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Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mir483 is a conserved and highly expressed microRNA in placental mammals, embedded within the Igf2 gene. Its expression is dysregulated in a number of human diseases, including metabolic disorders and certain cancers. Here, we investigate the developmental regulation and function of Mir483 in vivo. We find that Mir483 expression is dependent on Igf2 transcription and the regulation of the Igf2/H19 imprinting control region. Transgenic Mir483 overexpression in utero causes fetal, but not placental, growth restriction through insulin-like growth factor 1 (IGF1) and IGF2 and also causes cardiovascular defects leading to fetal death. Overexpression of Mir483 post-natally results in growth stunting through IGF1 repression, increased hepatic lipid production, and excessive adiposity. IGF1 infusion rescues the post-natal growth restriction. Our findings provide insights into the function of Mir483 as a growth suppressor and metabolic regulator and suggest that it evolved within the INS-IGF2-H19 transcriptional region to limit excessive tissue growth through repression of IGF signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments., Methods: We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1 -/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan., Findings: We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure., Conclusion: This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them., Funding: This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant., Competing Interests: Declaration of interests J.S. is the deputy CEO and 50% owner of BLUsang AB. He holds patents on Vel genotyping (inventors: Jill Storry, Magnus Jöud, Björn Nilsson, and Martin L. Olsson). J.S. has received speaker fees, royalties, and honoraria from the following companies: Grifols Diagnostic Solutions, QuidelOrtho Inc., and Biorad Laboratories. J.S. receives an honorarium for Section Editor work, Vox Sanguinis from John Wiley & Sons Ltd. J.S. is Vice President of the International Society of Blood Transfusion and married to Professor M.L.O. M.L.O. is CEO and 50% owner of BLUsang AB. M.L.O. holds patents on Vel genotyping (inventors: Jill Storry, Magnus Jöud, Björn Nilsson, and Martin L. Olsson). M.L.O. received speaker fees, royalties, and honoraria from the following companies: Grifols Diagnostic Solutions, QuidelOrtho Inc., and Biorad Laboratories. M.L.O. is married to Adjunct Professor J.S. W.N.E. is chair of the International Council for Standardization in Haematology. W.N.E. works as advisor for Scorpio Labs and is on the editorial board of the Journal of Clinical Pathology. W.H.O. is chair of the Blood Transfusion Genomics Consortium. W.H.O. is in receipt of an educational/research grant from Thermo Fisher Scientific. N.G. offers scientific consulting services to Thermo Fisher Scientific., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hypothalamic neural circuits regulate instinctive behaviors such as food seeking, the fight/flight response, socialization, and maternal care. Here, we identified microdeletions on chromosome Xq23 disrupting the brain-expressed transient receptor potential (TRP) channel 5 (TRPC5). This family of channels detects sensory stimuli and converts them into electrical signals interpretable by the brain. Male TRPC5 deletion carriers exhibited food seeking, obesity, anxiety, and autism, which were recapitulated in knockin male mice harboring a human loss-of-function TRPC5 mutation. Women carrying TRPC5 deletions had severe postpartum depression. As mothers, female knockin mice exhibited anhedonia and depression-like behavior with impaired care of offspring. Deletion of Trpc5 from oxytocin neurons in the hypothalamic paraventricular nucleus caused obesity in both sexes and postpartum depressive behavior in females, while Trpc5 overexpression in oxytocin neurons in knock-in mice reversed these phenotypes. We demonstrate that TRPC5 plays a pivotal role in mediating innate human behaviors fundamental to survival, including food seeking and maternal care., Competing Interests: Declaration of interests I.S.F. has consulted for a number of companies developing weight loss drugs (including Eli Lilly, Novo Nordisk, and Rhythm Pharmaceuticals) and investors (Goldman Sachs, SV Health). I.S.F. is a member of the Advisory Board of Cell. J.R.B.P. is an employee and shareholder of Insmed, receives research funding from GSK, and is a paid consultant for WW International. K.W.W. holds shares in Novo Nordisk and Eli Lilly., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity causes significant morbidity and mortality globally. Research in the last three decades has delivered a step-change in our understanding of the fundamental mechanisms that regulate energy homeostasis, building on foundational discoveries in mouse models of metabolic disease. However, not all findings made in rodents have translated to humans, hampering drug discovery in this field. Here, we review how studies in mice and humans have informed our current framework for understanding energy homeostasis, discuss their challenges and limitations, and offer a perspective on how human studies may play an increasingly important role in the discovery of disease mechanisms and identification of therapeutic targets in the future., Competing Interests: Declaration of interests I.S.F. is on the Editorial Board of Cell., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Deficiency of the epigenome modulator histone deacetylase 3 (HDAC3) in brown adipose tissue (BAT) impairs the ability of mice to survive in near-freezing temperatures. Here, we report that short-term exposure to mild cold temperature (STEMCT: 15°C for 24 h) averted lethal hypothermia of mice lacking HDAC3 in BAT (HDAC3 BAT KO) exposed to 4°C. STEMCT restored the induction of the thermogenic coactivator PGC-1α along with UCP1 at 22°C, which is greatly impaired in HDAC3-deficient BAT, and deletion of either UCP1 or PGC-1α prevented the protective effect of STEMCT. Remarkably, this protection lasted for up to 7 days. Transcriptional activator C/EBPβ was induced by short-term cold exposure in mouse and human BAT and, uniquely, remained high for 7 days following STEMCT. Adeno-associated virus-mediated knockdown of BAT C/EBPβ in HDAC3 BAT KO mice erased the persistent memory of STEMCT, revealing the existence of a C/EBPβ-dependent and HDAC3-independent cold-adaptive epigenomic memory., Competing Interests: Declaration of interests M.A.L. is an advisory board member for Pfizer Inc. and is an advisory board member and co-founder of Flare Therapeutics. S.K. serves on Scientific Advisory Boards for Merck, Alnylum, AbbVie, and Boehringer Ingelheim., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Brown adipose tissue (BAT) in rodents appears to be an important tissue for the clearance of plasma branched-chain amino acids (BCAAs) contributing to improved metabolic health. However, the role of human BAT in plasma BCAA clearance is poorly understood. Here, we evaluate patients with prostate cancer who underwent positron emission tomography-computed tomography imaging after an injection of 18 F-fluciclovine (L-leucine analog). Supraclavicular adipose tissue (AT; primary location of human BAT) has a higher net uptake rate for 18 F-fluciclovine compared to subcutaneous abdominal and upper chest AT. Supraclavicular AT 18 F-fluciclovine net uptake rate is lower in patients with obesity and type 2 diabetes. Finally, the expression of genes involved in BCAA catabolism is higher in the supraclavicular AT of healthy people with high BAT volume compared to those with low BAT volume. These findings support the notion that BAT can potentially function as a metabolic sink for plasma BCAA clearance in people., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Approximately 1 in 4 people worldwide have non-alcoholic fatty liver disease (NAFLD); however, there are currently no medications to treat this condition. This study investigated the role of adiposity-associated orphan G protein-coupled receptor 75 (GPR75) in liver lipid accumulation. We profiled Gpr75 expression and report that it is most abundant in the brain. Next, we generated the first single-cell-level analysis of Gpr75 and identified a subpopulation co-expressed with key appetite-regulating hypothalamic neurons. CRISPR-Cas9-deleted Gpr75 mice fed a palatable western diet high in fat adjusted caloric intake to remain in energy balance, thereby preventing NAFLD. Consistent with mouse results, analysis of whole-exome sequencing data from 428,719 individuals (UK Biobank) revealed that variants in GPR75 are associated with a reduced likelihood of hepatic steatosis. Here, we provide a significant advance in understanding of the expression and function of GPR75, demonstrating that it is a promising pharmaceutical target for NAFLD treatment., Competing Interests: Declaration of interests E.J.G. and J.R.B.P. are employees of and hold shares in Adrestia Therapeutics., (Copyright © 2024. Published by Elsevier Inc.)

The obesity epidemic is principally driven by the consumption of more calories than the body requires. It is therefore essential that the mechanisms underpinning feeding behavior are defined. Neurons within the brainstem dorsal vagal complex (DVC) receive direct information from the digestive system and project to second-order regions in the brain to regulate food intake. Although γ-aminobutyric acid is expressed in the DVC (GABA DVC ), its function in this region has not been defined. In order to discover the unique gene expression signature of GABA DVC cells, we used single-nucleus RNA sequencing (Nuc-seq), and this revealed 19 separate clusters. We next probed the function of GABA DVC cells and discovered that the selective activation of GABA DVC neurons significantly controls food intake and body weight. Optogenetic interrogation of GABA DVC circuitry identified GABA DVC → hypothalamic arcuate nucleus (ARC) projections as appetite suppressive without creating aversion. Electrophysiological analysis revealed that GABA DVC → ARC stimulation inhibits hunger-promoting neuropeptide Y (NPY) neurons via GABA release. Adopting an intersectional genetics strategy, we clarify that the GABA DVC → ARC circuit curbs food intake. These data identify GABA DVC as a new modulator of feeding behavior and body weight and a controller of orexigenic NPY neuron activity, thereby providing insight into the neural underpinnings of obesity., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although a high amount of brown adipose tissue (BAT) is associated with low plasma triglyceride concentration, the mechanism responsible for this relationship in people is not clear. Here, we evaluate the interrelationships among BAT, very-low-density lipoprotein triglyceride (VLDL-TG), and free fatty acid (FFA) plasma kinetics during thermoneutrality in women with overweight/obesity who had a low (<20 mL) or high (≥20 mL) volume of cold-activated BAT (assessed by using positron emission tomography in conjunction with 2-deoxy-2-[ 18 F]-fluoro-glucose). We find that plasma TG and FFA concentrations are lower and VLDL-TG and FFA plasma clearance rates are faster in women with high BAT than low BAT volume, whereas VLDL-TG and FFA appearance rates in plasma are not different between the two groups. These findings demonstrate that women with high BAT volume have lower plasma TG and FFA concentrations than women with low BAT volumes because of increased VLDL-TG and FFA clearance rates. This study was registered at ClinicalTrials.gov (NCT02786251)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Investigations of memory mechanisms have been, thus far, neuron centric, despite the brain comprising diverse cell types. Using rats and mice, we assessed the cell-type-specific contribution of hippocampal insulin-like growth factor 2 (IGF2), a polypeptide regulated by learning and required for long-term memory formation. The highest level of hippocampal IGF2 was detected in pericytes, the multi-functional mural cells of the microvessels that regulate blood flow, vessel formation, the blood-brain barrier, and immune cell entry into the central nervous system. Learning significantly increased pericytic Igf2 expression in the hippocampus, particularly in the highly vascularized stratum lacunosum moleculare and stratum moleculare layers of the dentate gyrus. Igf2 increases required neuronal activity. Regulated hippocampal Igf2 knockout in pericytes, but not in fibroblasts or neurons, impaired long-term memories and blunted the learning-dependent increase of neuronal immediate early genes (IEGs). Thus, neuronal activity-driven signaling from pericytes to neurons via IGF2 is essential for long-term memory., Competing Interests: Declaration of interests C.M.A. is an inventor of patents filed by NYU entitled on the use of IGF2 receptor agonist ligands for treatment of neurodevelopmental disorders and neurodegenerative diseases. C.M.A. is a founder of a company seeking to develop compounds covered by the patents., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The long-term clinical outcomes of severe obesity due to leptin signaling deficiency are unknown. We carry out a retrospective cross-sectional investigation of a large cohort of children with leptin (LEP), LEP receptor (LEPR), or melanocortin 4 receptor (MC4R) deficiency (n = 145) to evaluate the progression of the disease. The affected individuals undergo physical, clinical, and metabolic evaluations. We report a very high mortality in children with LEP (26%) or LEPR deficiency (9%), mainly due to severe pulmonary and gastrointestinal infections. In addition, 40% of surviving children with LEP or LEPR deficiency experience life-threatening episodes of lung or gastrointestinal infections. Although precision drugs are currently available for LEP and LEPR deficiencies, as yet, they are not accessible in Pakistan. An appreciation of the severe impact of LEP or LEPR deficiency on morbidity and early mortality, educational attainment, and the attendant stigmatization should spur efforts to deliver the available life-saving drugs to these children as a matter of urgency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response., Competing Interests: Declaration of interests R.K.G. has received honoraria for consulting and educational activities from Gilead, GSK, Janssen, and Moderna., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

New approaches are needed to treat people whose obesity and type 2 diabetes (T2D) are driven by specific mechanisms. We investigate a deletion on chromosome 16p11.2 (breakpoint 2-3 [BP2-3]) encompassing SH2B1, a mediator of leptin and insulin signaling. Phenome-wide association scans in the UK (N = 502,399) and Estonian (N = 208,360) biobanks show that deletion carriers have increased body mass index (BMI; p = 1.3 × 10 -10 ) and increased rates of T2D. Compared with BMI-matched controls, deletion carriers have an earlier onset of T2D, with poorer glycemic control despite higher medication usage. Cystatin C, a biomarker of kidney function, is significantly elevated in deletion carriers, suggesting increased risk of renal impairment. In a Mendelian randomization study, decreased SH2B1 expression increases T2D risk (p = 8.1 × 10 -6 ). We conclude that people with 16p11.2 BP2-3 deletions have early, complex obesity and T2D and may benefit from therapies that enhance leptin and insulin signaling., Competing Interests: Declaration of interests I.S.F. has consulted for a number of companies developing weight-loss drugs, including Eli Lilly, Novo Nordisk, and Rhythm Pharmaceuticals., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to profound epigenetic changes and the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The extent to which chromatin remodeling shapes this anti-oxidant response is currently unknown. Here, we explored the effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the remodeled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor that regulates anti-oxidant response genes and subsequent metabolic rewiring cooperating without direct interaction with the anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides additional insights into the molecular mechanisms behind cell responses to fumarate accumulation and potentially provides further avenues for therapeutic intervention for HLRCC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Maternal-offspring interactions in mammals involve both cooperation and conflict. The fetus has evolved ways to manipulate maternal physiology to enhance placental nutrient transfer, but the mechanisms involved remain unclear. The imprinted Igf2 gene is highly expressed in murine placental endocrine cells. Here, we show that Igf2 deletion in these cells impairs placental endocrine signaling to the mother, without affecting placental morphology. Igf2 controls placental hormone production, including prolactins, and is crucial to establish pregnancy-related insulin resistance and to partition nutrients to the fetus. Consequently, fetuses lacking placental endocrine Igf2 are growth restricted and hypoglycemic. Mechanistically, Igf2 controls protein synthesis and cellular energy homeostasis, actions dependent on the placental endocrine cell type. Igf2 loss also has additional long-lasting effects on offspring metabolism in adulthood. Our study provides compelling evidence for an intrinsic fetal manipulation system operating in placenta that modifies maternal metabolism and fetal resource allocation, with long-term consequences for offspring metabolic health., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The relevance of extracellular matrix (ECM) remodeling is reported in white adipose tissue (AT) and obesity-related dysfunctions, but little is known about the importance of ECM remodeling in brown AT (BAT) function. Here, we show that a time course of high-fat diet (HFD) feeding progressively impairs diet-induced thermogenesis concomitantly with the development of fibro-inflammation in BAT. Higher markers of fibro-inflammation are associated with lower cold-induced BAT activity in humans. Similarly, when mice are housed at thermoneutrality, inactivated BAT features fibro-inflammation. We validate the pathophysiological relevance of BAT ECM remodeling in response to temperature challenges and HFD using a model of a primary defect in the collagen turnover mediated by partial ablation of the Pepd prolidase. Pepd-heterozygous mice display exacerbated dysfunction and BAT fibro-inflammation at thermoneutrality and in HFD. Our findings show the relevance of ECM remodeling in BAT activation and provide a mechanism for BAT dysfunction in obesity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Adipose tissue from pheochromocytoma patients acquires brown fat features, making it a valuable model for studying the mechanisms that control thermogenic adipose plasticity in humans. Transcriptomic analyses revealed a massive downregulation of splicing machinery components and splicing regulatory factors in browned adipose tissue from patients, with upregulation of a few genes encoding RNA-binding proteins potentially involved in splicing regulation. These changes were also observed in cell culture models of human brown adipocyte differentiation, confirming a potential involvement of splicing in the cell-autonomous control of adipose browning. The coordinated changes in splicing are associated with a profound modification in the expression levels of splicing-driven transcript isoforms for genes involved in the specialized metabolism of brown adipocytes and those encoding master transcriptional regulators of adipose browning. Splicing control appears to be a relevant component of the coordinated gene expression changes that allow human adipose tissue to acquire a brown phenotype., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Cachexia, a systemic wasting condition, is considered a late consequence of diseases, including cancer, organ failure, or infections, and contributes to significant morbidity and mortality. The induction process and mechanistic progression of cachexia are incompletely understood. Refocusing academic efforts away from advanced cachexia to the etiology of cachexia may enable discoveries of new therapeutic approaches. Here, we review drivers, mechanisms, organismal predispositions, evidence for multi-organ interaction, model systems, clinical research, trials, and care provision from early onset to late cachexia. Evidence is emerging that distinct inflammatory, metabolic, and neuro-modulatory drivers can initiate processes that ultimately converge on advanced cachexia., Competing Interests: Declaration of interests S.O. receives remuneration for scientific advisory services provided to Pfizer, AstraZeneca, Courage Therapeutics, and Third Rock Ventures. M.J.-H. has consulted for, and is a member of, the Achilles Therapeutics Scientific Advisory Board and Steering Committee, has received speaker honoraria from Pfizer, Astex Pharmaceuticals, Oslo Cancer Cluster, and is co-inventor on a European patent application relating to methods to detect lung cancer (PCT/US2017/028013). All authors, other than J.G. and D.M., are members of the CANCAN Grand Challenge Team funded by Cancer Research UK and the National Cancer Institute. The funders had no role in writing or reviewing the manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation., Competing Interests: Declaration of interests M.K. is a founder and K.K. is an employee of BitBio. M.K. holds patents on the methods for generation of the OptiOx cell line., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

During pre-implantation stages of mammalian development, maternally stored material promotes both the erasure of the sperm and oocyte epigenetic profiles and is responsible for concomitant genome activation. Here, we have utilized single-cell methylome and transcriptome sequencing (scM&T-seq) to quantify both mRNA expression and DNA methylation in oocytes and a developmental series of human embryos at single-cell resolution. We fully characterize embryonic genome activation and maternal transcript degradation and map key epigenetic reprogramming events in developmentally high-quality embryos. By comparing these signatures with early embryos that have undergone spontaneous cleavage-stage arrest, as determined by time-lapse imaging, we identify embryos that fail to appropriately activate their genomes or undergo epigenetic reprogramming. Our results indicate that a failure to successfully accomplish these essential milestones impedes the developmental potential of pre-implantation embryos and is likely to have important implications, similar to aneuploidy, for the success of assisted reproductive cycles., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously. In white adipocytes, LDs can reach 100 μm in diameter, occupying >90% of the cell. Cidec, which is strictly required for the formation of large LDs, is concentrated at interfaces between adjacent LDs and facilitates directional flux of neutral lipids from the smaller to the larger LD. The mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs remains elusive. Here we visualize interfaces between LDs by electron cryo-tomography and analyze the kinetics of lipid transfer by quantitative live fluorescence microscopy. We show that transfer occurs through closely apposed monolayers, is slowed down by increasing the distance between the monolayers, and follows exponential kinetics. Our data corroborate the notion that Cidec facilitates pressure-driven transfer of neutral lipids through two "leaky" monolayers between LDs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.)

At the moment of union in fertilization, sperm and oocyte are transcriptionally silent. The ensuing onset of embryonic transcription (embryonic genome activation [EGA]) is critical for development, yet its timing and profile remain elusive in any vertebrate species. We here dissect transcription during EGA by high-resolution single-cell RNA sequencing of precisely synchronized mouse one-cell embryos. This reveals a program of embryonic gene expression (immediate EGA [iEGA]) initiating within 4 h of fertilization. Expression during iEGA produces canonically spliced transcripts, occurs substantially from the maternal genome, and is mostly downregulated at the two-cell stage. Transcribed genes predict regulation by transcription factors (TFs) associated with cancer, including c-Myc. Blocking c-Myc or other predicted regulatory TF activities disrupts iEGA and induces acute developmental arrest. These findings illuminate intracellular mechanisms that regulate the onset of mammalian development and hold promise for the study of cancer., Competing Interests: Declaration of interests The authors declare no conflicting interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Insulin secretion is critical for glucose homeostasis, and increased levels of the precursor proinsulin relative to insulin indicate pancreatic islet beta-cell stress and insufficient insulin secretory capacity in the setting of insulin resistance. We conducted meta-analyses of genome-wide association results for fasting proinsulin from 16 European-ancestry studies in 45,861 individuals. We found 36 independent signals at 30 loci (p value < 5 × 10 -8 ), which validated 12 previously reported loci for proinsulin and ten additional loci previously identified for another glycemic trait. Half of the alleles associated with higher proinsulin showed higher rather than lower effects on glucose levels, corresponding to different mechanisms. Proinsulin loci included genes that affect prohormone convertases, beta-cell dysfunction, vesicle trafficking, beta-cell transcriptional regulation, and lysosomes/autophagy processes. We colocalized 11 proinsulin signals with islet expression quantitative trait locus (eQTL) data, suggesting candidate genes, including ARSG, WIPI1, SLC7A14, and SIX3. The NKX6-3/ANK1 proinsulin signal colocalized with a T2D signal and an adipose ANK1 eQTL signal but not the islet NKX6-3 eQTL. Signals were enriched for islet enhancers, and we showed a plausible islet regulatory mechanism for the lead signal in the MADD locus. These results show how detailed genetic studies of an intermediate phenotype can elucidate mechanisms that may predispose one to disease., Competing Interests: Declaration of interests J.B.M. is an academic associate for Quest Diagnostics Endocrine R&D. M.E.K. is employed by SYNLAB Holding Deutschland GmbH. C.M.L. receives grants from Bayer Ag and Novo Nordisk and her husband works for Vertex. B.Z. is employed at the Swedish Medical Products Agency, SE-751 03 Uppsala, Sweden; the views expressed in this paper are the personal views of the authors and not necessarily the views of the Swedish government agency. B.Z. has not received any funding or benefits from any sponsor for the present work. J.C.F. receives consulting honoraria from Goldfinch Bio and AstraZeneca and speaker honoraria from Novo Nordisk, AstraZeneca, and Merck for research lectures over which he had full control on content. D.A.L. has received support from Medtronics Ltd and Roche Diagnostics for research unrelated to this paper. W.M. reports grants and personal fees from Siemens Diagnostics, grants and personal fees from Aegerion Pharmaceuticals, grants and personal fees from AMGEN, grants and personal fees from AstraZeneca, grants and personal fees from Danone Research, grants and personal fees from Sanofi, personal fees from Hoffmann LaRoche, personal fees from MSD, grants and personal fees from Pfizer, personal fees from Synageva, grants and personal fees from BASF, grants from Abbott Diagnostics, and grants and personal fees from Numares, outside the submitted work. W.M. is employed by Synlab Holding Deutschland GmbH. R.W. reports lecture fees from Novo Nordisk and Sanofi and served on an advisory board for Akcea Therapeutics, Daiichi Sankyo, Sanofi, and Novo Nordisk. E.W. is now an employee of AstraZeneca., (Copyright © 2023 American Society of Human Genetics. All rights reserved.)

In the mouse, feto-placental endothelial cells (FPEC) line the inner surface of the feto-placental blood vessels located within placental labyrinthine zone and play critical roles in placental development and function. Here, we present a detailed protocol for isolation and culture of primary mouse FPEC, as well as two complementary methods (immunohistochemistry staining and flow cytometry analysis) to assess their purity. These cells are suitable for downstream ex vivo studies to investigate their functional properties, both in normal and pathological contexts. For complete details on the use and execution of this protocol, please refer to Sandovici et al. (2022)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

This protocol outlines a translational lipidomic approach to discover lipid biomarkers that could predict morphometric body and histological organ measurements (e.g., weight and adiposity gains) during specific stages of life (e.g., early life). We describe procedures ranging from animal experimentation and histological analyses to downstream analytical steps through lipid profiling, both in mice and humans. This protocol represents a reliable and versatile approach to translate and validate candidate lipid biomarkers from animal models to a human cohort. For complete details on the use and execution of this protocol, please refer to Olga et al. (2021)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics., Competing Interests: Declaration of interests P.W.A. receives royalties from the Wistar Institute for sales of antibodies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field., Competing Interests: Declaration of interests S.W.S. is on the scientific advisory council of the Lewy Body Dementia Association and the MSA Coalition. S.W.S. is an editorial board member for the Journal of Parkinson Disease and JAMA Neurology. S.W.S. received research support from Cerevel Therapeutics. M.K. serves on the scientific advisory boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, and Alector and is a consultant to Modulo Bio and Recursion Therapeutics. Participation by researchers from Data Tecnica International, LLC in this project was part of a competitive contract awarded to Data Tecnica International, LLC by the National Institutes of Health to support open science research. M.A.N. also currently serves on the scientific advisory board for Clover Therapeutics and is an advisor to Neuron23 Inc. E.A. is founder, shareholder, and scientific advisor of Cholestenix, Ltd., (Published by Elsevier Inc.)

Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent disease that is associated with multiple metabolic disturbances, yet the metabolic pathways underlying its progression are poorly understood. Here, we studied metabolic pathways of the human liver across the full histological spectrum of NAFLD. We analyzed whole liver tissue transcriptomics and serum metabolomics data obtained from a large, prospectively enrolled cohort of 206 histologically characterized patients derived from the European NAFLD Registry and developed genome-scale metabolic models (GEMs) of human hepatocytes at different stages of NAFLD. We identified several metabolic signatures in the liver and blood of these patients, specifically highlighting the alteration of vitamins (A, E) and glycosphingolipids, and their link with complex glycosaminoglycans in advanced fibrosis. Furthermore, we derived GEMs and identified metabolic signatures of three common NAFLD-associated gene variants (PNPLA3 , TM6SF2 , and HSD17B13) . The study demonstrates dysregulated liver metabolic pathways which may contribute to the progression of NAFLD., Competing Interests: J.M.S. declares Consultancy: Boehringer Ingelheim, BMS, Genfit, Gilead Sciences, Intercept Pharmaceuticals, Madrigal, Novartis, Novo Nordisk, Nordic Bioscience, Pfizer, Roche, Sanofi, Siemens Healthcare GmbH. Research Funding: Gilead Sciences, Boehringer Ingelheim. Speakers Bureau: Falk Foundation MSD Sharp & Dohme GmbH. M.A. reports consultancy/advisory with MedImmune/Astra Zeneca and E3Bio, as well as honoraria from Intercept and grant support from GSK and Takeda. E.B. reports advisory/consulting for BMS, Genfit SA, Gilead, Intercept, and Novartis. Q.M.A. reports grants, personal fees and other from Allergan/Tobira, other from E3Bio, other from Eli Lilly & Company Ltd, other from Galmed, grants, personal fees and other from Genfit SA, personal fees and other from Gilead, other from Grunthal, other from Imperial Innovations, grants and other from Intercept Pharma Europe Ltd, other from Inventiva, other from Janssen, personal fees from Kenes, other from MedImmune, other from NewGene, grants, personal fees and other from Pfizer Ltd, other from Raptor Pharma, grants and other from Novartis Pharma AG, grants from Abbvie, personal fees and other from BMS, grants from GSK, other from NGMBio, other from Madrigal, other from Servier, other from EcoR1, other from 89Bio, other from Altimmune, grants and other from AstraZeneca, other from Axcella, other from Blade, other from BNN Cardio, other from Celgene, other from Cirius, other from CymaBay, other from Genentech, other from HistoIndex, other from Indalo, other from IQVIA, other from Metacrine, other from North Sea Therapeutics, personal fees and other from Novo Nordisk, other from Poxel, other from Terns, other from Viking Therapeutics, grants from Glympse Bio, other from PathAI, outside the submitted work. All other authors declare that they have no competing interests., (© 2022 The Author(s).)

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Nausea is a discomforting sensation of gut malaise that remains a major clinical challenge. Several visceral poisons induce nausea through the area postrema, a sensory circumventricular organ that detects bloodborne factors. Here, we use genetic approaches based on an area postrema cell atlas to reveal inhibitory neurons that counteract nausea-associated poison responses. The gut hormone glucose insulinotropic peptide (GIP) activates area postrema inhibitory neurons that project locally and elicit inhibitory currents in nausea-promoting excitatory neurons through γ-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to poisons in wild-type mice, with protection eliminated by targeted area postrema neuron ablation. These findings provide insights into the basic organization of nausea-associated brainstem circuits and reveal that area postrema inhibitory neurons are an effective pharmacological target for nausea intervention., Competing Interests: Declaration of interests S.D.L. is a consultant for Kallyope, Inc., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metabolite levels measured in the human population are endophenotypes for biological processes. We combined sequencing data for 3,924 (whole-exome sequencing, WES, discovery) and 2,805 (whole-genome sequencing, WGS, replication) donors from a prospective cohort of blood donors in England. We used multiple approaches to select and aggregate rare genetic variants (minor allele frequency [MAF] < 0.1%) in protein-coding regions and tested their associations with 995 metabolites measured in plasma by using ultra-high-performance liquid chromatography-tandem mass spectrometry. We identified 40 novel associations implicating rare coding variants (27 genes and 38 metabolites), of which 28 (15 genes and 28 metabolites) were replicated. We developed algorithms to prioritize putative driver variants at each locus and used mediation and Mendelian randomization analyses to test directionality at associations of metabolite and protein levels at the ACY1 locus. Overall, 66% of reported associations implicate gene targets of approved drugs or bioactive drug-like compounds, contributing to drug targets' validating efforts., Competing Interests: Declaration of interests John Danesh reports grants, personal fees, and non-financial support from Merck Sharp & Dohme (MSD); grants, personal fees, and non-financial support from Novartis; grants from Pfizer; and grants from AstraZeneca outside the submitted work. John Danesh sits on the International Cardiovascular and Metabolic Advisory Board for Novartis (since 2010); the Steering Committee of UK Biobank (since 2011); the MRC International Advisory Group (ING) member, London (since 2013); the MRC High Throughput Science ‘Omics Panel Member, London (since 2013); the Scientific Advisory Committee for Sanofi (since 2013); the International Cardiovascular and Metabolism Research and Development Portfolio Committee for Novartis; and the AstraZeneca Genomics Advisory Board (2018). Adam Butterworth reports institutional grants from AstraZeneca, Bayer, Biogen, BioMarin, Bioverativ, Merk and Sanofi. During the course of the project Praveen Surendran became an employee of GSK, Lorenzo Bomba became an employee of BioMarin, Mohd Karim became an employee of Variant Bio and Qi Guo became an employee of BenevolentAI., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The common loss-of-function mutation R577X in the structural muscle protein ACTN3 emerged as a potential target of positive selection from early studies and has been the focus of insightful physiological work suggesting a significant impact on muscle metabolism. Adaptation to cold climates has been proposed as a key adaptive mechanism explaining its global allele frequency patterns. Here, we re-examine this hypothesis analyzing modern (n = 3,626) and ancient (n = 1,651) genomic data by using allele-frequency as well as haplotype homozygosity-based methods. The presented results are more consistent with genetic drift rather than selection in cold climates as the main driver of the ACTN3 R577X frequency distribution in human populations across the world. This Matters Arising paper is in response to Wyckelsma et al. (2021), 1 published in The American Journal of Human Genetics. See also the response by Wyckelsma et al. (2022), 2 published in this issue., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The suprachiasmatic nucleus (SCN) integrates light and systemic signals from peripheral tissues to coordinate physiology and behavior daily rhythms. However, the contribution that nutrients and feeding patterns provide to the SCN network regulation remains controversial. Here, we found that time-restricted feeding (TRF) in ZT0-4 (Zeitgeber Time) generates a robust and long-term shift in locomotor behavior and increased wakefulness. Intracellular Ca 2+ signals in SCN GABAergic neurons of freely moving mice showed significant activation after ZT0-4 TRF treatment. Furthermore, RNA-seq profiling of SCN showed that TRF during ZT0-4 increased Insulin-like Growth Factor 2 ( Igf2 ) expression and dysregulated ion transporters, including the downregulation of Kcc2 . SCN neuron-specific loss of function of Kcc2 amplified ZT0-4 TRF induced aftereffect. Moreover, overexpression of IGF2 in SCN GABAergic neurons extended the locomotion range, mirroring the TRF aftereffect. In summary, our study showed that the IGF2-KCC2 pathway plays an important role for TRF induced behavior changes., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Despite their widespread use in research, there has not yet been a systematic genomic analysis of human embryonic stem cell (hESC) lines at a single-nucleotide resolution. We therefore performed whole-genome sequencing (WGS) of 143 hESC lines and annotated their single-nucleotide and structural genetic variants. We found that while a substantial fraction of hESC lines contained large deleterious structural variants, finer-scale structural and single-nucleotide variants (SNVs) that are ascertainable only through WGS analyses were present in hESC genomes and human blood-derived genomes at similar frequencies. Moreover, WGS allowed us to identify SNVs associated with cancer and other diseases that could alter cellular phenotypes and compromise the safety of hESC-derived cellular products transplanted into humans. As a resource to enable reproducible hESC research and safer translation, we provide a user-friendly WGS data portal and a data-driven scheme for cell line maintenance and selection., Competing Interests: Declaration of interests K.E. is a founder of Q-State Biosciences, Quralis, and Enclear Therapies; an employee and shareholder of BioMarin; and a member of Cell Stem Cell’s advisory board. K.J.K. is a consultant for Vor Biopharma, and C.O. is now an employee of 54gene., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here, we demonstrate, through fetal, endothelial, hematopoietic, and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The angiocrine effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signaling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypothalamic pro-opiomelanocortin (POMC) neurons are known to trigger satiety. However, these neuronal cells encompass heterogeneous subpopulations that release γ-aminobutyric acid (GABA), glutamate, or both neurotransmitters, whose functions are poorly defined. Using conditional mutagenesis and chemogenetics, we show that blockade of the energy sensor mechanistic target of rapamycin complex 1 (mTORC1) in POMC neurons causes hyperphagia by mimicking a cellular negative energy state. This is associated with decreased POMC-derived anorexigenic α-melanocyte-stimulating hormone and recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Electrophysiology and optogenetic studies further reveal that pharmacological blockade of mTORC1 simultaneously activates POMC/GABAergic neurons and inhibits POMC/glutamatergic ones, implying that the functional specificity of these subpopulations relies on mTORC1 activity. Finally, POMC neurons with different neurotransmitter profiles possess specific molecular signatures and spatial distribution. Altogether, these findings suggest that mTORC1 orchestrates the activity of distinct POMC neurons subpopulations to regulate feeding behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The mediobasal hypothalamus (MBH; arcuate nucleus of the hypothalamus [ARH] and median eminence [ME]) is a key nutrient sensing site for the production of the complex homeostatic feedback responses required for the maintenance of energy balance. Here, we show that refeeding after an overnight fast rapidly triggers proliferation and differentiation of oligodendrocyte progenitors, leading to the production of new oligodendrocytes in the ME specifically. During this nutritional paradigm, ME perineuronal nets (PNNs), emerging regulators of ARH metabolic functions, are rapidly remodeled, and this process requires myelin regulatory factor (Myrf) in oligodendrocyte progenitors. In genetically obese ob/ob mice, nutritional regulations of ME oligodendrocyte differentiation and PNN remodeling are blunted, and enzymatic digestion of local PNN increases food intake and weight gain. We conclude that MBH PNNs are required for the maintenance of energy balance in lean mice and are remodeled in the adult ME by the nutritional control of oligodendrocyte differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The 2021 Gairdner Prize is awarded to Daniel Drucker, Joel Habener, and Jens Juul Holst for the discovery of novel peptides encoded in the proglucagon sequence and the establishment of their physiological roles. These discoveries underpinned the development of therapeutics that are now benefiting patients with type 2 diabetes and other disorders worldwide., Competing Interests: Declaration of interests S.O. provides or has provided remunerated consultancy services to a number of pharmaceutical and biotechnology companies including Pfizer, AstraZeneca Ltd., GSK, Novo-Nordisk, and ERX pharmaceuticals., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Obese non-diabetic patients receiving semaglutide, an injectable long-acting GLP-1 receptor agonist, in a large randomized placebo-controlled trial, lost and maintained ∼15% of their body weight for over a year (Wilding et al., 2021). This impressive result is likely to usher in a new era of anti-obesity drugs based on hormones that suppress food intake, largely through acting on the brain., Competing Interests: Declaration of interests S.O.R. has provided remunerated consultancy services to a number of pharmaceutical and biotechnology companies including Pfizer, AstraZeneca Ltd, GSK, Novo-Nordisk, and ERX pharmaceuticals., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gα s protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, β-arrestin recruitment, and/or coupling to Gα s , establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig -/- ) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig -/- mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Increasing brown adipose tissue (BAT) mass and activation is a therapeutic strategy to treat obesity and complications. Obese and diabetic patients possess low amounts of BAT, so an efficient way to expand their mass is necessary. There is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Accessing human BAT is challenging, given its low volume and anatomical dispersion. These constraints make detailed BAT-related developmental and functional mechanistic studies in humans virtually impossible. We have developed and characterized functionally and molecularly a new chemically defined protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs) that overcomes current limitations. This protocol recapitulates step by step the physiological developmental path of human BAT. The BAs obtained express BA and thermogenic markers, are insulin sensitive, and responsive to β-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A growing body of evidence indicates that obesity is strongly and independently associated with adverse outcomes of COVID-19, including death. By combining emerging knowledge of the pathological processes involved in COVID-19 with insights into the mechanisms underlying the adverse health consequences of obesity, we present some hypotheses regarding the deleterious impact of obesity on the course of COVID-19. These hypotheses are testable and could guide therapeutic and preventive interventions. As obesity is now almost ubiquitous and no vaccine for COVID-19 is currently available, even a modest reduction in the impact of obesity on mortality and morbidity from this viral infection could have profound consequences for public health., Competing Interests: S.O. is an employee of the University of Cambridge and has provided remunerated consultancy services to the following pharmaceutical and biotechnology companies: AstraZeneca, eRx Network, Glaxo-Smith-Kline, Novo Nordisk, and Pfizer., (Crown Copyright © 2020 Published by Elsevier Inc.)

This protocol describes the peptidomic analysis of organoid lysates, FACS-purified cell populations, and 2D culture secretions by liquid chromatography mass spectrometry (LC-MS). Currently, most peptides are quantified by ELISA, limiting the peptides that can be studied. However, an LC-MS-based approach allows more peptides to be monitored. Our group has previously used LC-MS for tissue peptidomics and secretion of enteroendocrine peptides from primary culture. Now, we extend the use to organoid models. For complete details on the use and execution of this protocol, please refer to Goldspink et al. (2020)., Competing Interests: The F.R./F.M.G. lab receives additional grant support from AstraZeneca and Eli Lilly for unrelated work. F.M.G. is a consultant for Kallyope (New York, USA)., (© 2020 The Authors.)

Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein ( SNCA ) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)