Chronic kidney disease (CKD) is commonly linked to hematopoietic abnormalities beyond the well-recognized anemia, with underlying mechanisms poorly understood. Here, we show that asymmetric dimethylarginine (ADMA), which accumulates in the CKD microenvironment, perturbs hematopoietic stem cell (HSC) homeostasis. Clinical data analysis reveals that serum ADMA levels rise with CKD progression and inversely correlate with the myeloid-to-lymphoid ratio. Notably, ADMA compromises HSC quiescence and long-term repopulating capacity, as observed in CKD mouse models established by 5/6 nephrectomy. Further investigations demonstrate that ADMA hyperactivates mammalian target of rapamycin (mTOR) signaling, promoting the transition from glycolysis to oxidative phosphorylation and inducing reactive oxygen species (ROS) overproduction in HSCs. Mechanistically, ADMA binds with high affinity to protein phosphatase 2 regulatory subunit Bβ (PPP2R2B), relieving its inhibition of mTOR signaling. Inhibition of mTOR signaling by rapamycin or clearance of ROS by N-acetyl-L-cysteine (NAC) reverses ADMA-induced HSC functional defects. Collectively, our findings identify ADMA as a key mediator of CKD-associated hematopoietic dysfunction via the PPP2R2B-mTOR-mitochondria axis, providing a potential therapeutic target.

Mechanical stimulation plays a pivotal role in bone remodeling by increasing interstitial hydrostatic pressure and generating compressive stress within the bone microenvironment. However, the effects of different loading conditions on osteoblast responses and the underlying mechanotransduction mechanisms remain incompletely understood. In the present study, we utilized the MechanoCulture TR hydrostatic stimulation system to evaluate the effects of different hydrostatic compressive stress magnitudes and loading durations on MC3T3-E1 cells and elucidate the role of the ROCK signaling pathway in stress-induced proliferation. Among the conditions tested at a fixed frequency of 0.05 Hz, hydrostatic compressive stress at 5 kPa for 45 min elicited the strongest proliferative response and was accompanied by F-actin reorganization. Furthermore, compressive loading significantly upregulated the expression of ROCK, Cyclin D1, and CDK4. Conversely, inhibition of ROCK with Y-27632 reduced ROCK activity, altered F-actin organization, suppressed cell proliferation, and attenuated compression-induced upregulation of Cyclin D1 and CDK4. In conclusion, hydrostatic compressive stress promotes MC3T3-E1 cell proliferation through ROCK-dependent regulation of Cyclin D1 and CDK4. These findings support an important role for ROCK in the proliferative response of osteoblasts to hydrostatic compressive stress.

Innervation is essential for tissue healing, yet strategies to enhance nerve growth during repair remain limited. Here, we show that chemically modified messenger RNA (cmRNA) encoding nerve growth factor (NGF) can stimulate neurogenic responses in vitro. NGF cmRNA was delivered to human bone marrow-derived mesenchymal stem cells using lipid carriers, leading to efficient NGF production. In co-culture, NGF-secreting stem cells induced differentiation of neural crest-derived PC-12 cells, as evidenced by increased neurite outgrowth and branching. Proteomic analyses revealed activation of neurogenesis-related pathways in both cell types. Notably, NGF cmRNA-transfected stem cells produced lower NGF levels than recombinant protein yet achieved comparable neurite-outgrowth response, demonstrating the efficiency of the transcript-based approach. These findings demonstrate that NGF cmRNA can induce NGF production by hMSCs and promote neurite outgrowth in NGF-responsive PC12 cells in vitro. This approach may provide the neurotrophic component of future regenerative strategies combining NGF cmRNA with osteogenic signals; however, its effects on functional tissue innervation and bone healing remain to be established in vivo.

Tumor metastasis and drug resistance are the leading causes of mortality in patients with colorectal cancer (CRC). Protein palmitoylation exerts a pivotal role in the metabolic reprogramming across various malignancies. However, the precise mechanisms underlying its contribution to CRC metastasis and drug resistance via lipid metabolism reprogramming remain elusive. Therefore, this study aimed to elucidate the specific role of ZDHHC9 in conferring Cetuximab resistance in CRC. Initial phenotypic screening with the broad-spectrum inhibitor 2-BP implicated palmitoylation in Cetuximab resistance. Subsequently, targeted genetic experiments identified ZDHHC9 as the specific molecular driver. Furthermore, ZDHHC9 was significantly upregulated in CRC tissues and strongly correlated with poor patient prognosis. Subsequent in vitro and in vivo experiments demonstrated that ZDHHC9 functionally promoted Cetuximab resistance in CRC. Mechanistically, ZDHHC9 physically interacted with ACSL3 and enhanced its palmitoylation, thereby driving the malignant progression of CRC. Moreover, we identified cysteine 468 (C468) as the critical residue responsible for the palmitoylation of ACSL3. Notably, ectopic overexpression of ACSL3 effectively rescued the suppression of CRC cell proliferation induced by ZDHHC9 depletion. In conclusion, our findings establish the ZDHHC9-ACSL3 axis as a preclinically validated resistance driver and an actionable metabolic vulnerability for overcoming Cetuximab resistance in CRC.

Irregular circadian rhythm drives tumor malignant progression by governing a range of cellular processes, but its roles in gallbladder carcinoma (GBC) remain unclear. As a potential tumor suppressor gene, Per3 was down-regulated in GBC with a poor prognosis. Overexpression of Per3 inhibited GBC growth and metastasis in vitro and in vivo. Mechanistically, Per3 combined with P4HA1 to promote degradation of HIF-1α protein, consequently, HIF-1α-targeted genes were transcriptionally down-regulated to inhibit glycolytic capacity. Meanwhile, the key inducers of epithelial-mesenchymal transition (EMT), TGFB1 and TGFBI were transcriptionally repressed by Per3 combining at the promoters, subsequently reducing the mRNA levels of EMT-related genes and blocking EMT progression. The decrease of Per3 was determined at protein post-translational modification (PTM), not at transcription. Mass spectrometry identified that CKII, not CKI or other kinases, phosphorylated Per3 at S1048, which promoted ubiquitination-dependent degradation of Per3, leading to loss of its tumor-suppressive function. Collectively, these results reveal the mechanisms of Per3 S1048 phosphorylation-driven degradation and the roles of Per3 in regulating HIF-1α/glycolysis and TGF-β/EMT in gallbladder carcinoma.

Drosophila development is a highly regulated process governed by the precise timing and spatial cues of hormones and signaling pathways. The follicular epithelium of the Drosophila egg chamber has been a well-studied model system for identifying key molecular regulators of morphological changes and cell migration. Broad (Br), a zinc finger transcription factor, is a well-known early response gene of the steroid hormone ecdysone, which is essential for the initiation of metamorphosis. Our previous findings have shown that Br is a target of Notch signaling, and its suppression is required for proper epithelial cell cuboidal-to-squamous transition during Drosophila oogenesis. Here, we report that proper border cell (BC) migration requires the suppression of Br expression during Drosophila oogenesis. Br expression can be induced by Notch signaling as early as stage 4, but its expression decreases in BCs during cell migration at stage 9. Further findings revealed that JAK/STAT and ecdysone signaling are highly active during the onset of BC migration and contribute to Br suppression. We demonstrated that JAK/STAT suppresses Br expression to facilitate proper BC migration. In addition, we analyzed single-cell RNA sequencing data to highlight the shift in gene expression while Br was suppressed and BCs became mobile. The analysis confirmed the high expression of known BC markers, including slbo and sn, and identified br as specifically repressed in BCs, supporting the quality of the dataset and our experimental findings. Gene Ontology enrichment analysis revealed significant overrepresentation of biological processes related to cell migration, morphogenesis, and cell junction assembly, all of which are important for BC morphological transition and migration.

Hair follicle stem cells (HFSCs) reside in a relatively hypoxic niche and preferentially use glycolytic metabolism, but how metabolic adaptation is coupled to epigenetic regulation of HFSC function remains incompletely understood. Since oxidative phosphorylation produces more reactive oxygen species (ROS) than glycolysis, and excessive ROS production eliminates various stem cell properties including quiescence and self-renewal. Stem cells prefer glycolysis as their main energy source. Here, cultured human HFSCs were exposed to 2%O2. Hypoxia increased HIF-1α expression, glycolysis-related gene expression, intracellular H3K18 histone lactylation (H3K18la), EdU incorporation, colony-forming efficiency, and HFSC-associated markers CK15, CK19, and CD34 expression. Separate treatment with the glycolysis inhibitors 2-deoxy-D-glucose (2-DG) or oxamate reduced lactate accumulation, H3K18la, and HFSC-associated marker expression. Integrated H3K18la CUT&Tag and RNA sequencing identified 822 genes with concordantly increased H3K18la peaks and mRNA levels, with ubiquitin-mediated proteolysis among the enriched pathways. FBXW11 was prioritized as a candidate E3 ubiquitin ligase target. IGV visualization and H3K18la ChIP-qPCR showed increased H3K18la enrichment at the FBXW11 locus under hypoxia, whereas 2-DG or the p300 inhibitor C646 attenuated H3K18la enrichment and FBXW11 expression. FBXW11 overexpression increased EdU incorporation, colony formation, and HFSC-associated marker expression, while FBXW11 knockdown attenuated hypoxia-induced clonogenic growth and proliferation. In an ex vivo whole-hair-follicle culture model, hypoxia enhanced hair shaft elongation, and this effect was reduced by FBXW11 knockdown. Together, these findings support a hypoxia-glycolysis/lactate-H3K18la-FBXW11 pathway that contributes to proliferative and self-renewal-associated phenotypes of cultured HFSCs and to hypoxia-responsive hair shaft growth ex vivo. Further in vivo studies are required to establish its role in physiological hair cycle.

Pyroptosis is a gasdermin-mediated form of inflammatory cell death that may alter tumour-immune interactions. Its canonical, non-canonical and granzyme-associated pathways, together with NINJ1-dependent terminal membrane rupture, are reviewed here. Whether these mechanisms promote antitumour immunity or sustained inflammation depends on biological context. Pediatric ovarian and adnexal neoplasms include germ-cell tumours (GCTs), sex cord-stromal tumours (SCSTs), and less-common epithelial or other lesions. Histology, age, pubertal status, hormonal secretion, stage, and treatment exposure vary substantially across patients. Direct studies of pyroptosis and immune remodelling in these paediatric histologies remain scarce; evidence from adult epithelial ovarian cancer, non-ovarian tumours, cell lines, and mice cannot be treated as proof of a paediatric mechanism. This review retains a histology-specific framework to examine molecular pathways, the paediatric ovarian tumour immune microenvironment (TIME), candidate therapeutic approaches, and biomarkers. Established molecular or clinical observations are separated from cross-tumour extrapolations and untested hypotheses. Particular attention is paid to developmental immunity, endocrine context, on-target and off-target inflammation, and evidence required before clinical translation.

Steroid-induced osteonecrosis of the femoral head (SONFH) is characterized by marrow adiposity, trabecular deterioration, and impaired bone repair, but the progenitor-level regulatory mechanisms underlying this process remain incompletely defined. Here, we investigated whether dysregulated bone marrow stromal cell (BMSC) lineage commitment contributes to SONFH progression and identified a nucleus-associated miRNA mechanism that promotes adipogenic skew. Human femoral heads from patients with femoral neck fracture or SONFH showed increased empty osteocytic lacunae, trabecular rarefaction, and Perilipin-1-positive marrow adiposity. Patient-derived SONFH BMSCs displayed enhanced adipogenic potential and impaired osteogenic differentiation, accompanied by increased C/EBPα/PPARγ and reduced RUNX2/OCN expression. Cross-source miRNA prioritization and validation identified miR-661 as an upregulated candidate associated with this lineage imbalance. miR-661 overexpression promoted lipid accumulation, suppressed mineral deposition, and activated a pro-adipogenic transcriptional program. Mechanistically, miR-661 showed nuclear enrichment and did not induce a detectable change in β-catenin expression or nuclear localization, directing our focus to the C/EBPα-PPARγ axis. ChIP-qPCR, promoter DNA pull-down, dual-luciferase reporter assays, and C/EBPα RIP-qPCR showed that miR-661 was associated with increased C/EBPα occupancy at the PPARG promoter. Importantly, C/EBPα knockdown attenuated miR-661-induced PPARG, FABP4, and adipogenic activation, whereas C/EBPα rescue restored these effects. Nuclear AGO2-RIP-qPCR further demonstrated enrichment of miR-661 in nuclear AGO2-containing complexes, and AGO2 ChIP-qPCR showed miR-661-dependent AGO2 occupancy at the PPARG promoter, which was reduced by miR-661 inhibition and lost with a seed-mutant miR-661 mimic. In vivo, local antagomiR-661 delivery alleviated steroid-induced osteonecrosis-like bone loss and marrow adiposity. These findings identify miR-661 as a nuclear, AGO2-associated regulator of the C/EBPα-PPARγ axis that drives BMSC adipogenic lineage bias in SONFH.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related death, and systemic therapy with sorafenib is limited by primary and acquired resistance. Copper is now recognized both as a growth-promoting cofactor in HCC and as the trigger of cuproptosis, a copper-dependent form of regulated cell death executed through ferredoxin-1 (FDX1)-mediated reduction of Cu[2+], aggregation of lipoylated tricarboxylic acid (TCA) cycle proteins such as dihydrolipoamide S-acetyltransferase (DLAT), loss of Fe-S cluster proteins and proteotoxic mitochondrial stress. This review is deliberately restricted to a single mechanistic axis - copper handling, cuproptosis competence and sorafenib response in HCC - and grades the evidence supporting it. We distinguish direct experimental demonstrations of cuproptosis from copper overload, oxidative stress and mitochondrial injury, which are frequently but incorrectly treated as equivalent, and we apply the same standard to the noncoding RNA and immune literature, in which most cuproptosis-related lncRNA and miRNA signatures remain computationally derived and lack mechanistic proof or external validation. Current data indicate that sorafenib can create an intracellular environment permissive for cuproptosis, mainly through glutathione depletion and altered copper-transporter expression; a direct, sorafenib-specific action on the cuproptosis machinery, including the proposed stabilization of FDX1, is plausible but not yet established. We further summarize the emerging determinants of cuproptosis resistance and the translational barriers - systemic and hepatic copper toxicity, cirrhosis-related vulnerability, tumor heterogeneity, unfavorable pharmacokinetics of copper ionophores and chelators, and the absence of validated predictive biomarkers - that must be addressed before cuproptosis-directed strategies can reasonably be tested in patients with HCC.

Chronic vascular endothelial inflammation plays a pivotal role in the pathogenesis of coronary artery disease (CAD). N[6]-methyladenosine (m[6]A), the predominant epitranscriptomic modification in messenger RNA (mRNA), regulates pro-atherogenic inflammatory responses in coronary endothelial cells. This study explores the role of the m[6]A methyltransferase METTL3 in CAD-associated endothelial inflammation through its regulation of DNA methyltransferase 1 (DNMT1) mRNA stability. Using cultured human coronary artery endothelial cells (HCAECs), an apolipoprotein E-deficient (ApoE[-/-]) mouse model of atherosclerosis, and clinical atherosclerotic specimens from CAD patients, a marked increase in METTL3 expression was observed in CAD endothelium and within plaques. METTL3 knockdown strongly suppressed tumor necrosis factor-α (TNF-α)-induced expression of pro-inflammatory mediators interleukin-6 (IL-6) and interleukin-1β (IL-1β), while upregulating anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10). Mechanistically, METTL3 bound to and deposited m[6]A modifications in the 3' untranslated region (3'UTR) of DNMT1 mRNA, thereby enhancing its association with the m[6]A reader protein YTH N6-methyladenosine RNA binding protein 1 (YTHDF1). This interaction increased DNMT1 mRNA stability and elevated its protein levels. Furthermore, endothelial-specific Mettl3 deletion significantly reduced aortic atherosclerotic lesion area and inflammatory infiltration in ApoE[-/-] mice. In summary, METTL3-mediated m[6]A modification promotes DNMT1 expression by stabilizing its mRNA, thereby aggravating vascular endothelial inflammation in CAD. The METTL3-m[6]A-DNMT1 axis may represent a potential therapeutic target for CAD.