Adenosine deaminase acting on RNA 1 (ADAR1) regulates mRNA fate and function through adenosine-to-inosine (A-to-I) RNA editing and RNA-binding activities. While its role in innate immunity is established, the broader regulatory functions of ADAR1 in macrophages remain poorly defined. Here, we systematically profiled ADAR1 expression across human immune cells and identified marked enrichment in macrophages, driven by selective usage of an alternative transcription start site during monocyte-to-macrophage differentiation. ADAR1 binds, edits, and modulates key macrophage targets involved in efferocytosis, endocytosis, lysosomal processing, lipid metabolism, and proliferation in an isoform-specific manner. We further demonstrate that ADAR1 levels and activity are dynamically regulated in adipose tissue and liver during the progression of metabolic disease. Linked to this, macrophage-specific ablation of ADAR1 co-cultured in organotypic 3D primary human liver spheroids and exposed to metabolic stress resulted in an exacerbated lipid accumulation phenotype. Finally, we identify a lipid-associated macrophage-specific upregulation of ADAR1 in adipose tissue following weight loss interventions, mechanistically driven by free fatty acids. These findings uncover a previously unrecognized role for ADAR1 in lipid-buffering, scavenging, and proliferative macrophage functions, extending its biological relevance beyond canonical interferon-mediated immunity and establishing ADAR1 as a key regulator of macrophage adaptation in metabolic disease.

Neuroblastoma, the most prevalent extracranial pediatric solid tumor, arises from neural crest progeny cells. It exhibits substantial developmental plasticity and intratumoral heterogeneity, leading to survival rates below 50% in high-risk cases. The regulatory mechanisms underlying this plasticity remain largely elusive. In this integrative study, we used single-cell MultiOmics from a mouse spontaneous tumor model and spatial transcriptomics from human patient samples to dissect the transcriptional and epigenetic landscapes that govern developmental states in neuroblastoma. We identified developmental intermediate states in high-risk neuroblastomas critical for malignant transitions and uncovered extensive epigenetic priming with latent capacity for diverse state transitions. Furthermore, we mapped enhancer gene regulatory networks (eGRNs) and tumor microenvironments sustaining these aggressive states. State transitions and malignancy could be interfered with by targeting transcription factors controlling the eGRNs.

While single-cell multi-omics has advanced our understanding of cellular heterogeneity, the complexity of these datasets remains a barrier for non-computational users. Here, we introduce GUANACO, a Dash-based Python package for interactive, code-free visualization of single-cell RNA-seq and ATAC-seq data. GUANACO integrates matrix- and genome-track views, supporting flexible cell/gene selection, statistical testing, and transcription factor binding site exploration. Its user-friendly interface offers colorblind-friendly palettes, intuitive controls, and options for generating publication-ready figures. With a low memory requirement and cost-effective deployment, GUANACO facilitates seamless sharing and reproducible research, empowering researchers to explore, interpret, and communicate single-cell insights without extra coding. ### Competing Interest Statement The authors have declared no competing interest. Research Council of Finland, 357061 Sigrid Jusélius Foundation, 240021 Swedish Research Council, 2024-03620 CIMED Region Stockholm, FoUI-976025 Karolinska Institutet Research Grant, 2024-02719 Åke Wibergs Stiftelse, M23-0072, M24-0263

Background Branching morphogenesis orchestrates organogenesis in many tissues including kidney, where ureteric bud branching determines kidney size and nephron number. Defects in branching morphogenesis result in congenital renal anomalies which manifest as deviations in size, function, and nephron number thus critically compromising the lifelong renal functional capacity established during development. Advances in the genetic and molecular understanding of ureteric bud branching regulation have proved insufficient to improve prognosis of congenital renal defects. Thus, we addressed mechanisms regulating three-dimensional (3D) ureteric bud epithelial cell morphology and cell shape changes during novel branch initiation to uncover the contributions of cellular mechanics on cellular functions and tissue organization in normal and branching-compromised bud tips.Methods We explored epithelial cell behavior at all scales by utilizing a combination of mouse genetics and a custom machine-learning segmentation pipeline in MATLAB. Ureteric bud epithelial cell shapes and sizes were quantified in 3D wholemount kidneys. A combination with live imaging of fluorescently labelled UB cells, traction force microscopy, and primary UB cells were used to determine how basic cellular features and niche biomechanics contribute to complex novel branch point determination in the process that aims at gaining optimal growth and epithelial density in a limited space.Results Machine learning-based segmentation of tip epithelia identified geometrical round-to-elliptical transformation as a key cell shape change facilitating shifts in growth direction that enable propitious branching complexity. Cell shape and molecular analyses in branching-compromised epithelia demonstrated a failure to condense cell size and conformation. Analysis of branching-compromised ureteric bud derived epithelial cells demonstrated disrupted E-CADHERIN and PAXILLIN mediated adhesive forces and defective cytoskeletal dynamics as detected by fluorescent labelling of actin in primary ureteric bud epithelial cells. Branching-compromised ureteric bud epithelial cells showed wrinkled nuclear shapes and alterations in MYH9-based microtubule organization, which suggest a stiff cellular niche with disturbed sensing of and response to biomechanical cues.Conclusions Our results indicate that the adhesive forces within the epithelium and towards the niche composed of nephron progenitors must dynamically fluctuate to allow complexity in arborization during new branch formation. The data collectively propose a model where epithelial cell crowding in tandem with stretching transforms individual cells into elliptical and elongated shapes. This creates local curvatures that drive new branch formation during the ampulla-to-asymmetric ampulla transition of ureteric bud.### Competing Interest StatementThe authors have declared no competing interest.

Aging clocks, built from comprehensive molecular data, have emerged as promising tools in medicine, forensics, and ecological research. However, few studies have compared the suitability of different molecular data types to predict age in the same cohort and whether combining them would improve predictions. Here, we explored this at the level of proteins and small RNAs in 103 human blood plasma samples. First, we used a two-step mass spectrometry approach measuring 612 proteins to select and quantify 21 proteins that changed in abundance with age. Notably, proteins increasing with age were enriched for components of the complement system. Next, we used small RNA sequencing to select and quantify a set of 315 small RNAs that changed in abundance with age. Most of these were microRNAs (miRNAs), downregulated with age, and predicted to target genes related to growth, cancer, and senescence. Finally, we used the collected data to build age-predictive models. Among the different types of molecules, proteins yielded the most accurate model (R² = 0.59 ± 0.02), followed by miRNAs as the best-performing class of small RNAs (R² = 0.54 ± 0.02). Interestingly, the use of protein and miRNA data together improved predictions (R2 = 0.70 ± 0.01). Future work using larger sample sizes and a validation dataset will be necessary to confirm these results. Nevertheless, our study suggests that combining proteomic and miRNA data yields superior age predictions, possibly by capturing a broader range of age-related physiological changes. It will be interesting to determine if combining different molecular data types works as a general strategy to improve future aging clocks.

ABSTRACT Perturbed secretion of insulin and other pancreatic islet hormones is the main cause of type 2 diabetes (T2D). The islets harbor five cell types that are potentially altered differently by T2D. Whole-islet transcriptomics and single-cell RNA-sequencing (scRNAseq) studies have revealed differentially expressed genes without reaching consensus. Here, we demonstrate that unprecedented insights into disease mechanisms can be obtained by network-based analysis of scRNAseq data. We developed differential gene coordination network analysis (dGCNA) and analyzed islet scRNAseq data from 16 T2D and 16 non-T2D individuals. dGCNA revealed T2D-induced cell type-specific networks of dysregulated genes with remarkable ontological specificity, thus allowing for a comprehensive and unbiased functional classification of genes involved in T2D. In beta cells eleven networks of genes were detected, revealing that mitochondrial electron transport chain, glycolysis, cytoskeleton organization, cell proliferation, unfolded protein response and three networks of beta cell transcription factors are perturbed, whereas exocytosis, lysosomal regulation and insulin translation programs are instead enhanced in T2D. Furthermore, we validated the ability of dGCNA to reveal disease mechanisms and predict the functional context of genes by showing that TMEM176A/B regulates the beta cell cytoskeleton and that CEPBG is a key regulator of the unfolded protein response. In addition, comparing beta- and alpha and cells, we found substantial differences, reproduced across independent datasets, confirming cell type-specific alterations in T2D. We conclude that analysis of networks of differentially coordinated genes provides outstanding insight into cell type-specific gene function and T2D pathophysiology.

Although multiple populations of macrophages have been described in the human liver, their function and turnover in patients with obesity at high risk of developing non-alcoholic fatty liver disease (NAFLD) and cirrhosis are currently unknown. Herein, we identify a specific human population of resident liver myeloid cells that protects against the metabolic impairment associated with obesity. By studying the turnover of liver myeloid cells in individuals undergoing liver transplantation, we find that liver myeloid cell turnover differs between humans and mice. Using single-cell techniques and flow cytometry, we determine that the proportion of the protective resident liver myeloid cells, denoted liver myeloid cells 2 (LM2), decreases during obesity. Functional validation approaches using human 2D and 3D cultures reveal that the presence of LM2 ameliorates the oxidative stress associated with obese conditions. Our study indicates that resident myeloid cells could be a therapeutic target to decrease the oxidative stress associated with NAFLD.

Supplementary Figure S1. Outcome of PSFinder. Supplementary Figure S2. Kaplan-Meier survival curves. Supplementary Figure S3. Kaplan-Meier survival curves on time to progression in month. Supplementary Figure S4. Expression boxplot of validated transcript markers. Supplementary Figure S5. Promoter methylation changes. Supplementary Figure S6. Expression of genes affected by promoter hyper- and hypo- methylation. Supplementary Figure S7. DNMT3B expression. Supplementary Figure S8. Decitabine treatment result. Supplementary Figure S9. Kaplan-Meier survival curves on overall survival time. Tables Supplementary Table S1. PSFinder identified 61 transcripts at isoform-level for Poor I, Poor II and Good prognostic patient characterization. Supplementary Table S2. PSFinder identified 32 genes at gene-level for Poor I, Poor II and Good prognostic patient characterization. Supplementary Table S3. CpG loci with promoter hypermethylation (N=32) and hypomethylation (N=1) in Poor I patients. Supplementary Table S4. Primers and probes used in Taqman qRT-PCR. Supplementary Table S5. PSFinder identified subgroups and BRCA1/2 mutation status in patients from the TCGA discovery set. Supplementary Table S6. LPS predicted subgroups and BRCA1/2 mutation status in patients from the TCGA validation set.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Adipocyte hypertrophy and expression of adipokines in subcutaneous adipose tissue (SAT) have been linked to steatosis, nonalcoholic steatohepatitis (NASH) and fibrosis in morbidly obese (BMI ≥ 40 kg/m2) subjects. It is unknown if this is also true for subjects with NAFLD with lesser degrees of obesity (BMI < 35 kg/m2). Thirty-two subjects with biopsy-proven NAFLD and 15 non-diabetic controls matched for BMI underwent fine-needle biopsies of SAT. Adipocyte volume was calculated. RNA-sequencing of SAT was performed in a subset of 20 NAFLD patients. Adipocyte volume and gene expression levels were correlated to the presence of NASH or significant fibrosis. Subjects with NAFLD had larger adipocyte volume compared with controls, (1939 pL, 95% CI 1130–1662 vs. 854 pL, 95% CI 781–926, p < 0.001). There was no association between adipocyte volume and the presence of NASH. Gene expression of adipokines previously described to correlate with NASH in morbid obesity, was not associated with NASH or fibrosis. Our results suggest that persons with NAFLD have larger SAT adipocytes compared with controls and that adipocytes are involved in the pathophysiology of hepatic steatosis in NAFLD. However, adipocyte volume was not associated with NASH or fibrosis in NAFLD subjects with varying degrees of obesity.

pup growth, serum BCAA levels through mass spectrometry, DBT protein expression via immunoblotting and vector genome copy via digital droplet PCR.Immunohistochemistry was used to assess presence of DBT protein in liver sections.As neonatal vector administration improved survival but did not rescue all pups, the timing of vector delivery was suspected to be critical, and the same vector was administered in utero to pups on E15.Equivalent studies are being initiated to compare outcomes following in utero treatment.Results: Gene addition with a codon optimised DBT human transgene significantly improved survival, growth and BCAA levels ( p < 0.05) compared to untreated knockout pups when administered on D0 of life.DBT protein in vector treated homozygous knock out pups was expressed at 2.5 times higher than untreated wild type pups.Immunohistochemistry demonstrated presence of DBT protein in vector treated pups (Figure 1).We will also present the results from the in utero vector administration.Figure 1.Presence of DBT protein ( purple) in murine liver cells in A) untreated DBT -/-and B) D0 vector treated DBT -/-mouse pups.Hepatocyte nuclei were co-stained (blue, DAPI).Conclusion: Neonatal murine liver transduction with a human DBT transgene significantly improves the disease phenotype in a murine neonatal lethal model of MSUD.Liver directed AAV mediated gene therapy represents a potential novel treatment for MSUD.

Macrophages have diverse phenotypes and functions due to differences in their origin, location and pathophysiological context. Although their main role in the liver has been described as immunoregulatory and detoxifying, changes in macrophage phenotypes, diversity, dynamics and function have been reported during obesity-related complications such as non-alcoholic fatty liver disease (NAFLD). NAFLD encompasses multiple disease states from hepatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocarcinoma. Obesity and insulin resistance are prominent risk factors for NASH, a disease with a high worldwide prevalence and no approved treatment. In this Review, we discuss the turnover and function of liver-resident macrophages (Kupffer cells) and monocyte-derived hepatic macrophages. We examine these populations in both steady state and during NAFLD, with an emphasis on NASH. The explosion in high-throughput gene expression analysis using single-cell RNA sequencing (scRNA-seq) within the last 5 years has revolutionized the study of macrophage heterogeneity, substantially increasing our understanding of the composition and diversity of tissue macrophages, including in the liver. Here, we highlight scRNA-seq findings from the last 5 years on the diversity of liver macrophages in homeostasis and metabolic disease, and reveal hepatic macrophage function beyond their classically described inflammatory role in the progression of NAFLD and NASH pathogenesis.

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206loESAM- population (KC1) and a minor CD206hiESAM+ population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions.

[This corrects the article DOI: 10.7150/thno.15007.].

Abstract Rectal indomethacin and diclofenac are promising drugs for prevention of post‐endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP). However, their prophylactic effect on PEP in average‐risk patients remains controversial. We performed a systematic review and meta‐analysis to assess the efficacy and safety of rectal indomethacin and diclofenac in average‐risk patients, and to indirectly compare the prophylactic effect of the two drugs. A comprehensive search of the PubMed, EMBASE, and Cochrane Library databases was performed to identify randomized controlled trials (RCTs) on rectal indomethacin or diclofenac for prophylaxis against PEP. Fixed‐ and random‐effects models weighted by the Mantel–Haenszel method were used for direct comparisons. The adjusted indirect treatment comparison method was used to indirectly compare the efficacy of indomethacin and diclofenac. A total of 10 RCTs, including 2928 patients, met our inclusion criteria. No significant publication bias was identified. Pooled estimates showed that rectal indomethacin and diclofenac were associated with a significant reduction in the overall risk of PEP compared with control intervention [relative risk (RR) = 0.62; 95% confidence interval (CI): 0.46–0.83] in average‐risk patients. Subgroup analyses showed that both rectal indomethacin (RR = 0.67; 95% CI: 0.49–0.94) and diclofenac (RR = 0.42; 95% CI: 0.23–0.75) were effective in the prevention of PEP. Indirect comparison showed no significant difference between the effectiveness of the two drugs in the prevention of PEP (RR = 1.607; 95% CI: 0.824–3.136). The updated meta‐analysis suggests that both drugs provide equivalent protection against PEP in average‐risk patients.

Obesity is considered an important factor for many chronic diseases, including diabetes, cardiovascular disease and cancer. The expansion of adipose tissue in obesity is due to an increase in both adipocyte progenitor differentiation and mature adipocyte cell size. Adipocytes, however, are thought to be unable to divide or enter the cell cycle. We demonstrate that mature human adipocytes unexpectedly display a gene and protein signature indicative of an active cell cycle program. Adipocyte cell cycle progression associates with obesity and hyperinsulinemia, with a concomitant increase in cell size, nuclear size and nuclear DNA content. Chronic hyperinsulinemia in vitro or in humans, however, is associated with subsequent cell cycle exit, leading to a premature senescent transcriptomic and secretory profile in adipocytes. Premature senescence is rapidly becoming recognized as an important mediator of stress-induced tissue dysfunction. By demonstrating that adipocytes can activate a cell cycle program, we define a mechanism whereby mature human adipocytes senesce. We further show that by targeting the adipocyte cell cycle program using metformin, it is possible to influence adipocyte senescence and obesity-associated adipose tissue inflammation.

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb -expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.

Large-scale sequencing of RNA from individual cells can reveal patterns of gene, isoform and allelic expression across cell types and states1. However, current short-read single-cell RNA-sequencing methods have limited ability to count RNAs at allele and isoform resolution, and long-read sequencing techniques lack the depth required for large-scale applications across cells2,3. Here we introduce Smart-seq3, which combines full-length transcriptome coverage with a 5′ unique molecular identifier RNA counting strategy that enables in silico reconstruction of thousands of RNA molecules per cell. Of the counted and reconstructed molecules, 60% could be directly assigned to allelic origin and 30–50% to specific isoforms, and we identified substantial differences in isoform usage in different mouse strains and human cell types. Smart-seq3 greatly increased sensitivity compared to Smart-seq2, typically detecting thousands more transcripts per cell. We expect that Smart-seq3 will enable large-scale characterization of cell types and states across tissues and organisms. Smart-seq3 enables isoform- and allele-specific reconstruction of RNA molecules.