Congenital heart disease (CHD) combined with anomalies of the kidney and urinary tract (CAKUT) belongs to severe clinical conditions that represent isolated cases or can be part of complex inherited syndromes. To date, knowledge of the genetic basis of cardio-renal birth defects is very limited. Using chromosomal microarray analysis and exome sequencing, we investigated the spectrum of copy number variations (CNVs) and point genetic variants in a pediatric cohort of 30 patients presenting with combined CHD and CAKUT. Two patients possessed a pathogenic deletion in the 22q11.2 genomic region, well-known as the DiGeorge/velocardiofacial syndrome locus. Four other patients harbored rare copy number gains, which included one of the dosage-sensitive genes: DHFR (5q14.1), NPHP1 (2q13), NNT (5p12), and MCTP2 (15q26.2). In six children, novel or previously described causative variants were detected in the genes associated with orphan monogenic disorders such as Kabuki (KMT2D), Noonan (PTPN11), Adams-Oliver (NOTCH1), and Alagille (JAG1) syndromes, as well as SALL4- and MED13-related syndromes. Furthermore, potentially disease-contributing variants were identified in the genes that primarily code for cardiac transcription factors, chromatin remodeling proteins, Notch- or BMP-signaling molecules, and components of cilia. Notably, two children had loss-of-function variants in ROBO1, encoding one of the receptors of the SLIT/ROBO signaling pathway. In vitro studies on cardiac mesenchymal cells demonstrated a dramatically diminished expression level of ROBO1 in a patient with novel compound heterozygous variants c.541_542del (p.Val181CysfsTer18) and c.657+4A>G and a moderately reduced ROBO1 expression in a patient with a heterozygous stop-gain variant c.2758C>T (p.Arg920Ter), which suggests a haploinsufficiency mechanism. Together, our findings underlined the utility of early cytogenetic screening for the 22q11.2 deletion among children with cardio-renal malformations and confirmed the importance of rare single-nucleotide variants in the genes involved in cardiogenesis and kidney development. In particular, ROBO1 can be regarded as a candidate gene associated with combined heart and kidney structural defects and should be included in appropriate diagnostic gene-sequencing panels. The results support the usefulness of WES and array-based comparative genomic hybridization (array-CGH) analysis for new candidate gene identification, more accurate diagnosis, and family genetic counseling.
IntroductionCurrent tumor classification systems based on histology and molecular subtypes inadequately capture the functional complexity of cancer biology. Tumors frequently reactivate embryonic developmental programs, becoming dependent on master regulators that govern pluripotency, differentiation and tissue morphogenesis. This developmental reactivation creates architectural vulnerabilities in gene regulatory networks that can be therapeutically exploited.MethodsWe propose DNV-TC, a four-dimensional classification system that stratifies tumors according to the reactivated developmental program, network architectural vulnerability, cascade expansion phenotype and metastatic propensity. The system integrates developmental biology with network medicine through three quantitative indices computed on multi-layer gene regulatory networks: the tumor developmental regulatory impact (T-DRI), tumor disease network vulnerability (TD-NV) and cascade expansion index-tumor (CEI-T). DNV-TC was applied retrospectively to representative tumor types using published molecular and clinical data.ResultsTumors with high network vulnerability showed marked responses to targeted therapies (87% response rate) but rapidly developed resistance (median 6–8 months), while tumors with reactivated pluripotency programs displayed cancer stem-cell characteristics and therapeutic resistance. Network vulnerability class retained independent prognostic value after adjustment for stage, grade and age (HR 2.3, 95% CI 1.8–2.9, p < 0.001), and metastatic propensity class outperformed TNM staging for 5-year metastasis-free survival prediction (AUC 0.842 versus 0.712, p < 0.001).DiscussionDNV-TC provides a mechanistically grounded classification system that bridges developmental biology and precision oncology, thus enabling rational selection of therapeutic targets and combination strategies. The present analyses are retrospective and in silico, and prospective external validation is required.
BackgroundLiver cancer has high incidence and mortality worldwide, and timely identification is important for improving prognosis. However, prediction models based on routinely available clinical data remain insufficiently evaluated in hospitalized real-world populations. This study aimed to develop and interpret a machine-learning model for liver cancer prediction using multidimensional clinical and laboratory features.MethodsThis retrospective real-world cohort included 9,284 hospitalized patients from Hainan General Hospital between January 2020 and December 2023. Patients were divided into a training set (n = 6,426) and an internal validation set (n = 2,858). Seventy-four demographic, clinical, and laboratory variables were collected. Feature selection was performed using least absolute shrinkage and selection operator regression in the training set. Six models were developed: logistic regression, random forest, support vector machine, k-nearest neighbors, extreme gradient boosting (XGBoost), and Elastic Net. Discrimination was assessed primarily using the area under the receiver operating characteristic curve (AUC). Decision curve analysis evaluated clinical net benefit, and SHapley Additive exPlanations (SHAP) were used for model interpretation.ResultsLiver cancer accounted for 48.7% of the training cohort and 48.8% of the validation cohort. LASSO retained 57 nonzero model terms at the minimum-error penalty. XGBoost achieved the highest AUC among all models, with an AUC of 0.909 (95% CI, 0.903–0.915) in the training set and 0.802 (95% CI, 0.788–0.817) in the validation set. At a threshold of 0.5, validation accuracy, sensitivity, specificity, and F1 score were 0.730, 0.758, 0.698, and 0.753, respectively. Decision curve analysis showed that XGBoost provided the greatest net clinical benefit across a broad range of threshold probabilities. SHAP analysis identified serum sialic acid, the aspartate aminotransferase-to-alanine aminotransferase ratio, alkaline phosphatase, basophil percentage, and monocyte-to-lymphocyte ratio as the leading predictors.ConclusionXGBoost showed good discrimination and interpretability for liver cancer prediction in a hospitalized real-world cohort using routinely available clinical and laboratory data. The findings support the feasibility of leveraging large-scale inpatient laboratory data for risk-stratification model development. Serum sialic acid, the aspartate aminotransferase-to-alanine aminotransferase ratio, and alkaline phosphatase were important predictors. Prospective validation in outpatient, first-visit, high-risk, and external populations is required before broader clinical or screening application.
BackgroundProgenitor cells generate multiple cells during development and are thought to be involved in tissue repair. The widespread distribution of Kidney-specific cadherin-positive (KSP+) cells in the kidney suggests they play an important role in renal development. However, direct evidence demonstrating that KSP+ cells function as progenitor cells contributing to renal development and repair is lacking.MethodsTo obtain direct evidence for the differentiation and regenerative potential of KSP+ cells, we performed in vivo lineage tracing using Ksp-Cre and Rosa-LSL-tdTomato (RFPf/f) transgenic mice, combined with thymidine analog (5-chloro-2′-deoxyuridine, CldU) labeling in adult wild-type (WT) mice. Lineage tracing was analyzed from embryonic day 14.5 (E14.5) to adulthood by triple immunofluorescence staining for KSP, red fluorescent protein (RFP), and nephron segment-specific markers. Unilateral ureteral obstruction (UUO) was performed to investigate the regenerative potential of KSP+ cells following renal injury. Proliferative capacity was assessed by CldU/Ki67/PCNA labeling from days 2–14 post-surgery. To explore the molecular mechanism potentially underlying KSP+ cell-mediated repair, we analyzed Notch signaling pathway in UUO-subjected WT mice.ResultsThe Ksp-Cre: RFPf/f lineage-tracing model exhibited high efficiency and specificity, with RFP expression strictly confined to KSP+ cells at E14.5. Lineage tracing revealed that embryonic KSP+ cells function as progenitor cells, giving rise to cells in the formation of multiple nephron segments, including proximal tubules (PTs), loops of Henle (HLs), distal convoluted tubules (DCTs), and collecting ducts (CDs) during kidney development. Comparison of the differentiation capacity of KSP+ cells during embryonic versus postnatal development indicated that KSP+ cells preferentially contribute to postnatal kidney development. In adult kidneys under homeostatic conditions, KSP+ cells showed limited proliferative potential. However, following UUO injury, KSP+ cells exhibited markedly increased proliferation. Notably, the proliferative response of KSP+ cells was closely correlated with Notch activation.ConclusionOur lineage tracing data supports the KSP+ lineage behaves as a multipotent progenitor population. These cells give rise to cells of multiple nephron segments and preferentially drive postnatal kidney development. In adult kidneys, KSP+ cells are quiescent under homeostasis but become highly proliferative after renal injury, and this proliferative response is closely associated with activation of the Notch signaling pathway.
BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterized by marked molecular, cellular, and clinical heterogeneity. Chaperone-mediated autophagy (CMA) supports adaptation to metabolic and environmental stress, but its cell type-specific distribution and prognostic relevance in PDAC remain unclear.MethodsSingle-cell RNA sequencing data from GSE212966 were analyzed to characterize CMA-related transcriptional states in PDAC and adjacent non-tumor tissues. Bulk transcriptomic data from TCGA-PAAD were used for differential expression analysis, weighted gene co-expression network analysis, and molecular model development, while ICGC PACA-CA and PACA-AU served as independent validation cohorts. Multiple survival machine-learning approaches were compared to establish a CMA-related prognostic model. Hallmark pathway activity, immune infiltration, and predicted drug sensitivity were evaluated between risk groups. KRT19, the highest-weighted model gene, was selected for in vitro validation. In parallel, an independent single-center cohort of 468 patients was analyzed using eight survival machine-learning methods to identify clinical prognostic factors and construct a nomogram.ResultsCMA-related transcriptional activity varied among cell types, with macrophages showing prominent scores and PDAC-derived macrophages exhibiting higher CMA scores than those from adjacent tissues. Integration of TCGA differential expression analysis and WGCNA identified 105 candidate genes. The StepCox [forward] plus random survival forest model showed favorable overall performance, with C-index values of 0.903, 0.678, and 0.733 in the TCGA, PACA-CA, and PACA-AU cohorts, respectively. High molecular risk was associated with enhanced glycolytic, proliferative, and cell cycle-related signaling, increased M0 macrophages, reduced CD8+ T cells, and differential predicted drug sensitivity. KRT19 overexpression promoted PDAC cell proliferation, colony formation, migration, and invasion. In the single-center cohort, N stage, CA125, vascular tumor thrombus, and total bilirubin ranked highest in weighted prognostic importance. The clinical nomogram achieved AUC values of 0.661 and 0.750 for 1- and 3-year overall survival, respectively.ConclusionThis study identified CMA-related cellular heterogeneity, established a molecular prognostic model that retained prognostic discrimination in two independent validation cohorts, demonstrated the functional relevance of KRT19, and developed an independent clinical prediction tool. These molecular and clinical models provide complementary perspectives on PDAC prognosis and warrant further evaluation in matched prospective cohorts.
Graphical AbstractInfographic illustrating liquid biopsy as a dynamic window into cancer, highlighting multiple biomarker sources such as tumor cells, DNA, RNA, extracellular vesicles, immune and stromal cells, and platelets. A workflow depicts integrated, longitudinal sampling, multi-omics monitoring, data interpretation, and timely decision-making in precision oncology. Clinical impact box lists benefits: better detection, risk stratification, treatment guidance, and outcomes. Banner below states liquid biopsy advances from detecting cancer signals to providing dynamic, actionable insights for guiding oncology and improving care.
IntroductionTrauma is the leading cause of non-obstetrical maternal death and is linked to adverse pregnancy outcomes, including placental abruption, preterm birth, and stillbirth. Although placental injury is a known consequence of maternal trauma, understanding its direct effect on trophoblast function remains limited. This study aims to examine the cellular effects of mechanical trauma on human trophoblasts in vitro.MethodsHuman trophoblast cells (HTR-8/SVneo) were cultured and exposed to controlled mechanical trauma using a fluid percussion injury (FPI) model, previously validated in traumatic brain injury research. Cell viability was measured 48 h after injury with a fluorometric assay kit. Immunofluorescence and Western blot analyses were carried out to assess the levels of mitochondrial markers (TOM20, COX1, VDAC1) and the antiangiogenic factor sFlt-1.ResultsTrophoblasts subjected to trauma exhibited reduced viability compared with controls. Expression of sFlt-1 and SDHA increased in injured cells, indicating a cellular stress response. Western blot results revealed a twofold decrease in TOM20 levels and a slight reduction in VDAC1 levels, while COX1 levels remained unchanged. Moreover, next-generation sequencing of trophoblasts after trauma showed altered gene expression involving several injury pathways, including mitochondrial stress response, immune dysregulation, cytoskeletal changes, endothelial and angiogenic signaling pathways, calcium signaling, metabolic imbalance, and inflammation. These results suggest that mechanical trauma causes mitochondrial dysfunction and disrupts angiogenic signaling in trophoblasts.DiscussionTrauma induces significant mitochondrial changes and cellular stress, characterized by reduced viability, decreased TOM20 and VDAC1 levels, and increased sFlt-1 and SDHA levels. These patterns resemble those seen in hypoxia-related placental disorders such as preeclampsia, fetal distress, and placental abruption. Understanding these mechanisms may help clarify how maternal trauma contributes to adverse pregnancy outcomes. Further studies using in vivo models are required to explore the clinical relevance of these findings.
BackgroundCarpal tunnel syndrome (CTS) is a common compression neuropathy in which sustained median nerve ischemia drives progressive neuroinflammation, structural remodeling of the transverse carpal ligament (TCL), and functional nerve impairment. Current interventions typically address either the inflammatory or structural component of the disease, but rarely both simultaneously, and clinical evidence for combined approaches in moderate-to-severe CTS remains limited.ObjectiveTo evaluate the clinical efficacy of ultrasound-guided hydrodissection combined with acupotomy in moderate-to-severe CTS, and to explore whether improvements in electrophysiological and ultrasonographic outcomes are consistent with attenuation of neuroinflammatory processes, while acknowledging that direct molecular confirmation was not performed in this study.MethodsThis single-center retrospective cohort study enrolled 100 patients with moderate-to-severe CTS (February 2019–February 2025). The dual-axis group (n = 49) received hydrodissection combined with acupotomy; the single-axis group (n = 51) received hydrodissection alone. Primary outcomes were BCTQ-SSS and BCTQ-FSS scores; secondary outcomes included VAS, nerve electrophysiological biomarkers (SCV, DML, CMAP), ultrasonographic biomarkers (nerve CSA, TCL thickness), clinical efficacy grading, and adverse events at baseline, 1 week, 1 month, 3 months, and 6 months.ResultsBoth groups improved significantly from baseline (P < 0.05). The dual-axis group demonstrated significantly superior BCTQ-SSS, BCTQ-FSS, and VAS scores from 1 month onward (all Bonferroni-corrected P < 0.001). At 6 months, the dual-axis group showed markedly superior electrophysiological indicators of nerve functional recovery (SCV: 42.36 ± 4.87 vs. 38.92 ± 5.23 m/s; DML: 4.12 ± 0.65 vs. 4.58 ± 0.71 ms; CMAP: 7.85 ± 1.52 vs. 6.92 ± 1.64 mV; all P < 0.05) and greater reduction in ultrasonographic biomarkers of inflammatory edema (CSA: 10.35 ± 1.86 vs. 11.82 ± 2.14 mm2; TCL: 2.15 ± 0.38 vs. 2.68 ± 0.42 mm; all P < 0.001). The overall response rate was 93.9% vs. 80.4% (P = 0.031). No serious adverse events occurred.ConclusionUltrasound-guided hydrodissection combined with acupotomy demonstrated superior clinical, electrophysiological, and ultrasonographic outcomes compared with hydrodissection alone in moderate-to-severe CTS. Improvements in nerve conduction velocity, distal motor latency, CMAP amplitude, and median nerve cross-sectional area are consistent with, and may reflect, attenuation of underlying neuroinflammatory processes; however, direct molecular evidence for such a mechanistic connection was not obtained in this study and the association remains a hypothesis requiring prospective validation. These findings support the combined approach as a minimally invasive option for patients who are unable or unwilling to undergo surgical decompression, pending confirmation by prospective randomized trials with a surgical comparator arm.
BackgroundDoxorubicin (DOX) chemotherapy for hepatocellular carcinoma is hampered by tumor multidrug resistance and cumulative cardiotoxicity. Resveratrol (RES), a natural polyphenol with anti-tumor and antioxidant effects, may reverse DOX resistance and mitigate myocardial injury.ObjectiveTo investigate whether RES reverses DOX resistance and boosts anti-tumor activity in HepG2-R/SMMC-7721-R cells via apoptosis and EMT regulation, while protecting H9c2 cardiomyocytes; an H22 tumor-bearing mouse model was used to confirm the dual in vivo efficacy of combined RES + DOX therapy.MethodsIn vitro, CCK-8, morphology observation, wound-healing, Transwell and TUNEL assays detected cell proliferation, migration and apoptosis. Western blot measured apoptosis- and EMT-related proteins. Intracellular ROS, SOD and MDA were tested to clarify antioxidant cardioprotective mechanisms. In vivo, tumor volume, body weight, serum myocardial injury biomarkers (CK-MB, cTnI, BNP), oxidative-stress indicators, as well as H&E and Masson´s trichrome staining of myocardial tissue were detected to assess treatment efficacy and safety.ResultsDOX monotherapy barely inhibited resistant cell growth, while RES alone exerted weak anti-tumor activity; RES + DOX exerted obvious combined cytotoxicity to suppress proliferation, metastasis and induce tumor apoptosis. RES reversed EMT and enhanced the activation of Bax/Bcl-2/caspase-3 signaling. In H9c2 cells, RES eliminated DOX-triggered ROS overload, restored SOD, lowered MDA and suppressed cardiomyocyte apoptosis. In vivo, the combination achieved optimal tumor suppression, reversed DOX-induced weight loss, normalized serum cardiac-specific damage biomarkers and myocardial oxidative stress. Histological examinations demonstrated that RES alleviated DOX-provoked myocardial structural damage and interstitial fibrosis, and RES itself showed no intrinsic cardiac toxicity.ConclusionRES reverses DOX resistance and enhances anti-tumor effects; this finding is associated with the regulation of apoptosis and EMT signaling, and alleviates DOX-mediated cardiomyocyte oxidative injury through antioxidant pathways. This dual-action adjuvant strategy widens DOX´s therapeutic window for DOX-resistant hepatocellular carcinoma.
Poultry muscle growth and development critically depend on skeletal muscle formation during embryonic and hatching stages. However, early embryonic muscle development differences between pigeon breeds remain unclear. We used European meat pigeons and Shiqi pigeons, which differ in body size, as models. Pectoral muscles from both breeds were subjected to ATAC-seq and RNA-seq at embryonic day 14 (E14) and post-hatching day 1 (P1). At P1, Shiqi pigeons had a significantly greater proportion of myofibers with 1–50 μm diameters, higher myofiber density, and a predominant 50–100 μm distribution across developmental stages compared to European meat pigeons. Integrated ATAC-seq and RNA-seq analyses revealed developmental dynamics driven by muscle contraction, cardiac regulatory pathways, and PI3K-Akt/muscle remodeling signaling, as well as breed differences in chromosomal structure/cell cycle regulation, cancer-related pathways, metabolic homeostasis, and calcium/estrogen signaling. Furthermore, 62 chromatin accessibility-gene expression co-varying DEGs (59 upregulated) were enriched in insulin receptor binding and AMPK/cGMP-PKG pathways; key genes including Sln, MYOM2, Trim63, Fabp3, ATP1A2, KLF15, IRS2, and RYR3 showed RT-qPCR expression highly consistent with RNA-seq (r ≥ 0.8181). This study found that the number of pectoral myofibers in Shiqi pigeons was significantly greater than in European meat pigeons at P1. Chromatin accessibility was higher near the transcription start site than in other regions. Integrating proximal, distal, and transcriptomic data, key candidate genes were screened and validated. Subsequently, genes differentially expressed between breeds were validated by tissue expression profiling, and Sln was selected for functional study. Cellular and quantitative experiments showed that Sln positively regulates pigeon embryonic myoblast proliferation and differentiation by upregulating Myf5 during proliferation and Myog and MyH1 during differentiation. Western blot confirmed higher MyoD and MyHC protein levels in Sln-overexpressing versus knockdown groups. This study revealed significant differences in pectoral myofiber diameter, embryonic chromatin structure, and gene expression between Shiqi and European meat pigeons, providing molecular insights into divergent muscle growth and development. The findings provide a theoretical basis for understanding growth performance differences among pigeon breeds and a foundation for molecular marker-assisted breeding.
BackgroundPeriodontitis represents one of the most prevalent chronic inflammatory diseases, characterized by progressive periodontal tissue destruction driven by immune dysregulation. Emerging evidence establishes ferroptosis—an iron-dependent, lipid-peroxidation-driven form of regulated cell death—as a pivotal but underexplored driver of the pathological “cell death–inflammation–re-death” cycle in periodontitis. Ferroptotic cells release damage-associated molecular patterns (DAMPs) including oxidized lipids, HMGB1, and iron species that amplify inflammation. The spatiotemporal heterogeneity of ferroptosis susceptibility among periodontal cell subtypes, the identity of DAMPs-mediated intercellular signaling networks, and the epigenetic mechanisms governing ferroptosis-susceptibility transitions remain incompletely characterized.MethodsWe employed a single-arm cross-sectional design: a prospective single-cell RNA sequencing (scRNA-seq) study of gingival tissue from 6 periodontitis patients and 4 controls, integrating UMAP clustering, pseudotime trajectory, RNA velocity, CellChat-based ligand–receptor analysis, pySCENIC regulon inference, and epigenetic landscape analysis via published ATAC-seq/methylation dataset integration from independent cohorts. To functionally validate key transcriptomic findings, LPS-stimulated human cementoblast (HCEM) in vitro experiments with Ferrostatin-1 rescue were performed, and gene expression was quantified by RT-qPCR.ResultsThe scRNA-seq atlas resolved three cementoblast subtypes with distinct ferroptosis susceptibility profiles; ferroptosis-sensitive cementoblasts (FS-CEM) functioned as dominant DAMPs-releasing hubs engaging fibroblasts, endothelial cells, and immune cells via HMGB1–RAGE, IL-33–ST2, and MIF–CD74 axes that triggered secondary necroptotic and pyroptotic cascades. NFE2L2 (NRF2)–BACH1 was identified as the transcription factor master switch governing ferroptosis susceptibility. Epigenetic analysis, derived from integration with publicly available ATAC-seq and methylation datasets from independent cohorts (GEO: GSE194276; ENCODE), revealed hypermethylation of GPX4/SLC7A11 promoters and EZH2-mediated H3K27me3 enrichment at NFE2L2 target loci in ferroptosis-sensitive cells, representing hypothesis-generating epigenetic inferences requiring direct experimental validation. In vitro LPS stimulation of HCEM cells confirmed dose-dependent downregulation of GPX4 and SLC7A11 and upregulation of ACSL4, PTGS2, and HMGB1; these changes were significantly reversed by Ferrostatin-1 pre-treatment, validating the ferroptosis specificity of the identified molecular signatures.ConclusionThis study provides mechanistic evidence that ferroptosis drives the chronic inflammatory cycle in periodontitis, and proposes NFE2L2 activation, BACH1 inhibition, EZH2 inhibition, and HMGB1 neutralization as precision therapeutic targets to break the pathological cell death–inflammation cycle.
BackgroundGlioblastoma (GBM), IDH-wildtype, CNS WHO grade 4 has marked spatial heterogeneity, yet routine MRI-based prognostic assessment often relies on whole-tumor summaries. We developed a preoperative MRI habitat-analysis framework to quantify intratumoral and peritumoral spatial phenotypes and to evaluate their value for overall survival (OS).MethodsThis retrospective multicohort study included a development cohort of 473 patients assembled from the UCSF-PDGM-v5 dataset (n = 354) and Yantai Yuhuangding Hospital (n = 119), and an independent external testing cohort from the First Affiliated Hospital of Ningbo University (n = 82). All habitat generation, selection of the optimal habitat number, feature selection, and model development were performed using the pooled development cohort. Multiparametric preoperative MRI was partitioned into voxel-wise habitats using k-means clustering. Habitat-derived variables were selected and combined into a risk score using LASSO-Cox modeling. Baseline, habitat, and combined prognostic models were constructed using Cox proportional hazards regression and evaluated using the C-index, time-dependent AUC, calibration, prediction-error analysis, decision curve analysis, and Kaplan–Meier risk stratification.ResultsA four-habitat solution was stable and biologically interpretable. Necrotic-like and edema-dominant peripheral habitats showed the strongest adverse associations with OS. The habitat risk score remained independently prognostic after adjustment for the consistently available baseline variables of age, sex, and MGMT promoter methylation. In external testing, the combined model improved the external C-index from 0.604 to 0.701 and the 12-month AUC from 0.626 to 0.724, with reduced prediction error, improved calibration, and separated high- and low-risk groups.ConclusionMRI-derived habitat analysis captures survival-relevant spatial heterogeneity in GBM and provides an interpretable, noninvasive approach for risk stratification warranting prospective validation.
IntroductionExtraembryonic lineages, specifically the trophectoderm (TE) and primitive endoderm (PrE), are indispensable for early embryogenesis and successful pregnancy. Expanded potential stem cells (EPSCs) can contribute to both embryonic and extraembryonic fates, providing a platform to model early lineage specification.MethodsThis study investigated the effects of fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4) (2F condition) on lineage-associated priming in porcine EPSCs (pEPSCs).ResultsFollowing 2F treatment, cells maintained a normal karyotype and underwent morphological changes accompanied by downregulation of pluripotency markers (OCT4, SOX2) and upregulation of TE (CDX2 and GATA3) and PrE markers (GATA4, GATA6, SOX17, and HNF4A). Transcriptomic and immunofluorescence analyses consistently demonstrated activation of TE- and PrE-associated molecular programs, although markers of additional developmental lineages were also detected. These findings suggest that combined FGF2 and BMP4 treatment promotes extraembryonic lineage-associated priming in pEPSCs, and generates a heterogeneous population exhibiting features of both TE- and PrE-associated states.DiscussionCollectively, this study establishes a simple and defined in vitro platform for investigating signaling-dependent lineage priming and cell state remodeling in porcine EPSCs.
Extracellular purinergic signaling, mediated by nucleotides such as adenosine triphosphate (ATP) and adenosine, is a critical regulator of retinal homeostasis. Within the retina, this signaling pathway is indispensable for maintaining physiological functions. Dysregulation of extracellular purine balance is a key driver of retinal pathophysiology, contributing to inflammation, pathological angiogenesis, and neurodegeneration in diseases like diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma, and retinopathy of prematurity (ROP). The hydrolysis of extracellular ATP to adenosine, primarily orchestrated by the sequential actions of NTPDase1/ENTPD1 (CD39) and ecto-5′-nucleotidase/NT5E (CD73), represents a fundamental mechanism for calibrating purinergic signals and shifting the microenvironment from pro-inflammatory to anti-inflammatory and protective. CD39 and CD73 are pivotal enzymes in regulating retinal purinergic equilibrium. Despite their well-characterized roles in tumor biology and cerebral pathophysiology, the expression, physiological functions, and pathological implications of CD39/CD73 in the retina remain understudied and lack systematic synthesis. This review synthesizes current understanding of the roles of CD39 and CD73 in retinal physiology and pathology, with a focus on their implications in DR, AMD, glaucoma, and ROP. Critically, this review provides the first systematic integration of their layer-specific spatial compartmentalization, context-dependent functional duality, and emerging metabolic roles in photoreceptor homeostasis. We also discuss emerging therapeutic strategies that target this pathway, addressing the growing need for translational research to preserve vision.
Epidermal keratinocytes (KCs) undergo a tightly orchestrated differentiation programme that culminates in the formation of a protective barrier between the body and the environment. Although this process has been studied extensively, several regulators that coordinate KC survival, stratification, and terminal differentiation remain incompletely defined. To identify mechanisms governing epidermal homeostasis, we compared transcriptomic profiles of undifferentiated KCs, KCs differentiated in monolayer culture, and fully stratified KCs in three-dimensional organotypic skin equivalents. Both differentiation models showed downregulation of cell-cycle-associated transcripts and induction of KC differentiation genes, whereas genes involved in extracellular-matrix organisation were preferentially induced in skin equivalents. Transcription-factor activity inference based on target gene expression identified the transcriptional repressor and ubiquitin/SUMO E3 ligase TRIM28 as a candidate regulator of epidermal homeostasis. Single-cell RNA sequencing and immunostaining of human epidermis revealed strong TRIM28 expression throughout the living epidermal layers, with marked loss in the outermost terminally differentiated compartment. Functional depletion of TRIM28 in primary human KCs impaired epidermal development, increased apoptosis, and produced markedly thinner organotypic epidermis despite preserved stratification. TRIM28-deficient skin equivalents showed limited transcriptional reprogramming associated with reduced p53 ubiquitination, p53 accumulation, and apoptosis of basal KCs. Collectively, these findings identify TRIM28 as an important candidate regulator of epidermal homeostasis and suggest that TRIM28 supports epidermal expansion, at least in part, by contributing to p53 turnover.
Hyperoxia-induced acute lung injury (HALI) is a serious complication of excessive oxygen exposure, yet its pathogenesis and treatment remain incompletely understood. A growing body of evidence suggests that HALI results from the interplay of oxidative stress, inflammation, immune dysregulation, organelle dysfunction, alveolar barrier disruption, and regulated cell death. In this review, we summarize recent advances in our understanding of these processes and discuss therapeutic strategies that have been investigated in experimental models. These approaches include antioxidant and anti-inflammatory agents, metabolic and mitochondrial modulators, interventions targeting iron metabolism and ferroptosis, barrier-protective therapies, and cell- or extracellular vesicle-based treatments. We also consider the limitations of the current evidence, particularly the differences between experimental models and clinical settings and the safety, delivery, and reproducibility challenges associated with emerging therapies. Further studies are needed to define the key regulatory mechanisms and temporal features of HALI, establish clinically relevant experimental models, and generate stronger clinical evidence to determine whether promising experimental interventions can be translated into effective therapies.
BackgroundNonsteroidal anti-inflammatory drugs (NSAIDs) are a cornerstone of multimodal analgesia after total knee arthroplasty (TKA), but gastrointestinal, renal, cardiovascular, and bleeding risks may limit their perioperative use in some older adults. This study evaluated preoperative photobiomodulation therapy (PBMT) delivered with a stationary 810 nm semiconductor laser as a non-pharmacological pre-emptive analgesic strategy compared with celecoxib.MethodsIn this prospective, double-blind, double-dummy randomized controlled trial, 80 patients undergoing primary unilateral TKA were randomized 1:1 to preoperative PBMT (n = 40) or oral celecoxib (n = 40). The primary outcome was resting visual analogue scale (VAS) pain at 24 h after surgery. The non-inferiority margin was 1.0 VAS point and the assumed standard deviation for sample-size calculation was 1.5 points. Other pain time points, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), lower-limb swelling, early joint function, perioperative blood loss, and complications were also assessed.ResultsAt 24 h, resting VAS pain was 3.9 ± 1.1 with PBMT and 3.7 ± 1.0 with celecoxib (PBMT minus celecoxib mean difference, 0.20 points; 95% CI, −0.27–0.67); the upper confidence limit was below the 1.0-point margin. The 48-h WOMAC scores were 42.4 ± 5.8 and 41.8 ± 6.2, respectively (P = 0.655). PBMT was associated with smaller increases in thigh circumference on postoperative days 3 and 7, less hidden blood loss, and fewer reports of gastrointestinal discomfort.ConclusionPreoperative 810 nm semiconductor-laser PBMT met the stated non-inferiority criterion for 24-h resting pain compared with celecoxib and was associated with favorable swelling, blood-loss, and gastrointestinal outcomes.
The Wound Healing Assay is a standard technique for studying cell motility, yet it faces challenges in reproducibility and data interpretation. Here we present WoundPy, a Python-based executable software featuring a user-friendly interface for semi-automatic, researcher-supervised Region of Interest detection. WoundPy streamlines image analysis and management of replicates, providing rapid graphical outputs. A key pre-processing feature is the automated vertical wound alignment, which eliminates operator-dependent errors typical of optical microscopy. Velocity results are calculated as absolute values from multi-time-point imaging. The software was tested on three datasets from biological experiments, and a comparison with ImageJ Wound Healing Size tool revealed a significant underestimation of the wound area by the latter compared to WoundPy. In conclusion, this new software offers an extremely streamlined approach that easily enables to perform an analysis that is both accurate and fast, drastically reducing the time required for both numerical data acquisition and its subsequent analysis.
Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal interstitial lung disease with limited therapeutic options and a median survival of only 3–5 years. Although antifibrotic agents such as pirfenidone (PFD) and nintedanib can decelerate functional decline, they fail to reverse established fibrosis, underscoring an urgent need for novel therapeutic paradigms. Emerging evidence has positioned mitophagy—the selective autophagic clearance of damaged mitochondria—at the nexus of IPF pathogenesis. In this review, we systematically dissect the regulatory networks governing mitophagy, encompassing the canonical PINK1/Parkin pathway, receptor-mediated mechanisms (BNIP3/NIX/FUNDC1), and their intricate cross-talk with endoplasmic reticulum stress (ERS) and ferroptosis, highlighting how these interconnected pathways converge to determine alveolar epithelial cell (AEC) fate, fibroblast activation, and inflammatory reprogramming. Notably, the pathological impact of mitophagy is highly cell-type-specific and context-dependent, exhibiting protective functions in epithelial cells while paradoxically promoting pro-fibrotic phenotypes in macrophages under certain conditions, which poses both challenges and opportunities for therapeutic intervention. Furthermore, we critically evaluate emerging pharmacological and biological strategies targeting mitophagy, and propose that future combination regimens—guided by non-invasive mitophagy biomarkers—may overcome current clinical bottlenecks. By integrating mechanistic insights with translational perspectives, this review provides a roadmap for developing mitophagy-targeted interventions as a next-generation therapeutic paradigm for IPF.
