Peroxisomes are dynamic organelles essential for lipid metabolism, oxidative balance, and cellular stress responses. Their dysfunction contributes to various diseases, including metabolic and neurodegenerative disorders. Selective autophagy, or pexophagy, preserves peroxisomal quality by removing damaged or excess peroxisomes. Here, we propose a novel ATM-PINK1-STUB1-ABCD3-SQSTM1 signaling cascade that orchestrates pexophagy in response to peroxisomal impairment. Through siRNA screening, we find that PINK1 is a key regulator of pexophagy induced by PEX13 depletion. PINK1 phosphorylates STUB1, enhancing its E3 ligase activity to ubiquitinate ABCD3, which in turn recruits SQSTM1 for peroxisomal degradation. We further identify that ATM activates PINK1 under peroxisomal stress, linking cellular stress signaling to organelle quality control. These findings provide new insights into the molecular mechanisms underlying peroxisome turnover and may have implications for therapeutic strategies targeting diseases related to peroxisomal dysfunction.

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites play important roles in maintaining lipid homeostasis at the plasma membrane (PM), cellular calcium homeostasis, and cell signaling. Here, we show that MCTP1 and MCTP2 are at ER subdomains that form membrane contact sites (MCS) with multiple organelles using a proximity labeling assay. MCTPs are three C2 domain-containing transmembrane proteins. We show that upon overexpression, MCTPs promote ER-PM contact sites in a C2 domain-dependent manner. MCTP C2 domains bind to PI(4)P and PI(4,5)P2, phosphoinositides that are enriched in the PM. Furthermore, we show that deletion of MCTP1 or MCTP2 increases PI(4)P levels in the PM and promotes cell migration. Thus, our study identifies MCTPs as multiple ERorganelle contact site proteins and establishes its role at ER-PM contact sites in regulating lipid homeostasis and cell migration.

Acyl-CoA-binding domain-containing proteins (ACBDs) constitute a large and diverse multigene family involved in cellular lipid metabolism. Among them, the tail-anchored peroxisomal membrane protein ACBD5 has emerged as a key component of peroxisome–endoplasmic reticulum (ER) contact sites through its interaction with VAMP-associated proteins (VAPs). More recently, ACBD5 has also been implicated in peroxisome–mitochondria tethering under conditions of oxidative stress. Although initially characterized as a lipid-binding peroxisomal membrane protein, ACBD5 is increasingly recognized as a central mediator of inter-organelle cooperation. Here, we propose an expanded view of ACBD5 function, suggesting that it acts as a peroxisomal “master tether” that integrates intracellular organisation with metabolic and homeostatic regulation. By linking lipid sensing and metabolism to the formation and regulation of membrane contact sites, ACBD5 may serve as a key organizer of peroxisomal interactions within the cellular organelle network. We discuss the role of peroxisomes as interconnected hubs of cellular cooperation, examine the functions of ACBD5 at peroxisome–ER contact sites and beyond, highlight its relevance to human disease, and present a conceptual framework for understanding ACBD5 as a peroxisomal master tether.

Maintenance of mitochondrial redox homeostasis is of fundamental importance to cellular health. Mitochondria harbor a host of intrinsic antioxidant defenses, but the contribution of extrinsic, nonmitochondrial antioxidant mechanisms is less well understood. We found a direct role for peroxisomes in maintaining mitochondrial redox homeostasis through contact-mediated reactive oxygen species (ROS) transfer. We found that ACBD5 and PTPIP51 form a contact between peroxisomes and mitochondria. The percentage of these contacts increased during mitochondrial oxidative stress and helped to maintain mitochondrial health through the transfer of mitochondrial ROS to the peroxisome lumen. Our findings reveal a multiorganelle layer of mitochondrial antioxidant defense-suggesting a direct mechanism by which peroxisomes contribute to mitochondrial health-and broaden the scope of known membrane contact site functions.

Cancer cells undergo complex metabolic reprogramming, allowing them to survive and proliferate in their specific niche. Mitochondria play a key role in metabolism remodeling, and these changes can often lead to mitochondrial stress. However, the cancer cells must keep mitochondria “happy” due to their role in cellular death pathways. Mitochondria are the major producer of reactive oxygen species (ROS). Although mitochondrial ROS is required for cell signaling, excessive ROS production can activate various cell death pathways. Thus, understanding how cancer cells regulate mitochondrial ROS may provide insights into new therapeutic targets for cancer treatments. Yet, our understanding of how cells regulate mitochondria redox homeostasis is limited. Peroxisomes possess one of the most potent antioxidants, Catalase, and it has long been believed to regulate cellular redox homeostasis by scavenging cellular ROS. Interestingly, genetic mutations in peroxisomal genes called Zellweger Spectrum Disorders (ZSD) result in the loss of peroxisomal structures but not Catalase. However, in both patient tissues and animal models of ZSD, an accumulation of oxidatively damaged mitochondria was observed in almost all cell types. This talk will present published and unpublished work addressing how peroxisomes regulate mitochondria redox homeostasis. Our work suggests that peroxisomes directly interact with respiring mitochondria to regulate mitochondrial redox homeostasis. In the end, we will present a model by which peroxisomes regulate mitochondria health during conditions of cellular stress and its potential as a therapeutic target to induce cell death. Peter Kijun Kim. Keeping Mitochondria Happy: How peroxisomes regulate mitochondria oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr SY26-02.

INTRODUCTION:Cardiac adipose tissue, which directly interfaces with the myocardium and vasculature, has a pivotal role in obesity-related cardiovascular pathology through its metabolic activity. This tissue contributes to cardiac remodeling through its regulation of lipid metabolism. Among the key organelles involved, peroxisomes have a central role in lipid metabolism, yet their contribution to obesity-induced cardiac dysfunction remains poorly understood. OBJECTIVES:This study investigated whether peroxisomal dysfunction in cardiac adipose tissue drives obesity-associated cardiac hypertrophy. METHODS:Using a high-fat diet (HFD)-induced obese rat model, we evaluated changes in cardiac adipose tissues, focusing on their browning capacity and metabolic functions. To investigate mechanistic effects, H9C2 cardiomyocytes were exposed either to fatty acids extracted from cardiac adipose tissues or conditioned medium derived from adipocytes treated with mitochondrial and peroxisomal inhibitors (Mdivi-1 or 10,12-tricosadiynoic acid). RESULTS:HFD-fed obese rats exhibited significant expansion of cardiac adipose tissues and cardiac hypertrophy, driven by impaired lipid metabolism and loss of browning capacity in cardiac fat associated with peroxisomal dysfunction. Treatment of H9C2 cardiomyocytes with conditioned medium from adipocytes with peroxisomal dysfunction induced collagen accumulation, increased expression of pro-inflammatory cytokines, and cellular hypertrophy, which recapitulates key pathological features observed in vivo. CONCLUSION:Our findings demonstrate that peroxisomal dysfunction in cardiac adipose tissue drives lipid metabolic reprogramming and contributes to obesity-related cardiac hypertrophy. Targeting peroxisomal function in cardiac fat could be a novel therapeutic approach to mitigate obesity-induced cardiovascular remodeling.

Peroxisomes are essential for lipid metabolism and redox balance, with pexophagy playing a critical role in maintaining cellular homeostasis. However, the regulatory mechanisms of pexophagy remain unclear. Through functional screening, we identified MARCHF7 as a novel E3 ligase regulating pexophagy. MARCHF7 depletion impaired pexophagic flux under PEX1 knockdown conditions. MARCHF7 binds to PXMP4 and promotes its ubiquitination at lysine 20 in PEX1-deficient cells. Depletion of PXMP4 impairs pexophagy, and reconstitution with the PXMP4 lysine 20 ubiquitination-defective mutant failed to rescue pexophagy. PEX1 depletion also induces TBK1 phosphorylation at serine 172, activating TBK1, which subsequently phosphorylates MARCHF7. This activation is driven by ROS accumulation, which reduces PXMP4 ubiquitination and prevents peroxisome loss. Furthermore, downregulation of MARCHF7 or PXMP4 impairs NBR1 recruitment to peroxisomes, suggesting that ubiquitinated PXMP4 acts as a recognition signal for NBR1. Collectively, our findings establish the TBK1-MARCHF7-PXMP4-NBR1 axis as a key regulatory pathway for pexophagy in response to PEX1 depletion.

Mitochondria continually undergo fission to maintain their network and health. Nascent fission sites are marked by the ER, which facilitates actin polymerization to drive calcium flux into the mitochondrion and constrict the inner mitochondrial membrane. Septins are a major eukaryotic cytoskeleton component that forms filaments that can both directly and indirectly modulate other cytoskeleton components, including actin. Septins have been implicated in mitochondrial fission; however, a connection between septins and the regulation of cytoskeletal machinery driving fission is not known. We find that SEPTIN9 is present at mitochondrial fission sites from its early stages with the ER and prior to the fission factor dynamin-related protein 1 (DRP1). SEPTIN9 has an isoform-specific role in fission, dependent on its N-terminal interaction to activate a Rho guanine nucleotide exchange factor, ARHGEF18. Without SEPTIN9, mitochondrial calcium influx is impaired, indicating SEPTIN9-containing octamers play a critical role in the early stages of fission.

Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson’s Disease and Huntington’s Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.

Reactivation of BK polyomavirus (BKPyV) can cause significant kidney and bladder disease in immunocompromised patients. There are currently no effective, BKPyV-specific therapies. MAU868 is a novel, human immunoglobulin (Ig) G1 monoclonal antibody that binds the major capsid protein, VP1, of BKPyV with picomolar affinity, neutralizes infection by the 4 major BKPyV genotypes (EC50 ranging from 0.009-0.093 μg/mL; EC90 ranging from 0.102-4.160 μg/mL), and has comparable activity against variants with highly prevalent VP1 polymorphisms. No resistance-associated variants were identified in long-term selection studies, indicating a high in vitro barrier-to-resistance. The high-resolution crystal structure of MAU868 in complex with VP1 pentamer identified 3 key contact residues in VP1 (Y169, R170, and K172). A first-in-human study was conducted to assess the safety, tolerability, and pharmacokinetics of MAU868 following intravenous and subcutaneous administration to healthy adults in a randomized, placebo-controlled, double-blinded, single ascending dose design. MAU868 was safe and well-tolerated. All adverse events were grade 1 and resolved. The pharmacokinetics of MAU868 was typical of a human IgG, with dose-proportional systemic exposure and an elimination half-life ranging between 23 and 30 days. These results demonstrate the potential of MAU868 as a first-in-class therapeutic agent for the treatment or prevention of BKPyV disease.

Phosphoinositides are rare membrane lipids that mediate cell signaling and membrane dynamics. PI(4)P and PI(3)P are two major phosphoinositides crucial for endolysosomal functions and dynamics, making them the lipids of interest in many studies. The acute modulation of phosphoinositides at a given organelle membrane can reveal important insights into their cellular function. Indeed, the localized depletion of PI(4)P and PI(3)P is a viable tool to assess the importance of these phosphoinositides in various experimental conditions. Here, we describe a live imaging method to acutely deplete PI(4)P and PI(3)P on endolysosomes. The depletion assay utilizes the GAI-GID1 or the FRB-FKBP inducible dimerization system to target the catalytic domain of the PI(4)P phosphatase, Sac1, or the PI(3)P phosphatase domain of MTM1 to the endolysosome for localized depletion of these phosphoinositides. By using the fluorescently tagged biosensors, 2xP4M and PX, we can validate and monitor the depletion of PI(4)P and PI(3)P, respectively, on endolysosomes in real-time. We discuss a method for normalizing the fluorescence measurements to appropriate the relative amount of these phosphoinositides in the organellar membranes (endolysosomes), which is required for monitoring PI(4)P or PI(3)P levels during the acute depletion assay. Since the localization of the dimerization partners is specified by the membrane targeting signal, our protocol will be useful for studying the signaling and functions of phosphoinositides at any membrane.

Objectives To describe and compare liver mitochondrial and peroxisomal histopathology by nutritional status in children who died following hospitalization for acute illness in Malawi. Methods Liver tissue was collected using Minimally Invasive Tissue Sampling from eleven children under-five years old who died during hospitalization and were either non-wasted (n = 4), severely wasted (n = 4) or had edematous malnutrition (n = 3). Histology was assessed on hematoxylin and eosin stained slides. Mitochondrial and peroxisomal ultrastructural features were characterized using electron microscopy (EM) and immunofluorescence (IF). Results Hepatic steatosis was present in 50 % of non-wasted and severely wasted children and all children with edematous malnutrition. Edematous malnutrition was associated with 56 % and 45 % fewer mitochondria than severe wasting (p < 0.001) and no wasting (p = 0.006), respectively, and abnormal mitochondrial morphology compared to severe wasting (p = 0.002) and no wasting (p = 0.035). Peroxisomal abundance was reduced in edematous malnutrition compared to severe wasting (p = 0.005), but did not differ from no-wasting. Conclusion Edematous malnutrition is associated with reduced abundance and altered morphology of hepatic mitochondria and peroxisomes. Interventions targeting improvements in hepatic metabolic function may be beneficial in improving metabolism and reducing mortality in children with severe malnutrition, particularly in those with nutritional edema.

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier, where the cells play essential roles in the digestion of food, absorption of nutrients and water, protection from microbial infections, and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling, which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells, indeed, is linked to gastrointestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and, therefore, homeostasis. Here, we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol, which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover, analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn's Disease revealed that the dysregulation of peroxisome number, excessive cellular cholesterol, and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn's Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis.

Animal models have been utilized to understand the pathogenesis of Zellweger spectrum disorders (ZSDs); however, the link between clinical manifestations and molecular pathways has not yet been clearly established. We generated peroxin 5 homozygous mutant zebrafish (pex5−/−) to gain insight into the molecular pathogenesis of peroxisome dysfunction. pex5−/− display hallmarks of ZSD in humans and die within one month after birth. Fasting rapidly depletes lipids and glycogen in pex5−/− livers and expedites their mortality. Mechanistically, deregulated mitochondria and mechanistic target of rapamycin (mTOR) signaling act together to induce metabolic alterations that deplete hepatic nutrients and accumulate damaged mitochondria. Accordingly, chemical interventions blocking either the mitochondrial function or mTOR complex 1 (mTORC1) or a combination of both improve the metabolic imbalance shown in the fasted pex5−/− livers and extend the survival of animals. In addition, the suppression of oxidative stress by N-acetyl L-cysteine (NAC) treatment rescued the apoptotic cell death and early mortality observed in pex5−/−. Furthermore, an autophagy activator effectively ameliorated the early mortality of fasted pex5−/−. These results suggest that fasting may be detrimental to patients with peroxisome dysfunction, and that modulating the mitochondria, mTORC1, autophagy activities, or oxidative stress may provide a therapeutic option to alleviate the symptoms of peroxisomal diseases associated with metabolic dysfunction.

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the peroxisomal transporter ABCD1, resulting in the accumulation of very long-chain fatty acids (VLCFA). Strongly affected cell types, such as oligodendrocytes, adrenocortical cells and macrophages, exhibit high cholesterol turnover. Here, we investigated how ABCD1 deficiency affects cholesterol metabolism in human X-ALD patient-derived fibroblasts and CNS tissues of Abcd1-deficient mice. Lipidome analyses revealed increased levels of cholesterol esters (CE), containing both saturated VLCFA and mono/polyunsaturated (V)LCFA. The elevated CE(26:0) and CE(26:1) levels remained unchanged in LXR agonist-treated Abcd1 KO mice despite reduced total C26:0. Under high-cholesterol loading, gene expression of SOAT1, converting cholesterol to CE and lipid droplet formation were increased in human X-ALD fibroblasts versus healthy control fibroblasts. However, the expression of NCEH1, catalysing CE hydrolysis and the cholesterol transporter ABCA1 and cholesterol efflux were also upregulated. Elevated Soat1 and Abca1 expression and lipid droplet content were confirmed in the spinal cord of X-ALD mice, where expression of the CNS cholesterol transporter Apoe was also elevated. The extent of peroxisome-lipid droplet co-localisation appeared low and was not impaired by ABCD1-deficiency in cholesterol-loaded primary fibroblasts. Finally, addressing steroidogenesis, progesterone-induced cortisol release was amplified in X-ALD fibroblasts. These results link VLCFA to cholesterol homeostasis and justify further consideration of therapeutic approaches towards reducing VLCFA and cholesterol levels in X-ALD.

SLIT/ROBO signaling impacts many aspects of tissue development and homeostasis, in part, through the regulation of cell growth and proliferation. Recent studies have also linked SLIT/ROBO signaling to the regulation of diverse phagocyte functions. However, the mechanisms by which SLIT/ROBO signaling acts at the nexus of cellular growth control and innate immunity remain enigmatic. Here, we show that SLIT2-mediated activation of ROBO1 leads to inhibition of mTORC1 kinase activity in macrophages, leading to dephosphorylation of its downstream targets, including transcription factor EB and ULK1. Consequently, SLIT2 augments lysosome biogenesis, potently induces autophagy, and robustly promotes the killing of bacteria within phagosomes. Concordant with these results, we demonstrate decreased lysosomal content and accumulated peroxisomes in the spinal cords of embryos from Robo1 -/- , Robo2 -/- double knockout mice. We also show that impediment of auto/paracrine SLIT-ROBO signaling axis in cancer cells leads to hyperactivation of mTORC1 and inhibition of autophagy. Together, these findings elucidate a central role of chemorepellent SLIT2 in the regulation of mTORC1 activity with important implications for innate immunity and cancer cell survival.

Ig diversification occurs in peripheral lymphoid organs after establishment of central tolerance during B cell development. In germinal centers (GCs), somatic hypermutation of Ig genes occurs in dark zones, followed by selection of mutated clones in light zones (LZs). This generates high-affinity Ig receptors to pathogens but can also produce autoreactive Ig receptors, which are removed by selection mechanisms that are incompletely understood. The ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop potentially fatal autoimmune diseases; yet, how Itch regulates GC B cells is not well understood. By studying Itch-deficient mice, we have recently shown that Itch directly limits the magnitude of GC responses. Proteomic profiling of GC B cells uncovered that Itch-deficient cells exhibit high mTORC1 and Myc activity, hallmarks of positive selection. Bone marrow chimera and adoptive transfer experiments revealed that B cell Itch restricts noncycling LZ cells. These results support, to our knowledge, a novel role for Itch in skewing selection of GC B cells to restrict LZ accumulation and shape GC-derived humoral immunity. Determining how B cells integrate cues within GCs to navigate through LZs and dark zones will aid in understanding how autoreactive clones emerge from GCs in people with autoimmune disease.

Peroxisomes are rapidly degraded during amino acid and oxygen deprivation by a type of selective autophagy called pexophagy. However, how damaged peroxisomes are detected and removed from the cell is poorly understood. Recent studies suggest that the peroxisomal matrix protein import machinery may serve double duty as a quality control machinery, where they are directly involved in activating pexophagy. Here, we explored whether any matrix import factors are required to prevent pexophagy, such that their loss designates peroxisomes for degradation. Using gene editing and quantitative fluorescence microscopy on culture cells and a zebrafish model system, we found that PEX13, a component of the peroxisomal matrix import system, is required to prevent the degradation of otherwise healthy peroxisomes. The loss of PEX13 caused an accumulation of ubiquitinated PEX5 on peroxisomes and an increase in peroxisome-dependent reactive oxygen species that coalesce to induce pexophagy. We also found that PEX13 protein level is downregulated to aid in the induction of pexophagy during amino acid starvation. Together, our study points to PEX13 as a novel pexophagy regulator that is modulated to maintain peroxisome homeostasis.

Peroxisomes are essential for mitochondrial health, as the absence of peroxisomes leads to altered mitochondria. However, it is unclear whether the changes in mitochondria are a function of preserving cellular function or a response to cellular damage caused by the absence of peroxisomes. To address this, we developed conditional hepatocyte-specific Pex16 deficient ( Pex16 KO) mice that develop peroxisome loss and subjected them to a low-protein diet to induce metabolic stress. Loss of PEX16 in hepatocytes led to increased biogenesis of small mitochondria and reduced autophagy flux but with preserved capacity for respiration and ATP capacity. Metabolic stress induced by low protein feeding led to mitochondrial dysfunction in Pex16 KO mice and impaired biogenesis. Activation of PPARα partially corrected these mitochondrial disturbances, despite the absence of peroxisomes. The findings of this study demonstrate that the absence of peroxisomes in hepatocytes results in a concerted effort to preserve mitochondrial function, including increased mitochondrial biogenesis, altered morphology, and modified autophagy activity. Our study underscores the relationship between peroxisomes and mitochondria in regulating the hepatic metabolic responses to nutritional stressors.

Background The intestine of children with severe malnutrition (SM) shows structural and functional changes that are linked to increased infection and mortality. SM dysregulates the tryptophan-kynurenine pathway, which may impact processes such as SIRT1-and mTORC1-mediated autophagy and mitochondrial homeostasis. Using a mouse and organoid model of SM, we studied the repercussions of these dysregulations on malnutrition enteropathy and the protective capacity of maintaining autophagy activity and mitochondrial health.Methods SM was induced through feeding male weanling C57BL/6 mice a low protein diet (LPD) for 14-days. Mice were either treated with the NAD+-precursor, nicotinamide; an mTORC1-inhibitor, rapamycin; a SIRT1-activator, resveratrol; or SIRT1-inhibitor, EX-527. Malnutrition enteropathy was induced in enteric organoids through amino-acid deprivation. Features of and pathways to malnutrition enteropathy were examined, including paracellular permeability, nutrient absorption, and autophagic, mitochondrial, and reactive-oxygen-species (ROS) abnormalities.Findings LPD-feeding and ensuing low-tryptophan availability led to villus atrophy, nutrient malabsorption, and intestinal barrier dysfunction. In LPD-fed mice, nicotinamide-supplementation was linked to SIRT1-mediated activation of mitophagy, which reduced damaged mitochondria, and improved intestinal barrier function. Inhibition of mTORC1 reduced intestinal barrier dysfunction and nutrient malabsorption. Findings were validated and extended using an organoid model, demonstrating that resolution of mitochondrial ROS resolved barrier dysfunction.Interpretation Malnutrition enteropathy arises from a dysregulation of the SIRT1 and mTORC1 pathways, leading to disrupted autophagy, mitochondrial homeostasis, and ROS. Whether nicotinamide-supplementation in children with SM could ameliorate malnutrition enteropathy should be explored in clinical trials.Funding This work was supported by the Bill and Melinda Gates Foundation, the Sickkids Research Institute, the Canadian Institutes of Health Research, and the University Medical Center Groningen.Copyright (c) 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).