Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne Orthoflavivirus that circulates primarily in birds and pigs. Previous observations of vector-free transmission between pigs indicates the possibility of single-host cycling in swine. Therefore, the aim of this work was to investigate the evolutionary pressure of single host cycling using a relevant primary cell culture model. To investigate whether such single-host cycles affect viral infectivity, fitness and genomic adaptations, two strains and a reverse genetic cDNA-derived clone of JEV were serially passaged 12 times in primary porcine monocyte-derived macrophages (MDMs), in Aedes albopictus-derived C6/36 cells, and alternately between both cell types. Next-generation sequencing analysis was used to identify selected single nucleotide variants (SNVs) and haplotypes. Phenotype-to-genotype connections were confirmed using reverse genetics. For all viruses, serial passaging in MDMs - but not in C6/36 cells - led to a rapid increase in relative infectivity toward MDMs, accompanied by reduced plaque sizes in porcine endothelial cells. In contrast to C6/36 cells, MDM imposed a strong selective pressure, rapidly favoring selection of many SNVs and viral haplotypes. In addition, we identified a dominant selection of mutants with glutamic acid to lysine substitutions at positions 49 or 138 in the E protein, which explained the small plaque phenotype and caused viral sensitivity to heparin-mediated inhibition of attachment, indicating enhanced virus binding to glycosaminoglycans (GAG). The E138K mutant also explained the increased relative infectivity for MDM. This work demonstrates a high evolutionary pressure on JEV in MDM causing rapid selections of minor haplotypes. Furthermore, the efficient selection of E49K and E138K SNV, which were responsible for the phenotype, are likely caused by a selective pressure for GAG binding, observed in vitro with other mammalian cells.
Globally, colorectal cancer (CRC) stands as the second most lethal malignancy. Chemotherapeutic drugs have shown some limitations, including systemic toxicity and non-specificity. Nanocarriers such as chitosan/gold nanoparticles (CS/Au NPs) facilitate drug localization at tumor sites, thereby reducing off-target adverse effects. Doxorubicin-loaded CS/Au NPs were synthesized, and their physicochemical properties were analyzed using Fourier transform infrared (FT-IR) spectroscopy and dynamic light scattering (DLS). HCT116 cells were treated with both free doxorubicin and CS/Au-DOX NPs. In vitro cytotoxic effects were evaluated using the MTT assay. Additionally, apoptosis induction, cell cycle arrest, and cellular uptake assay were examined by flow cytometry. The scratch assay used to evaluate the effect of CS/Au-DOX NPs on the cell migration potential of HCT116, and the expression levels of CLMAT3 and ZNRD1-AS1 were determined by qRT-PCR. CS/Au-DOX NPs exhibited high encapsulation efficiency (78%) with favorable nanoscale properties, and were markedly more potent than free doxorubicin against HCT116 cells (IC50 0.5 vs. 3 µM). Flow cytometry revealed significant apoptosis induction, G2/M arrest, and enhanced cellular uptake in HCT116 following treatment with CS/Au-DOX NPs. These NPs also reduced the migration potential of CRC cells. In CRC tissues, CLMAT3 and ZNRD1-AS1 were upregulated, with CLMAT3 overexpression associated with lymph node metastasis, while both lncRNAs showed limited diagnostic value. Notably, CS/Au-DOX NPs downregulated CLMAT3 and ZNRD1-AS1 expression in HCT116 cells. CS/Au-DOX NPs substantially enhanced the anticancer efficacy of DOX in the HCT116 cell line. Furthermore, CLMAT3 and ZNRD1-AS1 exhibited only limited utility as diagnostic biomarkers in CRC, suggesting that their clinical applicability in early detection may be restricted.
Inflammatory bowel disease (IBD) predisposes to neuropsychiatric comorbidity and increases the risk of Parkinson's Disease (PD). Although the gut-immune-brain axis was proposed as a link between IBD and PD and a driver of PD immunopathogenesis, the regional pattern and single-cell landscape of the brain immune response during colitis and its contribution to PD pathology remain poorly defined. Here, we observe a loss of dopaminergic neurons and synuclein pathology in the substantia nigra pars compacta of adult mice with chronic colitis. By confocal microscopy and integrated multi-omics, we reveal a complex midbrain-specific immune response to chronic colitis. Single-cell mapping of the midbrain immune landscape showed an inflammatory shift of microglial clusters including an expansion of interferon-response microglia, CD8+ T cell extravasation, and increased numbers of vessel-associated neutrophils. Selective myeloid cell depletion using a colony stimulating factor 1 receptor (Csf1r) inhibitor after colitis onset reduced midbrain microglia by 67% and led to a complete rescue of dopaminergic neuron loss, without affecting mucosal pathology or T cell and neutrophil migration to the midbrain. Collectively, within the complex midbrain immune response to chronic colitis, we demonstrate a causal role of Csf1r-dependent myeloid cells for dopaminergic neurodegeneration. Thus, Csf1r inhibition in IBD may not locally ameliorate colitis, but provide neuroprotection to dopaminergic neurons.These results reveal a novel cellular link between chronic gut-derived peripheral inflammation and midbrain vulnerability and thereby substantially enhance our understanding of the risk for PD related to the gut-immune-brain axis.
Systemic lupus erythematosus (SLE) is an autoimmune disease posing a significant threat to human health. Regulatory T cells (Tregs) are crucial for immune homeostasis. Dysfunction of Tregs have been demonstrated in SLE, but underlying mechanisms remain largely unclear. Here, we observe that peripheral Treg numbers and SLC7A11 expression in Tregs are reduced in SLE, with SLC7A11 levels negatively correlates with disease activity. Treg-specific SLC7A11 deletion impairs their stability and differentiation, promotes a pro-inflammatory phenotype, thereby aggravating autoantibody production and lupus progression in mice. Mechanistically, mild SLC7A11 inhibition stabilizes HIF-1α, enhances glycolysis and increases pro-inflammatory cytokines in Tregs. Severe SLC7A11 inhibition causes excessive lipid peroxidation and ferroptosis. In summary, our findings suggest that SLE Tregs with reduced SLC7A11 expression may undergo varying degrees of damage. Specifically, mild SLC7A11 deficiency drives glycolysis and Treg instability, whereas severe deficiency triggers ferroptosis. Upregulating SLC7A11 expression may be a promising therapeutic strategy for SLE.
Bone defects remain a significant challenge in orthopedics, and despite the widespread use of mesenchymal stem cells (MSCs) in regenerative medicine, their therapeutic performance in vivo often falls short of expectations. Emerging evidence suggests that the local microenvironment particularly age-related or injury-induced cellular senescence may compromise MSC function. In this study, we investigated how a senescent vascular niche arising after bone injury influences MSC proliferation, differentiation, and its possible mechanisms involved in regeneration and clearing of the aging microenvironment. A femoral trochlear defect was created in 3-month-old SD rats (n = 6/group) to characterize temporal senescence changes using SA-β-Gal staining, p16/CD31 immunofluorescence, and expression of Cdkn1a/Cdkn2a. Primary endothelial cells (ECs) were isolated and senescence-induced with 400 µM H₂O₂ for 45 min. MSCs were co-cultured with senescent ECs (SnECs) in 3D collagen I hydrogels (2 mg/mL, ~ 5 kPa). Quercetin, selected from DrugAge screening (20 µM), was incorporated into a 4 wt% thermosensitive hydrogel (TSH-Q) to enable 7-day sustained release. Bone regeneration was assessed by µCT, histology, and immunofluorescence at 1 and 4 weeks post-injury. Bone defects triggered a biphasic senescence response: early senescence occurred predominantly in peri-defect osteocytes at 1 week, while robust senescence was later observed in neovascular endothelial cells by week 4. SnECs significantly impaired MSC biological functions, reducing migration, chondrogenic differentiation (Safranin O intensity), and mineralization (Alizarin Red) (all p < 0.01). Local delivery of quercetin via TSH-Q cleared approximately 81% of p16⁺ endothelial cells in vivo and enhanced bone repair, increasing BV/TV compared with unloaded TSH (p < 0.001). In the early stage of bone defects, aging cells mainly represent bone cells in the tissues surrounding the bone defect. At later staged, with no changes in the microenvironment, the aging of vascular endothelial cells in the new tissues and blood vessels was most significant. We successfully induced endothelial cell senescence and further explored the functional impact of SnECs on MSCs and found that aging ECs led to a decline in MSCs effects in aging, including reduced proliferation, chondrogenic differentiation, osteogenic differentiation, tissue repair, and mineralization. The therapeutic effects of MSCs and the repair of bone defects were effectively promoted by constructing a quercetin thermosensitive hydrogel sustained-release system to improve the aging microenvironment of bone defects. Bone injury generates a senescent vascular niche that markedly disrupts MSC-mediated regeneration. Targeted rejuvenation of this niche using sustained-release quercetin effectively restores MSC function and significantly accelerates bone reconstruction. These findings highlight the aging microenvironment as a key therapeutic target for improving MSC-based treatments for bone defects.
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disease caused by loss-of-function pathogenic variants in COL7A1 encoding type VII collagen (C7). Patients with RDEB suffer since birth from skin and mucosal blistering and develop severe local and systemic complications resulting in poor prognosis. Mesenchymal stromal cells (MSCs) have demonstrated their potential to enhance wound healing and reduce skin inflammation in RDEB patients due to their anti-inflammatory properties and capacity to express C7. We aim to optimize in vitro conditioning of human bone marrow-derived MSCs (BM-MSCs) to improve their limited survival following local injection in a murine model. BM-MSCs from healthy human donors were transduced with a lentiviral vector encoding firefly luciferase and mCherry reporter proteins and then subjected to various culture conditions: monolayer on plastic or spheroid culture, either in hypoxia (5% O2) or in normoxia (21% O2). These cells were subsequently injected intradermally (ID) in immunodeficient mice and their survival was assessed by in vivo imaging. BM-MSCs populations were analyzed prior to injection by single-cell RNA sequencing (scRNAseq). Murine skin injected with BM-MSCs were sampled two months post-injection and the surviving subpopulations were characterized by spatial transcriptomic. scRNAseq analysis revealed marked variations between monolayer and spheroid conditions, which were significantly impacted by oxygen level. Although most injected cells gradually died within the first 2 months in all tested conditions, 1% of live bioluminescent cells persisted for 54 up to 61 weeks post-injection. Spatial transcriptomics data analysis demonstrated that all surviving cells, regardless of their in vitro preconditioning, retained the expression of the MSC markers THY1, ENG, and NT5E, shared features with fibroblasts, and exhibited enriched expression in genes related to extracellular matrix and collagen fibril organization, which are key processes in wound healing. Spatial transcriptomic and scRNAseq data integration suggested that surviving BM-MSCs were initially present in the injected population, regardless of their culture condition. Remarkably, none of the in vitro preconditioning strategies appeared to affect their survival capacity or functional properties following local injection. The identification and characterization of a BM-MSC subpopulation capable of long-term survival following ID injection hold promise for the development of improved cell therapy protocols for RDEB.
Ischemic stroke remains the leading disease in terms of global disability and fatality rates. Cerebral ischemia-reperfusion injury (CIRI) and neuroinflammation induced by CIRI represents a challenge for clinical therapy of ischemic stroke. Epigallocatechin-3-gallate (EGCG), the predominant catechin in green tea, is known to mitigate ischemic stroke and inflammation, while its role in regulating neuroinflammation post-recanalization remains unclear. This study aimed to explore the effect and mechanism of EGCG on the regulation of CIRI. The CIRI in vivo model was established by middle cerebral artery occlusion/reperfusion (MCAO/R) in rats and EGCG was administered via intracerebroventricular injection before MCAO/R. The neurological scores, infarct volume, neuronal injury, and microglial biomarkers were evaluated. BV2 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to establish an in vitro CIRI model. After pretreatment with EGCG and/or ML385, OGD/R, cell viability, microglial biomarkers, pro-inflammatory and anti-inflammatory factors, and transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) were detected. Different conditioned media collected from BV2 cells were used for culturing the neuronal mimic cell line SH-SY5Y. EGCG administration in vivo significantly improved neurological deficits, infarct volume, and neuronal damage. Moreover, pretreatment with EGCG significantly increased BV2 cells viability and anti-inflammatory factors, and reduced toxicity and ferroptosis in SH-SY5Y cells. Furthermore, the upregulation of M2 markers was consistent with in vivo experiments. However, this protective effect was abolished by ML385, an Nrf2 inhibitor. EGCG upregulated Nrf2 and HO-1 protein expression, thus promoting microglial polarization toward the M2 phenotype. EGCG has a protective effect on CIRI by promoting the polarization of microglia toward the M2 phenotype via the Nrf2/HO-1 pathway to inhibit ferroptosis of neuron, which may provide a novel target for ischemic stroke treatment.
The high clinical recurrence rate of colorectal cancer (CRC) is driven by the survival of residual tumor cells that evade therapy-induced death by entering a dormant state. While dormancy is a recognized mechanism of treatment resistance, the molecular drivers governing this "quiescent reservoir" and its associated vulnerabilities remain poorly characterized, limiting the development of strategies to eradicate these dormant seeds. We developed a COAD-specific Dormancy Score (CADS) derived from NMF analysis of ~ 69,000 single cells to quantify and identify a dormant subpopulation at single-cell resolution. Mechanistically, the IFN-β/cDC1 axis and its downstream MEK/ERK dependency were validated using a GFP-p27K- dormancy reporter system, spatial transcriptomics, and CRISPR/Cas9-mediated Ifnar1 knockdown. Finally, the synergistic efficacy of anti-PD-1 combined with MEK inhibition (Trametinib) was evaluated in orthotopic CRC mouse models. The CADS effectively identified a distinct dormant subpopulation in CRC characterized by profound G0/G1 arrest, enhanced stemness, and multi-drug resistance. We uncovered a novel evasion mechanism mediated by the hijacking of IFN-β signaling. Conventionally recognized for its anti-proliferative roles, IFN-β signaling is exploited by surviving tumor cells to enter a deep quiescent state. This phenotype acts as a biological reservoir that fuels intratumoral heterogeneity and underpins the relapse of colorectal tumors by conferring resistance to conventional cytotoxic regimens. Effective anti-PD-1 therapy paradoxically enriches this dormant population via an enhanced IFN-β-conventional type 1 dendritic cell (cDC1) axis. Mechanistically, IFN-β-induced dormancy depends on MEK/ERK pathway activity, which sustains survival while suppressing apoptosis. This creates a synthetic lethal vulnerability: MEK inhibition (e.g., Trametinib) synergizes with IFN-β to re-sensitize dormant cells to apoptosis. Consequently, combining Trametinib with anti-PD-1 therapy overcomes this evasion mechanism, eliminates the dormant subpopulation, remodels the immune microenvironment, and shows strong synergistic efficacy in preclinical models. Our work redefines an immune-cell death paradox, revealing how tumors exploit IFN-β to evade therapy. We propose CADS as a translational biomarker for identifying tumors reliant on this pathway and validate a mechanism-based combination therapy that selectively targets dormancy-associated death resistance, offering a promising strategy to improve CRC outcomes.
In response to stress, cells exploit plasticity to self-activate and form protrusions that explore the environment and guide migration. While protrusion initiation is well characterized, their maturation and functional composition remain poorly understood. Here, using a new pooled genetic approach (ReGenT-seq) to interrogate chromatin factors controlling epidermal progenitor activation, we unexpectedly identified Cbx3 as a critical determinant of protrusion formation. Beyond its nuclear activities, we revealed a cytoplasmic moonlighting function of CBX3 that governs the subcellular localization of specific mRNAs within polarizing protrusions of migrating epidermal progenitor cells, as well as in invadopodia of squamous carcinoma cells. CBX3-dependent mRNA transport promotes the localized establishment of multiple organelles, including the endoplasmic reticulum and lysosomes, within maturing protrusions, and regulates focal adhesion dynamics. Together, our findings uncover a multilayered gene regulatory mechanism controlling cell motility through protrusionogenesis and identify a chromatin-to-cell-protrusion mRNA transport pathway that represents a potential therapeutic target for enhancing wound healing and limiting cancer invasion.
Non-allergic rhinitis syndrome (NAR) is a chronic rhinitis characterized by the significant absence of an allergy history, negative skin prick test results, and normal serum IgE levels. Nasal cytology is a valuable diagnostic method that enables qualitative and quantitative assessment of inflammatory cells - including eosinophils, neutrophils, mast cells, and lymphocytes - in the nasal mucosa. This study aimed to evaluate the levels of nasal eosinophilia in a pediatric NAR population and to evaluate the correlation of this local inflammatory biomarker with clinical severity scales such as ARIA and PRQLQ. This prospective, cross-sectional study included 103 children aged 5-18 years: 53 with NAR and 50 healthy controls. Symptom duration and severity were classified according to ARIA 2019 criteria. The Paediatric Rhinitis Quality of Life Questionnaire (PRQLQ) was used for quality of life assessment. Nasal cytology specimens were collected by nasal swab from the middle meatus and the eosinophil percentage was calculated by counting a total of 100 cells in the area of highest cell density. A total of 103 children were enrolled: 53 NAR patients and 50 healthy controls. Median age was 10 (8-13) years in the study group and 11 (8-14) years in the control group. Family history of allergy was significantly higher in the study group (30.2%) compared to controls (12.0%) (p = 0.024). Median nasal eosinophil level was 7.0 (3.5-15.5) in the study group and 0 (0-3.0) in controls; the difference was statistically significant (p < 0.001). The nasal eosinophil cut-off value was determined as 3.5%. No significant difference was found between nasal eosinophil groups (< 3.5% and ≥ 3.5%) and any PRQLQ subscale or total score (p > 0.05). Nasal cytology may serve as a simple, non-invasive diagnostic tool to identify NAR subtypes and to determine clinical severity in pediatric patients.
Retinal organoids represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but the capacity of host cone bipolar cells in the primate macula to rewire with transplanted photoreceptors has not been established. In this study, we transplanted genome-edited ISL1-/- human retinal organoids lacking ON-bipolar cells into an acute laser-induced macular photoreceptor ablation nonhuman primate model. Using immunohistochemistry, ultrastructural imaging, and focal macular electroretinography (FMERG), we demonstrate that host rod and cone bipolar cells actively extend dendrites toward grafted photoreceptors and form synaptic contacts, with evidence of functional signal transmission in a subset of transplanted eyes. Longitudinal, per-eye analyses revealed that host ON-bipolar responses improved in two of four eyes with ISL1-/- graft by up to 21.6% and remained stable for up to 2 years post-transplantation. Moreover, OFF-pathway connectivity showed potential progressive maturation, with delayed increase in d-wave after 13 months in one of those eyes. These findings provide the first demonstration of long-term anatomical host-graft synaptic integration in the primate macula, establishing that central cone bipolar circuits retain the capacity for durable rewiring with human stem cell-derived grafts. Our results highlight ISL1-/- retinal organoids as a promising approach for central vision restoration in macular degeneration.
Circ_0001313 is upregulated in colorectal cancer (CRC) and has been implicated in tumor progression, but its role in remodeling the immune microenvironment remains incompletely defined. This study investigated whether circ_0001313 contributes to CRC progression in association with granulocytic myeloid-derived suppressor cell (gMDSC)-like immunosuppression. Circ_0001313 expression was evaluated by qRT-PCR in CRC tissues and cell lines. Functional studies were performed using transient siRNA-mediated circ_0001313 knockdown in MC38 cells, a syngeneic murine CRC model suitable for immunocompetent C57BL/6J mice, followed by subcutaneous tumor growth and experimental lung metastasis assays. Tumor immune composition was analyzed by flow cytometry, Ly6G+ granulocytic myeloid cell-mediated suppression of CD8 + T-cell proliferation was assessed by coculture, and tumor explant supernatant (TES) assays, qPCR, ELISA, and GM-CSF neutralization were used to examine a GM-CSF-associated mechanism linked to gMDSC-like activation. Circ_0001313 was upregulated in CRC tissues and cell lines. Transient circ_0001313 knockdown suppressed primary tumor growth and prolonged survival in the lung metastasis model. Immune profiling showed increased intratumoral CD4 + and CD8 + T cells but reduced Ly6G+ granulocytic myeloid cell abundance after circ_0001313 silencing. Ly6G+ cells from circ_0001313-silenced tumors exhibited lower PD-L1 and arginase 1 expression and diminished suppression of CD8 + T-cell proliferation. Mechanistically, circ_0001313 depletion was associated with reduced tumor-derived GM-CSF expression, and GM-CSF neutralization attenuated TES-induced gMDSC-like activation. Circ_0001313 promotes CRC progression, at least in part, by remodeling the tumor immune microenvironment through a GM-CSF-associated granulocytic suppressor program, supporting further evaluation of this axis as a potential therapeutic target in CRC.
The CD74-ROS1 fusion protein is an aggressive oncogenic driver detected in non-small cell lung cancer (NSCLC) patients from stages I-IV. Despite its rare occurrence, CD74-ROS1 has a notable clinical value, with ROS1 inhibitors used as first-line therapeutics in precision medicine. Upon targeting the cancer with ROS1 tyrosine kinase inhibitors (TKIs), resistance occasionally emerges for reasons that are not well-understood. Therefore, analyzing the individual roles of CD74 and ROS1 in the context of the CD74-ROS1 fusion may reveal mechanistic insights that would guide the scientific community to more effective therapies. Here, we describe the development and characterization of A549-transfected CD74-ROS1K439M and CD74∆2-41-ROS1 variants, which together with wild-type CD74-ROS1 and control cells, enable interrogation of each protein partner. Expression analysis of 23,342 genes demonstrates distinct profiles among the variants, with 62 genes identified as differentially expressed between the control and CD74-ROS1 transfected cells. Further analysis designates 13 and 5 of these genes with expression patterns that were influenced by either truncation of the CD74 intracellular domain or the kinase inactivation mutant, respectively. From the genes specified, IL-6 stands out because of its well-established role in NSCLC and its association with CD74. In alignment with the mRNA findings, the phospho-kinase array results expose variant-mediated signaling events that were not previously linked with the functionality of CD74-ROS1. Taken altogether, this study provides an innovative view of CD74 as a fusion partner in CD74-ROS1 and contributes a list of novel molecular targets for mechanistic analysis and drug development.
Elucidating cellular hierarchies and regulatory mechanisms of placental development across gestation is critical for understanding pregnancy maintenance and improving reproductive outcomes in mammals, including cattle. However, a comprehensive, temporally resolved single-cell characterization of the bovine placenta has remained lacking. We construct a longitudinal single-nucleus transcriptomic atlas comprising 311,299 placental cells and nuclei across 13 developmental timepoints (E12, E14, E16, E18, E24, E30, E50, E60, E85, E110, E180, E240, and E280). We identify 13 major cell types and 14 trophoblast subtypes, revealing pronounced cellular heterogeneity and stage-specific transcriptional programs. Regulatory analyses highlight HAND1 and DLX5 as candidate key regulators of maternal recognition of pregnancy. Trajectory inference demonstrates that binucleate cells arise from specific uninucleate cell subpopulations around E24, with differentiation governed by genomic imprinting and metabolic reprogramming. Integration with genome-wide association study data identifies eight early trophoblast subtypes significantly associated with gestation length, along with candidate pathways and risk genes, including CYCS, HMGA1, and VDAC1, under strong evolutionary constraint. Additionally, we find that placental macrophages emerge from E30 in cattle and show significant associations with pregnancy loss in both cattle and humans, sharing conserved risk pathways. This study provides a comprehensive spatiotemporal single-cell atlas of bovine placental development, defining cellular hierarchies, lineage dynamics, and regulatory networks at the maternal-fetal interface. These findings offer a valuable resource and conceptual framework for understanding pregnancy maintenance and for improving reproductive traits in ruminants.
Cancer stem cells (CSCs) are a subpopulation with self-renewal and differentiation capacity that drive the progression, recurrence, and therapeutic resistance of patients with cervical cancer (CC). However, the complete set of genes that maintain stemness in CC remains incompletely defined. We aimed to identify key stemness-related genes and evaluate their prognostic utility, immune associations, and drug sensitivity. Through literature mining and CellMarker 2.0, we identified 1345 stemness-associated genes that overlapped with differentially expressed genes (DEGs) from the TCGA-CESC dataset (log2FC > 2, p < 0.05), yielding 216 stemness-related DEGs. A protein-protein interaction network (STRING) and CytoHubba (MCC algorithm) revealed ten hub genes (HGs): CCNB1, CCNA2, BUB1B, UBE2C, KIF11, CCNB2, KIF23, CDC20, CDC6, and FOXM1. Gene ontology and KEGG analyses revealed predominant enrichment in cell cycle progression. Cox regression and Kaplan‒Meier analyses identified BUB1B, CCNA2, CDC20, FOXM1, and KIF23 as risk factors for poor overall survival, with KIF11 emerging as an independent prognostic factor. HGs overexpression significantly correlated with altered infiltration of 15 immune cell types, including negative associations with CD8 + T and NK cells. We identified 661 unique drugs/chemicals targeting these HGs, including FDA-approved repurposed agents. Experimental validation via RT‒PCR confirmed significant overexpression of FOXM1 and KIF11 in CC tissues and cell lines compared with normal samples. These stemness-associated HGs, particularly FOXM1 and KIF11, may serve as potential prognostic biomarkers and therapeutic targets, warranting further investigation of stemness-driven CC progression.
Cancer-associated genetic, epigenetic, and microenvironmental factors impose shortages of tryptophan and arginine, which induce specific codon reassignments (substitutants) in their proteomes. Whether cancers are deprived of other amino acids is unknown. Histidine is an essential amino acid reported to be low in certain tumor types. Therefore, we investigated the potential for a histidine shortage in cancer. Using cultured cancer cells, we pinpointed histidine to glutamine (H>Q) substitutants as the most pronounced proteomic event following histidine deprivation. We then scanned cancer proteomes for H>Q proteins and observed a marked enrichment in pancreatic, uterine, and kidney cancers. Mechanistically, we propose that H>Q is a result of tRNA misalignment, and used tRNA glutamine (tRNA(Gln)) modifications at the wobble position to demonstrate it. We further show that URM1-mediated U34 thiolation boosts H>Q production and influences the survival of cancer cells following histidine deprivation, suggesting a potential role for H>Q. Additional characterization of H>Q substitutants revealed preferred sequences and the maintenance of H>Q host protein expression despite the absence of histidine, indicating cellular regulation and functional consequences. Thus, histidine shortage induces H>Q substitutants, a regulated mistranslation event that pinpoints histidine limitation in cancer and impacts cell survival.
Acupuncture, as a significant component of Traditional Chinese Medicine (TCM), has attracted increased attention for its mechanism of controlling the physiological functions of the human body and promoting the recovery of diseases by stimulating specific acupoints. Despite its long history and wide clinical application, the mechanism of action of acupuncture is still not fully understood. More research needs to be done on how acupuncture affects molecular communication and cellular function. Exosomes are nanoscale vesicles that rely on cellular multivesicular bodies (MVBs) fused with cell membranes to be released into the extracellular matrix. They are crucial for information transfer between cells and are currently a hot research topic in the world's cutting-edge life sciences. Previous research has demonstrated that the therapeutic effect of acupuncture may be related to stimulating certain cells to secrete exosomes, that exosomes released may contain "acupuncture information", and that manipulating the back-injection of exosomes to produces "acupuncture-like" effects. These findings suggest that exosomes could serve as a bridge between conventional acupuncture therapy and modern precision medicine. They also offer fresh prospects and difficulties for acupuncture translational medicine research. To better define the relationship between exosomes and acupuncture, we reviewed and systematized the literature on past studies related to exosomes and acupuncture. This paper provides a significant theoretical and experimental foundation for applying exosomes in precision medicine by summarizing and analyzing the relationship between acupuncture stimulation and exosome function. This is expected to promote the combination of traditional acupuncture therapy and modern biotechnology and bring innovation and progress to future medical practice.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for high-risk hematologic malignancies, yet its efficacy is limited by the onset of graft-versus-host disease (GVHD). Acute GVHD (aGVHD) primarily affects epithelial cells of the gastrointestinal tract, skin, and liver by the involvement of alloreactive T-cells and pro-inflammatory cytokines. The barrier-protective role of hypoxia-inducible factor (HIF), regulated by prolyl hydroxylase, has been significantly implicated in hypoxic inflammatory conditions. In this study, Roxadustat, a prolyl hydroxylase inhibitor, was tested in a murine model of aGVHD (C57BL/6J → BALB/cJ) to decrease hypoxia-mediated early gastrointestinal injury. Recipients were treated with roxadustat or vehicle intraperitoneally daily for the first week post-transplant, and then twice a week till day 42. Roxadustat significantly improves survival by day + 42 with reduced clinical GVHD symptoms. Reduced gut pathology with significant reduction of serum IFN- γ and TNF-α levels, suggesting a decreased systemic inflammation. Roxadustat significantly decreased T-cell infiltration and prevented TNF-α-induced intestinal epithelial cell apoptosis through HIF-1α-dependent repression of Fas-associated death domain protein (FADD) and cleaved caspase 3. In vitro, roxadustat suppressed TNF-α production by peritoneal macrophages and mixed lymphocyte reaction. These results suggest that roxadustat attenuates early gut injury, offering an alternative strategy for acute GVHD following allo-HSCT.
To explore the protective effects and mechanisms of Asiaticoside (AC) against diabetic kidney disease (DKD) through network pharmacology combined with in vitro and in vivoexperiments. Based on network pharmacology and RNA sequencing (RNA-Seq) analyses, combined with molecular docking, the core targets and signaling pathways of AC in the treatment of DKD were predicted and screened. Subsequently, the therapeutic effect of AC on DKD was validated in db/db mice and SV40-MES-13 cells, and its molecular mechanism was evaluated. Network pharmacology and protein-protein interaction analyses identified 11-beta hydroxysteroid dehydrogenase type 2 (HSD11B2) as a potential key target for AC treatment of DKD. Molecular docking indicated strong affinity between AC derivative and HSD11B2. RNA-Seq analysis showed that AC significantly regulated steroid metabolism under diabetic conditions. In db/db mice, AC reduced urinary protein excretion, improved renal morphology, and protected renal function. AC also dose-dependently inhibited excessive proliferation of SV40-MES-13 cells in a high-glucose environment. Further validation showed that AC upregulated Hsd11b2 mRNA expression and HSD11B2 protein levels. AC may alleviate DKD-related pathological processes by regulating Hsd11b2 gene expression and HSD11B2 protein levels.
Mitochondrial function plays a critical role in skin aging. Chlorogenic acid (CGA), a botanical compound, has demonstrated regulatory effects on mitochondrial function and senescence inhibition. However, whether the anti-aging effects of CGA are attributable to its regulation of mitochondrial function remains unclear. There is a need to investigate the anti-aging effect and mechanism of CGA by modulating mitochondrial function, particularly its mode of action on mitochondrial function. Normal human dermal fibroblasts and human epidermal keratinocytes were used to detect the regulation of collagen I production, mitochondrial functions, and anti-aging properties of CGA and confirmed by mitochondrial transplantation. The photo-aging mouse model was established by ultraviolet (UV) radiation, followed by the treatment of CGA-gel (1 mmol/kg/d) for 14 days. The skin tissues were collected and tested. CGA promotes collagen I (Col1) production by activating the TGF-β/Smad signaling pathway, while concurrently inhibiting cellular senescence. CGA administration significantly reduced the expression of p21, senescence-associated secretory phenotype (SASP) production, and SA-β-Gal activity in skin cells. Additionally, CGA treatment notably enhanced mitochondrial function, improving disrupted mitochondrial cristae in senescent cells and boosting the oxidative phosphorylation (OXPHOS) process. ATP levels increased by approximately 40-80% following CGA treatment. Mitochondrial transplantation further confirmed CGA's anti-aging effects are linked to mitochondrial function. CGA significantly mitigated UV-induced reduction in Col1, suppressed p21 expression and SASP production, and improved mitochondrial morphology and structure in vivo. CGA promotes Col1 production and attenuates skin cellular senescence, with these effects being directly associated with mitochondrial regulation. Thus, CGA holds promise as a potent agent for preventing cellular senescence.