient demographics, tumour histopathology, morbidity, mortality and patency were studied. Kaplan-Meier estimates were performed for primary venous patency.ResultsSixty-two patients underwent pancreaticoduodenectomy and 14 underwent total pancreatectomy. Forty-seven, 19 and 10 patients underwent primary repair, end-to-end anastomosis and interposition graft respectively. Major morbidity (Clavien-Dindo >grade 2) and 30-day mortality were 14/76(18.4%) and 1/76(1.3%) respectively. There were 12(15.8%) venous occlusion including 4(5.3%) acute occlusions. Overall 6-month, 1-year and 2-year primary patency was 89.1%, 92.5% and 92.3% respectively. 1-year primary patency of primary repair was superior to end-to-end anastomosis and interposition graft (primary repair 100%, end-to-end anastomosis 81.8%, interposition graft 66.7%, p = 0.045). Pairwise comparison also demonstrated superior 1-year patency of primary repair (adjusted p = 0.037). There was no significant difference between the cumulative venous patency for each venous reconstruction method: primary repair 84±6%, end-to-end anastomosis 75±11% and interposition graft 76±15% (p = 0.561).Conclusion1-year primary venous patency of primary repair is superior to end-to-end anastomosis and interposition graft.

Currently, molecular markers are not used when determining the prognosis and treatment strategy for patients with hepatocellular carcinoma (HCC). In the present study, we proposed that the identification of common pro‐oncogenic pathways in primary tumors (PT) and adjacent non‐malignant tissues (AT) typically used to predict HCC patient risks may result in HCC biomarker discovery. We examined the genome‐wide mRNA expression profiles of paired PT and AT samples from 321 HCC patients. The workflow integrated differentially expressed gene selection, gene ontology enrichment, computational classification, survival predictions, image analysis and experimental validation methods. We developed a 24‐ribosomal gene‐based HCC classifier (RGC), which is prognostically significant in both PT and AT. The RGC gene overexpression in PT was associated with a poor prognosis in the training (hazard ratio = 8.2, P = 9.4 × 10−6) and cross‐cohort validation (hazard ratio = 2.63, P = 0.004) datasets. The multivariate survival analysis demonstrated the significant and independent prognostic value of the RGC. The RGC displayed a significant prognostic value in AT of the training (hazard ratio = 5.0, P = 0.03) and cross‐validation (hazard ratio = 1.9, P = 0.03) HCC groups, confirming the accuracy and robustness of the RGC. Our experimental and bioinformatics analyses suggested a key role for c‐MYC in the pro‐oncogenic pattern of ribosomal biogenesis co‐regulation in PT and AT. Microarray, quantitative RT‐PCR and quantitative immunohistochemical studies of the PT showed that DKK1 in PT is the perspective biomarker for poor HCC outcomes. The common co‐transcriptional pattern of ribosome biogenesis genes in PT and AT from HCC patients suggests a new scalable prognostic system, as supported by the model of tumor‐like metabolic redirection/assimilation in non‐malignant AT. The RGC, comprising 24 ribosomal genes, is introduced as a robust and reproducible prognostic model for stratifying HCC patient risks. The adjacent non‐malignant liver tissue alone, or in combination with HCC tissue biopsy, could be an important target for developing predictive and monitoring strategies, as well as evidence‐based therapeutic interventions, that aim to reduce the risk of post‐surgery relapse in HCC patients.

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

This article presents the results of a study that assessed the relationship among romantic attachment style, conflict resolution, and sexual satisfaction. It was found that high ambivalent attachment predicted less sexual satisfaction. It was also found that those who scored high on ambivalent or avoidant attachment had lower relationship satisfaction, as well as those who used verbal aggression as a means of conflict resolution had lower relationship satisfaction. Therapeutic implications and areas for future research are addressed. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma has one of the poorest survival rates amongst cancers. Here, the authors highlight the intra-tumour heterogeneity of this disease, finding that spatially closer tumour sectors are genetically more similar and that intra-hepatic metastasis is accompanied by rapid genetic diversification.

Surgery is the primary curative option in patients with hepatocellular carcinoma (HCC). Current prognostic models for HCC are developed on datasets of primarily patients with advanced cancer, and may be less relevant to resectable HCC. We developed a postoperative nomogram, the Singapore Liver Cancer Recurrence (SLICER) Score, to predict outcomes of HCC patients who have undergone surgical resection.Records for 544 consecutive patients undergoing first-line curative surgery for HCC in one institution from 1992-2007 were reviewed, with 405 local patients selected for analysis. Freedom from relapse (FFR) was the primary outcome measure. An outcome-blinded modeling strategy including clustering, data reduction and transformation was used. We compared the performance of SLICER in estimating FFR with other HCC prognostic models using concordance-indices and likelihood analysis.A nomogram predicting FFR was developed, incorporating non-neoplastic liver cirrhosis, multifocality, preoperative alpha-fetoprotein level, Child-Pugh score, vascular invasion, tumor size, surgical margin and symptoms at presentation. Our nomogram outperformed other HCC prognostic models in predicting FFR by means of log-likelihood ratio statistics with good calibration demonstrated at 3 and 5 years post-resection and a concordance index of 0.69. Using decision curve analysis, SLICER also demonstrated superior net benefit at higher threshold probabilities.The SLICER score enables well-calibrated individualized predictions of relapse following curative HCC resection, and may represent a novel tool for biomarker research and individual counseling.

Background and Objectives: Housing insecurity is associated with adverse effects on child growth and development cross-sectionally; less is known about its cumulative, long-term effects. This study describes longitudinal experiences of housing insecurity during childhood from infancy (age 1 year) to adolescence (age 15 years) and examines their associations with adolescent health outcomes., Methods: Using data from the Future of Families and Child Wellbeing Study, we created a composite measure of housing insecurity using 5 indicators (eg, skipping a rent or mortgage payment, eviction) for participants at ages 1, 3, 5, 9, and 15 years. We used group-based trajectory modeling to identify distinct patterns of housing insecurity, sociodemographic predictors of these patterns, and how these patterns relate to adolescent health outcomes., Results: We identified 3 trajectories of housing insecurity from infancy to adolescence: secure, moderately insecure, and highly insecure. Adolescents who experienced moderately and highly insecure housing had decreased odds of excellent health (adjusted odds ratio, 0.81; 95% confidence interval [CI], 0.69-0.95; adjusted odds ratio, 0.67; 95% CI, 0.50-0.92, respectively) and more depressive symptoms (adjusted incidence rate ratio, 1.05; 95% CI, 1.02-1.08; 1.13; 95% CI, 1.08-1.19, respectively) than adolescents with secure housing. Adolescents who experienced highly insecure housing reported significantly higher anxiety symptoms (adjusted incidence rate ratio, 1.05; 95% CI, 1.003-1.113)., Conclusions: Housing insecurity starting in infancy was associated with poorer adolescent health outcomes. These longitudinal patterns emphasize the need for novel screening mechanisms to identify housing insecurity when it emerges, as well as policies to prevent housing insecurity and its associated health outcomes., (Copyright © 2024 by the American Academy of Pediatrics.)

Objective: To examine whether a cultural adaptation of an early childhood obesity prevention program promotes healthy infant feeding practices., Methods: Prospective quasi-experimental study of a community-engaged multiphasic cultural adaptation of an obesity prevention program set at a federally qualified health center serving immigrant Chinese American parent-child dyads (N = 298). In a group of historical controls, we assessed early infant feeding practices (breastfeeding, sugar-sweetened beverage intake) in 6-month-olds and then the same practices alongside early solid food feeding practices (bottle weaning, fruit, vegetable, sugary or salty snack consumption) in 12-month-olds. After implementation, we assessed these practices in an intervention cohort group at 6 and 12 months. We used cross-sectional groupwise comparisons and adjusted regression analyses to evaluate group differences., Results: At 6 months, the intervention group had increased odds of no sugar-sweetened beverage intake (aOR: 5.69 [95% confidence interval (CI): 1.65, 19.63], P = .006). At 12 months, the intervention group also had increased odds of no sugar-sweetened beverage intake (aOR: 15.22 [95% CI: 6.33, 36.62], P < .001), increased odds of bottle weaning (aOR: 2.34 [95% CI: 1.05, 5.23], P = .03), and decreased odds of sugary snack consumption (aOR: 0.36 [0.18, 0.70], P = .003). We did not detect improvements in breastfeeding, fruit, vegetable, or salty snack consumption., Conclusions: A cultural adaptation of a primary care-based educational obesity prevention program for immigrant Chinese American families with low income is associated with certain healthy infant feeding practices. Future studies should evaluate cultural adaptations of more intensive interventions that better address complex feeding practices, such as breastfeeding, and evaluate long-term weight outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.)

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal., Competing Interests: WM, LG, AT, SZ, JC, JP, KP, AC, CC, SG, VG No competing interests declared, (© 2023, Mitchell et al.)

Background: People who inject drugs (PWID) are a key population for treatment with direct-acting antiviral medications (DAAs) to eliminate hepatitis C virus (HCV). We developed a Pharmacist, Physician, and Patient Navigator Collaborative Care Model (PPP-CCM) for delivery of HCV treatment; this study describes clinical outcomes related to HCV treatment (initial evaluation, treatment initiation, completion, and cure), as well as patient satisfaction., Methods: We conducted a single-arm prospective pilot study of adult PWID living with HCV. Participants completed baseline and six-month follow-up surveys, and treatment and outcomes were abstracted from electronic health records. Primary outcome was linkage to pharmacist for HCV evaluation; secondary outcomes included DAA initiation, completion, and cure, as well as patient-reported satisfaction., Results: Of the 40 PWID enrolled, mean age was 43.6 years, 12 (30 %) were female, 20 (50 %) were non-white, and 15 (38 %) were unhoused. Thirty-eight (95 %) were successfully linked to the pharmacist for initial evaluation. Of those, 21/38 (55 %) initiated DAAs, and 16/21 (76 %) completed treatment. Among those completing treatment who had viral load data to document whether they achieved "sustained virologic response", i.e. cure, 10/11 (91 %) were found to be cured. There was high satisfaction with 100 % responding "agree or strongly agree" that they had a positive experience with the pharmacist., Conclusion: Nearly all participants in this pilot were successfully linked to the pharmacist for evaluation, and more than half were started on DAAs; results provide preliminary evidence of feasibility of pharmacist-led models of HCV treatment for PWID., Clinicaltrialsgov Registration Number: NCT04698629., Competing Interests: The authors declare that all procedures were performed in compliance with relevant laws and institutional guidelines. Appropriate ethical safeguards, including privacy rights of human participants, and ethical approvals were obtained through the University of Washington's Institutional Review Board., (© 2024 The Authors. Published by Elsevier B.V.)

Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA sequencing and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against lipid peroxides and ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity., Significance: HCC harbors abundant mitochondria, mitochondrial DNA mutations, and chromosomal losses. Using a CRISPR-Cas9 screen inspired by transcriptomic and metabolomic profiling, we identify molecular effectors essential for cell fitness. We uncover lipid peroxide stress as a vulnerability coupled to mitochondrial complex I loss in HCC. See related article by Frank et al., p. 1884. This article is highlighted in the In This Issue feature, p. 1749., (©2023 The Authors; Published by the American Association for Cancer Research.)

Lupus anti-coagulant hypoprothrombinemia syndrome (LAHPS) is a rare condition that can be difficult to treat. It increases the risk of thrombosis and bleeding due to the presence of lupus anti-coagulant and factor II deficiency, respectively. There are a limited number of cases described in the literature. Herein we describe a case of LAHPS with bleeding symptoms as a first clinical manifestation of systemic lupus erythematosus (SLE) in an 8-year-old female. She has had multiple recurrences of her bleeding symptoms, requiring treatment with steroids, cyclophosphamide, mycophenolate mofetil, and rituximab. Her course was later complicated by development of arthritis and lupus nephritis. Her complicated course provides a new perspective on the clinical course and treatment of LAHPS. We also present a comprehensive literature review which demonstrates the difficulty in treating patients with LAHPS with underlying SLE and the variability of the clinical course and management of LAHPS depending on the age at presentation., (© 2023. The Author(s), under exclusive licence to International League of Associations for Rheumatology (ILAR).)

One hour plasma glucose (1-hr PG) concentration during an oral glucose tolerance test (OGTT) is steadily emerging as an independent predictor of type 2 diabetes (T2D)., Methods: We applied the current cut off thresholds reported in the pediatric literature for the 1-hr PG, 132.5 (7.4 mmol/l) and 155 mg/dL (8.6 mmol/l) during an OGTT, to report abnormal glucose tolerance (AGT) using ROC curve analyses. We determined the empirical optimal cut point for 1-hr PG for our multi ethnic cohort using the Youden Index., Results: About 1-hour and 2-hours plasma glucose showed the highest predictive potential based on Areas under the curve (AUC) values of 0.91 [CI: 0.85, 0.97] and 1 [CI: 1, 1], respectively. Further comparison of the ROC curves of the 1-hour and 2-hour PG measurements as predictors of an abnormal OGTT showed that their associated AUCs differed significantly ( X 2 (1) = 9.25, P  < .05). Using 132.5 mg/dL as the cutoff point for plasma glucose at 1-hour yielded a ROC curve with an AUC of 0.796, a sensitivity of 88%, and a specificity of 71.2%. Alternatively, the cutoff point of 155 mg/dL resulted in a ROC AUC of 0.852, a sensitivity of 80%, and a specificity of 90.4%., Conclusion: Our cross-sectional study affirms that the 1-hr PG can identify obese children and adolescents at increased risk for prediabetes and/or T2D with almost the same accuracy as a 2-hr PG. In our multi-ethnic cohort, a 1-hr PG ⩾ 155 mg/dL (8.6 mmol/l) serves as an optimal cut-point, using the estimation of the Youden index with AUC of 0.86 and sensitivity of 80%.We support the petition to consider the 1-hr PG as integral during an OGTT, as this adds value to the interpretation of the OGTT beyond the fasting and 2-hr PG., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

How phosphate levels are detected in mammals is unknown. The bone-derived hormone fibroblast growth factor 23 (FGF23) lowers blood phosphate levels by reducing kidney phosphate reabsorption and 1,25(OH)2D production, but phosphate does not directly stimulate bone FGF23 expression. Using PET scanning and LC-MS, we found that phosphate increases kidney-specific glycolysis and synthesis of glycerol-3-phosphate (G-3-P), which then circulates to bone to trigger FGF23 production. Further, we found that G-3-P dehydrogenase 1 (Gpd1), a cytosolic enzyme that synthesizes G-3-P and oxidizes NADH to NAD+, is required for phosphate-stimulated G-3-P and FGF23 production and prevention of hyperphosphatemia. In proximal tubule cells, we found that phosphate availability is substrate-limiting for glycolysis and G-3-P production and that increased glycolysis and Gpd1 activity are coupled through cytosolic NAD+ recycling. Finally, we show that the type II sodium-dependent phosphate cotransporter Npt2a, which is primarily expressed in the proximal tubule, conferred kidney specificity to phosphate-stimulated G-3-P production. Importantly, exogenous G-3-P stimulated FGF23 production when Npt2a or Gpd1 were absent, confirming that it was the key circulating factor downstream of glycolytic phosphate sensing in the kidney. Together, these findings place glycolysis at the nexus of mineral and energy metabolism and identify a kidney-bone feedback loop that controls phosphate homeostasis.

In response to COVID-19, schools across the United States closed in early 2020; many did not fully reopen until late 2021. Although regular testing of asymptomatic students, teachers, and staff can reduce transmission risks, few school systems consistently used proactive testing to safeguard return to classrooms. Socioeconomically diverse public school districts might vary testing levels across campuses to ensure fair, effective use of limited resources. We describe a test allocation approach to reduce overall infections and disparities across school districts. Using a model of SARS-CoV-2 transmission in schools fit to data from a large metropolitan school district in Texas, we reduced incidence between the highest and lowest risk schools from a 5.6-fold difference under proportional test allocation to 1.8-fold difference under our optimized test allocation. This approach provides a roadmap to help school districts deploy proactive testing and mitigate risks of future SARS-CoV-2 variants and other pathogen threats.

Objective: Objective structured clinical exams (OSCE) are used to train and assess resident foundational family-centered communication skills incorporating key stakeholders. In 2020 encounters were conducted virtually. We sought to compare standardized patient (SP) and family faculty (FF) assessment across OSCE and virtual OSCE (VOSCE) formats., Methods: The intern classes of 2019 and 2020 completed a live OSCE and VOSCE respectively where they disclosed an error to an SP. The 10 minute encounter was observed by an FF and facilitator followed by a 20 minute debrief. The SP and FF completed a behavioral checklist to evaluate skill mastery., Results: Eighteen (90%) of the 20 interns completed the encounter each year. Total mastery scores were significantly higher for SP assessment than for FF assessment in both OSCE [68% vs 46% (z = -3.005, P < .05)] and VOSCE formats [68% vs 53% (z = -2.105, P < .05)]. Total mastery scores of SPs across OSCE and VOSCE formats did not significantly differ, nor did FF scores based on evaluation format., Conclusions: Our current experience suggests VOSCEs are a viable alternative to in person sessions given the comparable assessment across the two modalities. The ease of participating in virtual sessions may provide a way to more easily partner with key stakeholders., (Copyright © 2022 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.)

Background: Direct-acting antivirals (DAAs) offer an unprecedented opportunity to eliminate hepatitis C virus (HCV) infection, yet barriers among people who inject drugs (PWID) remain. Having pharmacists provide care through collaborative drug therapy agreements (CDTAs) offers a promising solution. We developed and piloted a Pharmacist, Physician, and Patient Navigator-Collaborative Care Model (PPP-CCM) which utilized pharmacists to directly deliver HCV care at community organizations serving PWID. We conducted formative evaluation of the PPP-CCM pilot to characterize implementation experiences., Methods: The PPP-CCM was implemented from November of 2020 through July of 2022. Formative evaluation team members observed implementation-related meetings and conducted multiple site visits, taking detailed fieldnotes. Fieldnotes were iteratively reviewed to identify barriers and facilitators to implementation and used to inform 7 key informant interviews conducted with programmatic staff at the end of the pilot. All data were analyzed using a Rapid Assessment Process (RAP) guided by the Consolidated Framework for Implementation Research (CFIR). The formative evaluation team shared results with program stakeholders (pharmacists, physicians, and other site staff) to verify and expand on learnings., Results: Evaluation of PPP-CCM revealed 5 themes, encompassing all CFIR domains: 1) PPP-CCM was feasible but challenging to deliver efficiently; 2) the pharmacist role and characteristics (e.g., being flexible, available, and patient-centered) were key to PPP-CCM successes; 3) the PPP-CCM team met challenges engaging patients over time, but some team-based strategies helped; 4) community site characteristics (e.g., existing trusting relationships with PWID and physical space that enabled program visibility) were important contributors; and 5) financial barriers may limit PPP-CCM scale-up and sustainability., Conclusion: PPP-CCM is a novel and promising approach to HCV care delivery for PWID who may previously lack engagement in traditional care models, but careful attention needs to be paid to financial barriers to ensure scalability and sustainability., Competing Interests: Declarations of Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J. Scott reports that in the past two years he has served as a paid consultant to Gilead Sciences for work related to COVID therapeutics and as a paid expert on the data adjudication committee for a cardiovascular medication made by Novo Nordisk. E. Tung discloses that she has served on Advisory Board and Speaker's Bureau for Gilead Sciences. Other authors declare no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Colonization of the intestine by oral microbes has been linked to multiple diseases such as inflammatory bowel disease and colon cancer, yet mechanisms allowing expansion in this niche remain largely unknown. Veillonella parvula, an asaccharolytic, anaerobic, oral microbe that derives energy from organic acids, increases in abundance in the intestine of patients with inflammatory bowel disease. Here we show that nitrate, a signature metabolite of inflammation, allows V. parvula to transition from fermentation to anaerobic respiration. Nitrate respiration, through the narGHJI operon, boosted Veillonella growth on organic acids and also modulated its metabolic repertoire, allowing it to use amino acids and peptides as carbon sources. This metabolic shift was accompanied by changes in carbon metabolism and ATP production pathways. Nitrate respiration was fundamental for ectopic colonization in a mouse model of colitis, because a V. parvula narG deletion mutant colonized significantly less than a wild-type strain during inflammation. These results suggest that V. parvula harness conditions present during inflammation to colonize in the intestine., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). VLDL receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids. Since fatty acid uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We found that excess circulating lipids restrained retinal autophagy, which contributed to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived fatty acid sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.

The Research Engagement Survey Tool (REST) examines the level of partner engagement in research studies. This study used mixed methods, including web-based surveys (N = 336), a modified Delphi process (N = 18), and cognitive response interviews (N = 16), with convenience sampling to develop and validate a short version of the REST. We conducted factor analysis and calculated internal consistency for the condensed REST. We validated the condensed REST against the comprehensive REST. All analyses were carried out on two scales (quality and quantity) based on Likert-type response options. We examined convergent validity with other measures theoretically associated with the REST (e.g., the Community Engagement Research Index and the Partnership Self-Assessment Tool). This study produced a 9-item condensed version of the REST. The condensed REST loads on 1 factor, has high internal consistency (Cronbach's alpha = 0.92 for the quantity scale; 0.94 for the quality scale), is significantly correlated (ρ = 0.97; p < 0.001 for both scales) with the comprehensive (32-item) REST, and has negligible, low, and moderate correlation with other measures (e.g., the Partnership Assessment In community-based Research, trust in medical researchers, and the Coalition Self-Assessment Survey). Use of the condensed REST will reduce participant burden and time to complete. This standardized and validated quantitative measure is useful to compare engagement across projects or within a project over time.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure 1 , where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment 2 . Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The novel coronavirus (SARS-CoV-2) emerged in late 2019 and spread globally in early 2020. Initial reports suggested the associated disease, COVID-19, produced rapid epidemic growth and caused high mortality. As the virus sparked local epidemics in new communities, health systems and policy makers were forced to make decisions with limited information about the spread of the disease. We developed a compartmental model to project COVID-19 healthcare demands that combined information regarding SARS-CoV-2 transmission dynamics from international reports with local COVID-19 hospital census data to support response efforts in three Metropolitan Statistical Areas (MSAs) in Texas, USA: Austin-Round Rock, Houston-The Woodlands-Sugar Land, and Beaumont-Port Arthur. Our model projects that strict stay-home orders and other social distancing measures could suppress the spread of the pandemic. Our capacity to provide rapid decision-support in response to emerging threats depends on access to data, validated modeling approaches, careful uncertainty quantification, and adequate computational resources.

Genetic variation of the 16p11.2 deletion locus containing the KCTD13 gene and of CUL3 is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of Kctd13 mutant ( Kctd13 -/- ) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In Kctd13 -/- neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in Kctd13 -/- neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human KCTD13 variants suggests that KCTD13 variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in Kctd13 -/- neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome., Competing Interests: J.M.M. and A.J.C. are inventors on pending U.S. Patent Application No. 16/843,790, which is owned by the Broad Institute and is directed to certain subject matter disclosed herein., (© 2020 The Author(s).)

Background: Whereas regular exercise is associated with lower risk of cardiovascular disease and mortality, mechanisms of exercise-mediated health benefits remain less clear. We used metabolite profiling before and after acute exercise to delineate the metabolic architecture of exercise response patterns in humans., Methods: Cardiopulmonary exercise testing and metabolite profiling was performed on Framingham Heart Study participants (age 53±8 years, 63% women) with blood drawn at rest (n=471) and at peak exercise (n=411)., Results: We observed changes in circulating levels for 502 of 588 measured metabolites from rest to peak exercise (exercise duration 11.9±2.1 minutes) at a 5% false discovery rate. Changes included reductions in metabolites implicated in insulin resistance (glutamate, -29%; P =1.5×10 -55 ; dimethylguanidino valeric acid [DMGV], -18%; P =5.8×10 -18 ) and increases in metabolites associated with lipolysis (1-methylnicotinamide, +33%; P =6.1×10 -67 ), nitric oxide bioavailability (arginine/ornithine + citrulline, +29%; P =2.8×10 -169 ), and adipose browning (12,13-dihydroxy-9Z-octadecenoic acid +26%; P =7.4×10 -38 ), among other pathways relevant to cardiometabolic risk. We assayed 177 metabolites in a separate Framingham Heart Study replication sample (n=783, age 54±8 years, 51% women) and observed concordant changes in 164 metabolites (92.6%) at 5% false discovery rate. Exercise-induced metabolite changes were variably related to the amount of exercise performed (peak workload), sex, and body mass index. There was attenuation of favorable excursions in some metabolites in individuals with higher body mass index and greater excursions in select cardioprotective metabolites in women despite less exercise performed. Distinct preexercise metabolite levels were associated with different physiologic dimensions of fitness (eg, ventilatory efficiency, exercise blood pressure, peak Vo 2 ). We identified 4 metabolite signatures of exercise response patterns that were then analyzed in a separate cohort (Framingham Offspring Study; n=2045, age 55±10 years, 51% women), 2 of which were associated with overall mortality over median follow-up of 23.1 years ( P ≤0.003 for both)., Conclusions: In a large sample of community-dwelling individuals, acute exercise elicits widespread changes in the circulating metabolome. Metabolic changes identify pathways central to cardiometabolic health, cardiovascular disease, and long-term outcome. These findings provide a detailed map of the metabolic response to acute exercise in humans and identify potential mechanisms responsible for the beneficial cardiometabolic effects of exercise for future study.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that controls blood phosphate levels by increasing renal phosphate excretion and reducing 1,25-dihydroxyvitamin D3 [1,25(OH)2D] production. Disorders of FGF23 homeostasis are associated with significant morbidity and mortality, but a fundamental understanding of what regulates FGF23 production is lacking. Because the kidney is the major end organ of FGF23 action, we hypothesized that it releases a factor that regulates FGF23 synthesis. Using aptamer-based proteomics and liquid chromatography-mass spectrometry-based (LC-MS-based) metabolomics, we profiled more than 1600 molecules in renal venous plasma obtained from human subjects. Renal vein glycerol-3-phosphate (G-3-P) had the strongest correlation with circulating FGF23. In mice, exogenous G-3-P stimulated bone and bone marrow FGF23 production through local G-3-P acyltransferase-mediated (GPAT-mediated) lysophosphatidic acid (LPA) synthesis. Further, the stimulatory effect of G-3-P and LPA on FGF23 required LPA receptor 1 (LPAR1). Acute kidney injury (AKI), which increases FGF23 levels, rapidly increased circulating G-3-P in humans and mice, and the effect of AKI on FGF23 was abrogated by GPAT inhibition or Lpar1 deletion. Together, our findings establish a role for kidney-derived G-3-P in mineral metabolism and outline potential targets to modulate FGF23 production during kidney injury.

Background: The pandemic of cardiovascular disease (CVD) and type 2 diabetes (T2D) requires the identification of new predictor biomarkers. Biomarkers potentially modifiable with lifestyle changes deserve a special interest. Our aims were to analyze: (a) The associations of lysine, 2-aminoadipic acid (2-AAA) or pipecolic acid with the risk of T2D or CVD in the PREDIMED trial; (b) the effect of the dietary intervention on 1-year changes in these metabolites, and (c) whether the Mediterranean diet (MedDiet) interventions can modify the effects of these metabolites on CVD or T2D risk., Methods: Two unstratified case-cohort studies nested within the PREDIMED trial were used. For CVD analyses, we selected 696 non-cases and 221 incident CVD cases; for T2D, we included 610 non-cases and 243 type 2 diabetes incident cases. Metabolites were quantified using liquid chromatography-tandem mass spectrometry, at baseline and after 1-year of intervention., Results: In weighted Cox regression models, we found that baseline lysine (HR +1 SD increase  = 1.26; 95% CI 1.06-1.51) and 2-AAA (HR +1 SD increase  = 1.28; 95% CI 1.05-1.55) were both associated with a higher risk of T2D, but not with CVD. A significant interaction (p = 0.032) between baseline lysine and T2D on the risk of CVD was observed: subjects with prevalent T2D and high levels of lysine exhibited the highest risk of CVD. The intervention with MedDiet did not have a significant effect on 1-year changes of the metabolites., Conclusions: Our results provide an independent prospective replication of the association of 2-AAA with future risk of T2D. We show an association of lysine with subsequent CVD risk, which is apparently diabetes-dependent. No evidence of effects of MedDiet intervention on lysine, 2-AAA or pipecolic acid changes was found. Trial registration ISRCTN35739639; registration date: 05/10/2005; recruitment start date 01/10/2003.

Our understanding of how the gut microbiome interacts with its human host has been restrained by limited access to longitudinal datasets to examine stability and dynamics, and by having only a few isolates to test mechanistic hypotheses. Here, we present the Broad Institute-OpenBiome Microbiome Library (BIO-ML), a comprehensive collection of 7,758 gut bacterial isolates paired with 3,632 genome sequences and longitudinal multi-omics data. We show that microbial species maintain stable population sizes within and across humans and that commonly used 'omics' survey methods are more reliable when using averages over multiple days of sampling. Variation of gut metabolites within people over time is associated with amino acid levels, and differences across people are associated with differences in bile acids. Finally, we show that genomic diversification can be used to infer eco-evolutionary dynamics and in vivo selection pressures for strains within individuals. The BIO-ML is a unique resource designed to enable hypothesis-driven microbiome research.

Objective: To identify prediagnostic plasma metabolomic biomarkers associated with amyotrophic lateral sclerosis (ALS)., Methods: We conducted a global metabolomic study using a nested case-control study design within 5 prospective cohorts and identified 275 individuals who developed ALS during follow-up. We profiled plasma metabolites using liquid chromatography-mass spectrometry and identified 404 known metabolites. We used conditional logistic regression to evaluate the associations between metabolites and ALS risk. Further, we used machine learning analyses to determine whether the prediagnostic metabolomic profile could discriminate ALS cases from controls., Results: A total of 31 out of 404 identified metabolites were associated with ALS risk ( p < 0.05). We observed inverse associations (n = 27) with plasma levels of diacylglycerides and triacylglycerides, urate, purine nucleosides, and some organic acids and derivatives, while we found positive associations for a cholesteryl ester, 2 phosphatidylcholines, and a sphingomyelin. The number of significant associations increased to 67 (63 inverse) in analyses restricted to cases with blood samples collected within 5 years of onset. None of these associations remained significant after multiple comparison adjustment. Further, we were not able to reliably distinguish individuals who became cases from controls based on their metabolomic profile using partial least squares discriminant analysis, elastic net regression, random forest, support vector machine, or weighted correlation network analyses., Conclusions: Although the metabolomic profile in blood samples collected years before ALS diagnosis did not reliably separate presymptomatic ALS cases from controls, our results suggest that ALS is preceded by a broad, but poorly defined, metabolic dysregulation years before the disease onset., (© 2019 American Academy of Neurology.)

Clinical genomic tests increasingly use a next-generation sequencing (NGS) platform due in part to the high fidelity of variant calls, yet rare errors are still possible. In germline DNA screening, failure to correct such errors could have serious consequences for patients, who may follow an unwarranted screening or surgical management path. It has been suggested that routine orthogonal confirmation by Sanger sequencing is required to verify NGS results, especially low-confidence positives with depressed allele fraction (<30% of alternate allele). We evaluated whether an alternative method of confirmation-software-assisted manual call review-performed comparably with Sanger confirmation in >15,000 samples. Licensed reviewers manually inspected both raw and processed data at the batch, sample, and variant levels, including raw NGS read pileups. Of ambiguous variant calls with <30% allele fraction (1707 total calls at 38 unique sites), manual call review classified >99% (n = 1701) as true positives (enriched for long insertions or deletions and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining <1% (n = 6) being mosaic. Critically, results from software-assisted manual review and retrospective Sanger sequencing were concordant for samples selected from all ambiguous sites. We conclude that the confirmation required for high confidence in NGS-based germline testing can manifest in different ways; a trained NGS expert operating platform-tailored review software achieves quality comparable with routine Sanger confirmation., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The human adaptive starvation response allows for survival during long-term caloric deprivation. Whether the physiology of starvation is adaptive or maladaptive is context dependent: activation of pathways by caloric restriction may promote longevity, yet in the context of caloric excess, the same pathways may contribute to obesity. Here, we performed plasma metabolite profiling of longitudinally collected samples during a 10-day, 0-calorie fast in humans. We identify classical milestones in adaptive starvation, including the early consumption of gluconeogenic amino acids and the subsequent surge in plasma nonesterified fatty acids that marks the shift from carbohydrate to lipid metabolism, and demonstrate findings, including (a) the preferential release of unsaturated fatty acids and an associated shift in plasma lipid species with high degrees of unsaturation and (b) evidence that acute, starvation-mediated hypoleptinemia may be a driver of the transition from glucose to lipid metabolism in humans.

Thermogenesis by brown and beige adipose tissue, which requires activation by external stimuli, can counter metabolic disease 1 . Thermogenic respiration is initiated by adipocyte lipolysis through cyclic AMP-protein kinase A signalling; this pathway has been subject to longstanding clinical investigation 2-4 . Here we apply a comparative metabolomics approach and identify an independent metabolic pathway that controls acute activation of adipose tissue thermogenesis in vivo. We show that substantial and selective accumulation of the tricarboxylic acid cycle intermediate succinate is a metabolic signature of adipose tissue thermogenesis upon activation by exposure to cold. Succinate accumulation occurs independently of adrenergic signalling, and is sufficient to elevate thermogenic respiration in brown adipocytes. Selective accumulation of succinate may be driven by a capacity of brown adipocytes to sequester elevated circulating succinate. Furthermore, brown adipose tissue thermogenesis can be initiated by systemic administration of succinate in mice. Succinate from the extracellular milieu is rapidly metabolized by brown adipocytes, and its oxidation by succinate dehydrogenase is required for activation of thermogenesis. We identify a mechanism whereby succinate dehydrogenase-mediated oxidation of succinate initiates production of reactive oxygen species, and drives thermogenic respiration, whereas inhibition of succinate dehydrogenase supresses thermogenesis. Finally, we show that pharmacological elevation of circulating succinate drives UCP1-dependent thermogenesis by brown adipose tissue in vivo, which stimulates robust protection against diet-induced obesity and improves glucose tolerance. These findings reveal an unexpected mechanism for control of thermogenesis, using succinate as a systemically-derived thermogenic molecule.

Renal oncocytomas are benign tumors characterized by a marked accumulation of mitochondria. We report a combined exome, transcriptome, and metabolome analysis of these tumors. Joint analysis of the nuclear and mitochondrial (mtDNA) genomes reveals loss-of-function mtDNA mutations occurring at high variant allele fractions, consistent with positive selection, in genes encoding complex I as the most frequent genetic events. A subset of these tumors also exhibits chromosome 1 loss and/or cyclin D1 overexpression, suggesting they follow complex I loss. Transcriptome data revealed that many pathways previously reported to be altered in renal oncocytoma were simply differentially expressed in the tumor's cell of origin, the distal nephron, compared with other nephron segments. Using a heuristic approach to account for cell-of-origin bias we uncovered strong expression alterations in the gamma-glutamyl cycle, including glutathione synthesis (increased GCLC ) and glutathione degradation. Moreover, the most striking changes in metabolite profiling were elevations in oxidized and reduced glutathione as well as γ-glutamyl-cysteine and cysteinyl-glycine, dipeptide intermediates in glutathione biosynthesis, and recycling, respectively. Biosynthesis of glutathione appears adaptive as blockade of GCLC impairs viability in cells cultured with a complex I inhibitor. Our data suggest that loss-of-function mutations in complex I are a candidate driver event in renal oncocytoma that is followed by frequent loss of chromosome 1, cyclin D1 overexpression, and adaptive up-regulation of glutathione biosynthesis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Background: Although metabolomic profiling offers promise for the prediction of coronary heart disease (CHD), and metabolic risk factors are more strongly associated with CHD in women than men, limited data are available for women., Methods: We applied a liquid chromatography-tandem mass spectrometry metabolomics platform to measure 371 metabolites in a discovery set of postmenopausal women (472 incident CHD cases, 472 controls) with validation in an independent set of postmenopausal women (312 incident CHD cases, 315 controls)., Results: Eight metabolites, primarily oxidized lipids, were significantly dysregulated in cases after the adjustment for matching and CHD risk factors in both the discovery and validation data sets. One oxidized phospholipid, C34:2 hydroxy-phosphatidylcholine, remained associated with CHD after further adjustment for other validated metabolites. Subjects with C34:2 hydroxy-phosphatidylcholine levels in the highest quartile had a 4.7-fold increase in CHD odds in comparison with the lowest quartile; C34:2 hydroxy-phosphatidylcholine also significantly improved the area under the curve ( P <0.01) for CHD. The C34:2 hydroxy-phosphatidylcholine findings were replicated in a third replication data set of 980 men and women (230 cardiovascular events) with a stronger association observed in women., Conclusions: These data replicate known metabolite predictors, identify novel markers, and support the relationship between lipid oxidation and subsequent CHD., (© 2018 American Heart Association, Inc.)

Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD., Competing Interests: The authors declare no conflict of interest.