Usutu virus (USUV) is an emerging mosquito-borne flavivirus known to induce neuroinvasive disease in birds, mice, and humans in European and African countries. The mechanisms of infection and dissemination remain poorly understood. Thus, elucidating how USUV spreads in a susceptible host is crucial for identifying therapeutic targets. To investigate host defenses against USUV, we generated an infectious clone of the TC508 isolate. After characterizing its replication dynamics in cultured cells from multiple species, we investigated its pathogenesis in an array of mice with genetic perturbations. Previous studies demonstrated that whole-body deletion of type I interferon (IFN) signaling led to widespread USUV infection and fatality in mice. Here, we observed the same lethal phenotype in STAT1-deficient mice and identified hematopoietic cells specifically as central to USUV pathogenesis in a mammalian host. Deletion of STAT1 in all hematopoietic subsets, but not hepatocytes, neurons, macrophages or conventional dendritic cells, was sufficient for systemic viral dissemination and ultimate fatality. Conversely, mice lacking functional B, T, and natural killer (NK) cells but with intact myeloid cells were resistant to USUV. Our findings provide new insights into the tissue-specific barriers that regulate USUV infection and underscore the importance of innate immunity in host defense for this important emerging flavivirus.

Inbred mice used for biomedical research display an underdeveloped immune system compared with adult humans, which is attributed in part to the artificial laboratory environment. Despite representing a central component of adaptive immunity, the impact of the laboratory environment on the B cell compartment has not been investigated in detail. Here, we performed an in-depth examination of B cells following rewilding, the controlled release of inbred laboratory mice into an outdoor enclosure. In rewilded mice, we observed B cells in circulation with increased signs of maturation, alongside heightened germinal center responses within secondary lymphoid organs. Rewilding also expanded B cells in the gut, which was accompanied by elevated systemic levels of immunoglobulin G (IgG) and IgM antibodies reactive to the microbiota. Our findings indicate that exposing laboratory mice to a more natural environment enhances B cell development to better reflect the immune system of free-living mammals.

BACKGROUND AND AIMS: Endoscopic sleeve gastroplasty (ESG) is a minimally invasive procedure for obesity, a risk factor for atherosclerotic cardiovascular disease (ASCVD). However, its impact on cardiovascular risk remains underexplored. This study evaluates ESG's effects on ASCVD risk and obesity-related comorbidities. METHODS: In this cohort study, 168 adults with obesity (aged 30-79 years; body mass index, >30 kg/ m2 or >27 kg/m2; with comorbidities) underwent ESG at a single tertiary care facility from 2013 to 2024. Patients were evaluated at baseline and 12 months after procedure using the Pooled Cohort Equations (PCEs) for patients aged 40-79 years and the Predicting Risk of Cardiovascular Disease Events (PRE-VENT) equations for those aged 30-79 years. Changes in body weight, glycemic control, blood pressure, lipid profile (total cholesterol, low-and high-density lipoprotein cholesterol, triglycerides), and liver enzymes (alanine aminotransferase) were also analyzed. RESULTS: Of the 168 patients (median age, 48 years; 74% female; body mass index, 36.0 kg/m2), 129 and 159 met inclusion criteria for PCEs and PREVENT equations, respectively. At 12 months, median PCEs ASCVD risk score decreased from 3.3% to 3.0% (21% reduction, P < 0.001), while the PREVENT 10-year cardiovascular disease risk score declined from 3.1% to 2.4% (20% reduction; P < 0.001), with similar reductions in 30-year risk scores and greater improvements in high/intermediate-risk patients and those aged >50 years. Patients achieved a median total body weight loss of 13.5% (IQR, 8.1%-18.6%), with significant metabolic improvements, and >80% experienced diabetes resolution. There was 1 (0.6%) moderate adverse event. CONCLUSION: Adults who underwent ESG showed significant improvements in ASCVD risk and obesity-related comorbidities at 12 months after procedure. These findings suggest that ESG may be an effective intervention for managing obesity and mitigating cardiovascular risk in patients with obesity. ClincialTrials.gov identifier: NCT04494048.

Chronic hepatitis B virus (HBV) infection is an incurable pathogen responsible for causing liver disease and hepatocellular carcinoma. During the genesis of infection, HBV establishes an independent minichromosome consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. The viral X gene must be expressed immediately upon infection to induce degradation of the host silencing factor, the Smc5/6 complex. However, the relationship between cccDNA chromatinization and X gene transcription remains poorly understood. By establishing a reconstituted viral minichromosome platform, we found that nucleosome occupancy in cccDNA regulates X transcription. We corroborated these findings in situ and further showed that the chromatin-destabilizing molecule CBL137 inhibits full-length X transcription and HBV infection in primary human hepatocytes. Our results shed light on a long-standing paradox and represent a potential therapeutic approach for the treatment of chronic HBV infection.

Orphan genes, which encode species-specific proteins, are common but are rarely investigated. The SARS-CoV-2 orphan gene, ORF10, has been understudied; in vitro work suggests it may modulate innate immunity. Whether ORF10 influences COVID-19 outcomes in humans remained unknown. Here, analyzing millions of SARS-CoV-2 genomes, we find ORF10 sequences are identical to ancestral Wuhan-Hu-1 haplotype. In all variants of concern, <5% of genomes carry any ORF10 mutation. Despite limited statistical power due to the sparsity of mutated sequences, four ORF10 mutations were associated with less severe clinical outcomes in COVID-19 patients: three affect protein structure, one alters RNA structural dynamics. No mutations were linked to increased severity. ORF10 transcript levels in humans and human models are conditionally discordant from other SARS-CoV-2 genes. ORF10 expression in A549 and 293T cells significantly perturbs oxidative phosphorylation gene expression, disrupts immune gene networks, and shifts expression of 14 novel human transcripts. ORF10 is present in multiple Betacoronavirus pandemicum strains, but absent from SARS-CoV-1-like strains. We propose that this strain-restricted orphan gene contributes to severe COVID-19 in humans, with implications for future therapeutic strategies. These findings underscore the importance of studying recently evolved, potentially overlooked, viral orphan genes as a standard approach in pandemic preparedness.

BACKGROUND AND AIMS:Aging-induced degenerative changes in the liver are not inherently pathologic but pose an increased risk for liver diseases. However, the molecular mechanisms underlying aging-induced hepatic dyshomeostasis remain incompletely characterized. Here, we investigate how aging alters liver architecture, cellular communication, and hepatocyte zonation. APPROACH AND RESULTS:Histological analyses of aged (>24-month-old) wild-type mouse livers showed no fibrosis, but a uniform cellular enlargement compared to young (2-month-old) mouse livers. For an unbiased characterization of aging-driven changes, we used single-nucleus RNA sequencing and found that aged livers had altered cell-cell interactions and hepatocyte zonation with zone-specific transcriptomic changes. Immunostaining confirmed aging-induced expansion of ASS1 + , CYP2E1 + , and GS + hepatic zones, and an aberrant expression of ASS1 + -GS + "bi-zonal" hepatocytes, causing loss of distinct zonation. Mechanistically, this breakdown was associated with downregulation of key zonation regulators ( Ctnnb1 , Foxo1 , Tcf7l2 ) and compensatory alterations in Wnt and Rspo3 signaling from non-parenchymal cells. To assess translational relevance, liver biopsies from young (≤25YO) and aged (>60YO) human donors were analyzed, revealing comparable zonal alterations and supporting the conservation of these aging-associated phenotypes across species. CONCLUSIONS:These findings reveal that aging causes loss of distinct hepatic zonation and alters intercellular communication through widespread transcriptional and architectural remodeling of liver cell types. The emergence of bi-zonal hepatocytes and expansion of hepatic zones in aged livers represent key hallmarks of hepatic aging. Our study provides new insights into mechanisms of liver aging and may inform therapeutic strategies targeting age-associated liver dysfunction.

BACKGROUND:Chronic alcohol drinking causes hepatic vitamin A (retinoids and derivatives) decreases, which correlate with the progression and severity of alcohol-associated liver disease (ALD). However, the effects of short-term ethanol (EtOH) intake on liver retinoids and ALD are still undefined. METHODS:Using high-performance liquid chromatography and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC, HPLC-MS/MS), and molecular biology techniques in mice and cultured human hepatocytes, we investigated the temporal EtOH effects on retinoids and ALD. RESULTS:In female and male mice, acute EtOH intake caused hepatic retinol (ROL) and retinyl palmitate (RP) decreases within hours, whereas it did not significantly change the retinoic acid (RA) levels, and those of the RA catabolism metabolite, 4-oxo-RA. After EtOH withdrawal, the liver recovered the ROL and RP levels within 48 h, whereas RA and 4-oxo-RA levels remained almost undetectable by this time point. Compared with control diet-fed mice, hepatic ROL and RP levels remained decreased in the 10-day and 3-week-long EtOH treatments, while retinyl oleate and linoleate increased. Interestingly, some of the RA signaling receptors, Rarβ, along with Cyp26a1, revealed dramatic transcript increases during the 10-day-long experiments that attenuated over time (up to 8 weeks), reflecting impaired RA signaling. Our work also showed that primary human hepatocytes serve as a model to better define the role of EtOH in retinoid biology. CONCLUSIONS:This work reveals that acute and short-term exposures to EtOH disrupt retinoid homeostasis, identifying key events in the early pathogenesis of ALD.

Despite ESG's efficacy in promoting weight loss, data on its impact on body composition is limited. This study evaluated ESG's effectiveness in improving body composition and metabolic parameters over 6 and 12 months using InBody analysis. We prospectively analyzed patients with obesity (BMI > 30 kg/m2 or > 27 with comorbidities) who underwent ESG between August 2021 and May 2024. Body composition was assessed via bioelectrical impedance analysis (InBody 770™). Thirty-six patients (81

RATIONALE: Seasonal and epidemic influenza is associated with particularly high mortality in elderly patients. Severe influenza infection is associated with multiorgan dysfunction, including liver injury, which portends increased risk of debility and death. We hypothesized that changes in hepatic lipid metabolism during influenza infection contribute to liver injury and metabolic derangements in the aged host. METHODS: We inoculated young and aged mice with influenza intranasally and assessed metabolic parameters, liver histology, and liver gene expression by RNA-seq in mice infected for up to 7 days. RESULTS: We found that influenza-infected mice underwent marked adaptations of liver metabolism that supported fatty acid uptake and beta oxidation, likely due to glucocorticoid coactivation of peroxisome proliferator-activated receptor alpha targets. In aged mice, this response was biased toward lipid storage over beta oxidation, and significant hepatic triglyceride accumulation developed. This was associated with lower serum triglyceride levels and a reduced hyperglycemic response to infection in aged mice, which also developed increased glucagon levels suggestive of a starvation response. Aged mice had mild baseline steatosis and a depleted Kupffer cell niche prior to infection, and influenza infection led to repopulation of the liver with recruited myeloid cells, which may have a role in modulating hepatocyte metabolism. CONCLUSION: Our findings suggest that influenza respiratory infection leads to a metabolic shift to fat uptake and oxidation in the liver, and this transition is impaired in aged mice leading to excess fat storage and inflammation. Aged mice develop markers of a starvation response and liver injury with myeloid cell recruitment, which may reflect the biological underpinnings of the accelerated catabolic response and extrapulmonary organ injury seen in severe influenza infection in the aged host.

The Spatial Atlas of Human Anatomy (SAHA) represents the first multimodal, subcellular-resolution reference of healthy adult human tissues across multiple organ systems. Integrating spatial transcriptomics, proteomics, and histological features across over 15 million cells from more than 100 donors, SAHA maps conserved and organ-specific cellular niches in gastrointestinal and immune tissues. High-resolution profiling using CosMx SMI, 10x Xenium, RNAscope, GeoMx DSP, and single-nucleus RNA-seq reveals spatially organized cell states, rare adaptive immune populations, and tissue-specific cell-cell interactions and ligand-receptor pairs. Comparative analyses with colorectal cancer and inflammatory bowel disease demonstrate the power of SAHA to detect disease-associated spatial disruptions, including crypt dedifferentiation, perineural invasion, and therapy-resistant immune remodeling. All data are openly accessible through a FAIR-compliant interactive portal to support exploration, benchmarking, and machine learning model training. Through SAHA, we provide a foundational framework for spatial diagnostics and next-generation precision medicine grounded in a comprehensive human tissue atlas, enabling the development of context-aware models that simulate tissue behavior, decode complex pathologies, and accelerate therapeutic innovation at unprecedented scale.

COVID-19 patients commonly present with signs of central nervous system and/or peripheral nervous system dysfunction. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively susceptible and permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 infection of DA neurons triggers an inflammatory and cellular senescence response. High-throughput screening in hPSC-derived DA neurons identified several FDA-approved drugs that can rescue the cellular senescence phenotype by preventing SARS-CoV-2 infection. We also identified the inflammatory and cellular senescence signature and low levels of SARS-CoV-2 transcripts in human substantia nigra tissue of COVID-19 patients. Furthermore, we observed reduced numbers of neuromelanin+ and tyrosine-hydroxylase (TH)+ DA neurons and fibers in a cohort of severe COVID-19 patients. Our findings demonstrate that hPSC-derived DA neurons are susceptible to SARS-CoV-2, identify candidate neuroprotective drugs for COVID-19 patients, and suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.

There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single-cell RNA sequencing (scRNA-seq) analysis of human islets exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and β cell pyroptosis. To distinguish viral versus proinflammatory-macrophage-mediated β cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both β cells and endothelial cells compared with separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced β cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory-macrophage-mediated β cell pyroptosis. This study established hPSC-derived VMI organoids as a valuable tool for studying immune-cell-mediated host damage and uncovered the mechanism of β cell damage during viral exposure.