Diabetic foot ulcer is one of the most debilitating complications of diabetes mellitus, primarily driven by persistent inflammation and impaired angiogenesis. This study investigates the role of the circadian clock protein, nuclear receptor subfamily 1 group D member 1 (NR1D1), in diabetic wound healing and elucidates its underlying mechanism. Clinical and experimental analyses revealed that NR1D1 levels were significantly reduced in both the serum and cutaneous wounds of diabetic patients and mice, with down-regulated NR1D1 correlating with delayed wound closure. Functionally, NR1D1 knockout markedly exacerbated wound healing impairment in diabetic mice, accompanied by elevated proinflammatory cytokines and dysfunctional angiogenesis. Conversely, NR1D1 overexpression suppressed IL-6 and IL-1β expression and rescued high-glucose-induced vascularization deficits in vitro. Additionally, diminished NR1D1 expression coincided with hyperactivated stimulator of interferon genes (STING) signaling in both diabetic skin wounds and high-glucose-treated human umbilical vein endothelial cells. Luciferase assays confirmed that NR1D1 directly represses human STING promoter activity. Taken together, the results indicated that NR1D1 deficiency severely deteriorates wound healing by provoking inflammation and dysregulating endothelial angiogenesis, which is driven by the high-glucose-triggered derepression of NR1D1-mediated transcriptional silencing of STING.

The incidence of inflammatory bowel disease (IBD) continues to increase annually, which severely threatens public health. Although estimates indicate that current therapies cause a 30% IBD clinical treatment failure, the precise pathogenesis remains unclear. Here, it was found that a key embryonic development regulator, potassium channel tetramerization domain-containing 10 (KCTD10), is correlated with the severity of IBD. Enhanced KCTD10 expression was observed during enterocolitis. Intestinal epithelial cell-specific knockout of KCTD10 in mice impaired intestinal epithelial barrier function, activated intestinal adaptive immune response, and disrupted intestinal homeostasis. Furthermore, in a dextran sodium sulfate-induced ulcerative colitis model, intestinal epithelial cell deletion of KCTD10 exacerbated colonic inflammation and impaired postinjury repair. Mechanistically, KCTD10 deletion down-regulated intestinal stem cell marker genes and affected their function. Importantly, KCTD10 stabilizes the transcription factor ATOH1 by regulating its ubiquitination, thereby determining the fate of intestinal progenitor cells derived from leucine-rich repeat-containing G-protein-coupled receptor 5[+] intestinal stem cells. The data thus revealed a previously undescribed function of KCTD10 in modulating ulcerative colitis by regulating intestinal stem cell function.

The question of whether the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein persists in angiotensin converting-enzyme 2-positive (ACE2[+]) endothelia in the central nervous system (CNS)/other sites and induces a prolonged microvasculature injury syndrome was addressed. People who died of COVID-19, with age-matched controls, and people with long COVID with symptoms persisting from 1 to 5 years, with controls of people who had recovered from COVID-19, were studied. Also compared were wild-type mice injected with the spike protein euthanized at day 5 versus 1.5 months. The ACE2[+] microvasculature density in the CNS increased 19-fold in the mice exposed to spike protein for 1.5 months. Human fatal COVID-19 cases exhibited an ACE2[+] CNS density of 13.2/3.9 versus 2.1/0.9 for the controls. Long COVID cases showed an ACE2 microvasculature density of 11.9/3.0 versus 3.5/0.9 for the people who recovered from COVID (each, P < 0.001). Viral spike microvasculature density in the CNS was similar in the fatal COVID-19 cases (6.9/2.1) and mice (7.0/4.0) and in the skin of long COVID cases (6.0/1.9) and less in people who recovered from COVID-19 (0.9/0.3) (each, P < 0.001). Microvasculature collagen I deposition was only evident in fatal COVID-19, long COVID, and in the mice exposed to the S1 subunit for 1.5 months. Thus, SARS-CoV-2 spike (S1) protein can persist for months to years with an associated microvasculature pro-inflammatory state and a restrictive fibrosing microangiopathy, and exposure to high-dose S1 markedly increases ACE2 endothelial density.

Although a subset of nephrotic syndrome has been reported to be caused by autoantibodies against nephrin, its etiology remains poorly understood. A previous study demonstrated that anti-nephrin antibodies phosphorylate not only nephrin but also ephrin-B1 at the slit diaphragm of podocytes. However, the pathogenic role of ephrin-B1 in nephrotic syndrome remains unclear. The present study, using cultured human podocytes, demonstrated that direct stimulation of ephrin-B1 with soluble EphB2 or an anti-ephrin-B1 antibody induced phosphorylation of both ephrin-B1 and nephrin. Neither EphB2 nor the anti-ephrin-B1 antibody induced calcium influx, whereas the anti-nephrin antibody did. Phosphorylated ephrin-B1 dissociated from Par6, and the released Par6 subsequently interacted with Cdc42. This interaction promoted Cdc42 activation, leading to altered regulation of Snail and STAT3. These alterations resulted in decreased expression of nephrin and ephrin-B1 and induced claudin-1 expression. A Cdc42 inhibitor prevented both the down-regulation of nephrin and ephrin-B1 and the induction of claudin-1 expression. In contrast, a calcineurin inhibitor and an angiotensin II type 1 receptor blocker prevented the down-regulation of nephrin and ephrin-B1 but failed to prevent claudin-1 induction. This study proposed a novel potential etiology of nephrotic syndrome caused by direct stimulation of ephrin-B1. Targeting Cdc42 may represent a more effective therapeutic strategy for restoring podocyte homeostasis.

Peroxisomal β-oxidation processes substrate that mitochondria cannot handle. For example, C27 bile acid intermediates are subjected to β-oxidation to form C24 bile acids in peroxisomes rather than in mitochondria. Peroxisome proliferation is regulated by peroxisome proliferator-activated receptor α (PPARα), which is mediated by peroxisome membrane protein PEX16. Hepatocyte-specific PEX16-deficient (Pex16[Alb-Cre]) mice exhibit up-regulated PPARα signaling and display hepatocyte proliferation. It is unclear how hepatocytes proliferate in Pex16[Alb-Cre] mice. In this study, Pex16[Alb-Cre] mice were treated with the PPARα agonist WY-14,643 and the intestinal bile acid sequestrant cholestyramine. In addition to these pharmacologic interventions, Pex16[Alb-Cre] mice were subjected to abrogation of PPARα and farnesoid X receptor (FXR) by crossing with PPARα-absent mice and FXR-absent mice, respectively. The results showed that WY-14,643 did not further increase hepatocyte proliferation in Pex16[Alb-Cre] mice, but PPARα-absent Pex16[Alb-Cre] mice (Pparα[-/-]/Pex16[Alb-Cre]) exhibited increased hepatocyte proliferation. In contrast, FXR-absent Pex16[Alb-Cre] mice (FXR[-/-]/Pex16[Alb-Cre]) displayed reduced hepatocyte proliferation. Bile acids are FXR agonists. Consistently, serum bile acid levels are elevated in Pex16[Alb-Cre] mice, and feeding cholestyramine decreased serum bile acid levels and inhibited hepatocyte proliferation in Pex16[Alb-Cre] mice. These results suggest that hepatocyte proliferation in Pex16[Alb-Cre] mice is mediated by FXR but inhibited by PPARα.

In experimental autoimmune uveitis (EAU) mice, immune cells with an immunoregulatory phenotype are recruited to the surface of the avascular lens, traveling across ciliary zonule fibrils from the vasculature-rich ciliary body. It was also discovered that the lens epithelium harbors a population of tissue-resident immune cells, a cell type associated with inducing immune responses in response to danger signals in their environment, including recruitment of immune cells that function in immune surveillance and resolution of inflammation. In this study, the phenotype and localization of lens-resident immune cells (LR-ICs) are examined, their presence in adult mouse lenses is quantified, and whether there is a relationship between recruitment of lens capsule-associated immune cells (LCA-ICs) and the activation of LR-ICs in EAU is investigated. This study focused on the time period between induction of uveitis and the height of inflammation in the eyes of EAU mice. High-resolution confocal microscopy was performed on mouse eyes immunolabeled for molecules that detect activated resident immune cells or LCA-ICs. Quantification revealed that few LCA-ICs are recruited to the lens surface before the height of EAU inflammation, with the correlation between recruitment of immune cells to the lens capsule surface and the activation of LR-ICs occurring at the height of inflammation. These findings suggest LR-IC activation may contribute to regulation of inflammatory responses in the eye.

Clear-cell renal cell carcinoma (ccRCC), the most prevalent form of kidney cancer, is associated with an increased risk of local and systemic thromboembolism and a worse prognosis. The mechanisms that drive the coagulation program in tumors are not known. Combining RNAscope with immunofluorescence, local transcription of the key coagulation factors X and V, as well as prothrombin and fibrinogen chains (α, β, and γ), across tumor cells, vascular endothelial cells, and T cells is significantly enhanced in ccRCC tumors compared with adjacent normal kidney tissue. Elevated intratumoral protein expression of factors X and V is accompanied by local thrombin generation, fibrin deposition, and the presence of intravascular microthrombi, which themselves harbor coagulation transcripts. Mechanistic analysis using ccRCC organ cultures reveals that selective engagement of tumor necrosis factor receptor-2 (TNFR2) by a R2TNF mutein is sufficient to significantly enhance the transcription and protein expression of the same coagulation program in tumor cells, endothelial cells, and T cells compared with adjacent normal kidney cultures, whereas TNFR2 knockdown attenuates this response. In addition, TNFR2 appears to link inflammatory signaling with activation of the local coagulation system through the phosphatidylinositol 3-kinase/Akt/MEK/NF-κB signaling axes. These data provide evidence that locally produced coagulation factors driven by TNFR2 signaling may contribute to intratumoral prothrombotic outcomes in ccRCC, highlighting a novel pathway with potential therapeutic relevance.

There are no targeted therapeutic options for severe acute pancreatitis, largely owing to an incomplete understanding of the mechanisms involved in the transition from tissue injury to repair. Neutrophils are central effectors of severe acute pancreatitis, but their temporal contributions to injury and repair remain unresolved. In this study in ROSA26iDTR/MRP8-Cre mice, neutrophil depletion was timed to defined disease stages. Cerulein was administered to induce pancreatitis; neutrophil depletion was achieved using diphtheria toxin administered 0, 1, or 2 days later. The efficiency of neutrophil depletion was measured by flow cytometry. Depletion initiated on day 0 (early injury phase) reduced pancreatic injury by day 2, and histologic recovery from days 4 to 8 was more complete than in controls, a difference best explained by a reduced initial damage burden rather than a direct effect on the repair mechanism. Depletion initiated on day 1 (late injury phase) altered neither injury nor recovery. In contrast, depletion initiated on day 2 (regenerative phase) impaired tissue repair, with persistent acinar-to-ductal metaplasia, prolonged CD45[+] infiltration, and an expansion of CD86[+] cells (proinflammatory macrophages). Based on these findings, neutrophils may function sequentially as mediators of early pancreatic injury and as required contributors to subsequent resolution, supporting that neutrophil-directed therapy should be timed to suppress pathogenic activity while preserving repair.

Prediction of breast cancer biomarkers from routine formalin-fixed, paraffin-embedded (FFPE) hematoxylin and eosin (H&E) images has been explored previously, but whether biomarker-associated morphologic signals can be reliably captured from artifact-prone intraoperative frozen-section (FS) specimens remains insufficiently studied. This retrospective proof-of-concept study developed a foundation model-based multiple-instance learning framework to predict estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 status directly from H&E-stained intraoperative FS whole slide images of 248 patients with breast cancer, using postoperative immunohistochemistry and fluorescence in situ hybridization as ground truth. Exploratory feature evaluation identified UNI2-h as a suitable encoder for downstream modeling. In fivefold cross-validation, areas under the receiver operating characteristic curve were 0.847 for ER, 0.906 for PR, 0.893 for HER2, and 0.799 for Ki-67, with moderate agreement for ER, PR, and HER2 and fair agreement for Ki-67. An exploratory within-cohort cross-preparation analysis showed limited FFPE-to-FS transferability, whereas the FS-trained model retained relatively favorable performance on FFPE images. Attention heat maps highlighted tumor regions with biologically plausible biomarker-associated morphologic patterns. These findings provide proof of concept that intraoperative FS H&E images retain learnable biomarker-associated morphologic signals despite preparation-related alterations.

Neutrophils, the most prevalent leukocytes in circulation, are integral to initiating and resolving inflammatory responses. Although traditionally viewed as a homogeneous population, emerging studies have revealed significant phenotypic heterogeneity and functional versatility within the neutrophil population. However, developing a consensus on neutrophil markers for precise and comprehensive identification of neutrophil phenotypes at baseline is essential to deepen understanding of their biology. In this study, a combination of single-cell RNA sequencing and advanced multiplex flow cytometry analyses was used to examine the transcriptional and proteomic landscapes of human neutrophils in healthy peripheral blood while using functional assays to assess their migratory capabilities and phenotypic alterations after transmigration. Through transcriptional and proteomic analyses, the findings identify three distinct neutrophil profiles, each characterized by specific effector functions and migratory patterns. These subsets demonstrated distinction in the expression of markers related to neutrophil maturation, aging, immune modulation, and inflammatory responses, suggesting a complex spectrum of neutrophil states in circulation and after vascular transmigration. The findings advance the field's understanding of neutrophil heterogeneity and offer a more nuanced view of their roles in homeostasis and the immune response.

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases from simple steatosis to metabolic dysfunction-associated steatohepatitis and its associated fibrosis, cirrhosis, and MASLD-associated hepatocellular carcinoma (MASLD-HCC). MASLD affects over one-third of the global adult population and is closely associated with insulin resistance, obesity, genetic factors, and, increasingly recognized, gut microbiome dysbiosis. The gut-liver axis, referring to the communication network between the intestinal microbiota and liver, is critical to the progression of MASLD. The gut microbiota composition and/or functions are disrupted, leading to intestinal barrier destruction, systemic inflammation, and modulation of hepatic metabolism and immune responses. This review aims to summarize the effects of gut microbiota on MASLD and its malignant transition to associated HCC based on the roles of microbial metabolites, immune regulation, and associated genetic factors, and to discuss the potential application of microbiome-targeted therapeutic strategies, including probiotics and prebiotics, synbiotics and postbiotics, fecal microbiota transplantation, engineered bacteria and bacteriophage therapy, small-molecule inhibitors, and microbiota-derived metabolites, with the aim of providing a new vision in the treatment of MASLD and MASLD-HCC. Finally, current challenges in basic and clinical research of the role of microbiome in MASLD are discussed, and future directions are proposed to drive progress in this field.

Cholestatic liver diseases are characterized by impaired bile flow. Repairing damage and reestablishing biliary function may slow disease progression and reduce complications. The Wnt/β-catenin pathway can facilitate biliary repair during cholestasis through multiple mechanisms. The impact of constitutive Wnt/β-catenin signaling during cholestasis was analyzed using S45D transgenic (TG) mice, which express nondegradable β-catenin. Compared with wild type (WT), TG mice had increased inflammation and decreased fibrosis during prolonged cholestasis. Furthermore, TG mice had increased ductular reaction and hepatocyte reprogramming, which may alleviate injury. Because both Wnt/β-catenin and Yes-associated protein (Yap) pathways are implicated in cell plasticity, whether β-catenin works in concordance with or independent of Yap was investigated. In vitro studies demonstrated that Yap was not required for Wnt-mediated hepatocyte reprogramming. Spatial transcriptomics demonstrated suppression of Wnt/β-catenin activity in pericentral hepatocytes, whereas periportal Yap activation increased in early cholestasis. A dual reporter mouse model showed enhanced Wnt activation in both pericentral and periportal TG hepatocytes compared with WT, whereas Yap activation was equivalent in WT and TG. Transcriptomic analysis of human livers demonstrated that Wnt/β-catenin and Yap target genes also map to distinct cell compartments in cholestatic patients. These findings suggest that enhanced Wnt/β-catenin activation in TG mice accelerates compensatory responses and attenuates disease progression. Activation of Wnt/β-catenin and Yap in different cell types indicates that β-catenin functions independently of Yap.

Delayed corneal wound healing and sensory neuropathy are hallmarks of diabetic keratopathy, yet the role of corneal epithelial cell (CEC)-derived exosomes in these processes remains unclear. This study characterized exosomes secreted by the mouse corneal epithelial progenitor cell line TKE2 at either quiescent state or scratch wounded. The isolated extrasellar vesicles were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot analysis. Exosomal cargo was analyzed by liquid chromatography-tandem mass spectrometry proteomics. The roles of exosome and selected proteins were assessed for effects on wounded healing of normoglycemic and streptozotocin-induced diabetic C57BL/6J mice. The isolated vesicles exhibited canonical exosomal features and were readily taken up by CEC at 1 dpw and trigeminal sensory neurons at 3 dpw. Exosomes derived from healing, but not quiescent, cultures accelerated delayed epithelial wound closure in diabetic corneas, while having little effect on normoglycemic corneas. High-temperature requirement A serine peptidase 1, enriched in healing exosomes, contributed to epithelial wound healing in normal corneas, as its inhibition attenuated wound closure in normoglycemic but not diabetic mice. In contrast, treating healing exosomes with a STAT3 inhibitor, which permanently blocks its activity without altering protein content, partially reduced exosome-mediated wound healing in diabetic corneas. Collectively, these findings support context-dependent effects of CEC-derived exosomes in corneal wound repair and suggest that exosomal components may contribute to the regulation of diabetic corneal wound healing.

Epithelial-mesenchymal transition (EMT) of medial edge epithelial cells is a critical mechanism underlying embryonic palatal fusion, driven by transforming growth factor-β3 (TGF-β3). However, its regulatory interplay with pluripotency factors (SOX2/OCT4) and reliance on downstream cascades remain poorly understood. This study investigates these networks to elucidate the mechanisms of cellular reprogramming during palatogenesis. Embryonic day 15 C57BL6/J mouse palatal epithelial cells were isolated to eliminate mesenchymal interference. Cells were subjected to recombinant TGF-β3 (1, 3, and 10 ng/mL) or targeted siRNA knockdowns of Tgfb3, Sox2, Oct4, and Smad2/3, and evaluated via RNA sequencing, quantitative RT-PCR, immunofluorescence, transepithelial electrical resistance, and flow cytometry. Treatment with 1 to 3 ng/mL TGF-β3 successfully induced EMT in vitro, characterized by decreased E-cadherin, increased fibronectin, diminished membrane resistance, and a fibroblast-like morphologic shift. This transition was predominantly associated with noncanonical pathways; phosphorylated Smad2/3 levels remained unchanged, whereas Smad-independent mediators (p38 mitogen-activated protein kinase and Mmp13) were up-regulated (P < 0.05). Furthermore, TGF-β3 down-regulated the pluripotency markers Sox2 and Oct4 (P < 0.05). Combined Sox2/Oct4 knockdown with TGF-β3 yielded the most pronounced EMT characteristic. Although Tgfb3 knockdown heavily attenuated EMT, partial mesenchymal transition persisted, indicating redundant compensatory networks. In conclusion, optimal TGF-β3 concentrations (1 to 3 ng/mL) shift EMT toward Smad-independent pathways during palatogenesis. This reprogramming requires TGF-β3 to suppress SOX2 and OCT4, underlining their roles as negative regulators of EMT during palatal fusion.