Mast cells (MCs) are widely distributed sentinel cells of the myeloid lineage with substantial migratory capacity. Their activation is initiated by receptor-mediated signaling and culminates in the release of prestored mediators and cytokine secretion, processes tightly controlled by the lipid phosphatase SH2-containing inositol 5'-phosphatase 1 (SHIP1). Although MCs frequently reside within hypoxic microenvironments, the impact of hypoxia/reoxygenation on their activation remains elusive. Here we investigated the hypoxia tolerance of MCs and assessed whether hypoxia (≤1% O2) or subsequent reoxygenation (21% O2) induces degranulation and/or cytokine production. We further assessed the role of SHIP1 in posthypoxic MC activation. Primary mouse bone marrow-derived MCs (BMMCs) from wildtype and Ship1[-/-]mice were subjected to defined periods of hypoxia and subsequent reoxygenation to meticulously analyze their viability, metabolic activity, degranulation, and cytokine secretion. BMMCs were much more resilient to hypoxia than other primary murine cell types, such as microglia, astrocytes, and fibroblasts. A 3 h hypoxic exposure induced sufficient hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors carbonic anhydrase IX and glucose transporter 1, with higher baseline expression in Ship1[-/-] BMMCs. Hypoxia increased secretion of IL-6 and TNF-α, but failed to trigger degranulation, even in Ship1[-/-] cells. Reoxygenation after 3 h of hypoxia further amplified cytokine secretion and HIF-1α response. Our data indicate that hypoxia activates BMMCs, causing enhanced cytokine secretion and HIF-1α pathway engagement. SHIP1 restrains hypoxia/reoxygenation-driven cytokine release, implicating PI3K/AKT signaling in this regulatory pathway. Recognizing this regulatory checkpoint may inform future efforts to modulate MC-driven cytokine responses in ischemia-reperfusion and other hypoxic inflammatory settings.

Arteriovenous fistula (AVF) is the preferred vascular access for patients with end-stage renal disease; however, its failure is primarily due to neointimal hyperplasia. Five patients who underwent initial AVF surgery served as the control group, and another five patients with failed AVF surgery served as the experimental group. Herein, we employed mass spectrometry (MS)-based quantitative proteomics coupled with tandem mass tag labeling to screen differentially expressed proteins (DEPs) in the anastomotic cephalic vein, followed by bioinformatics analyses and verification experiments. A total of 121 DEPs were identified in the failed AVF group. GO analysis was primarily enriched in protein binding, nucleic acid binding, enzyme binding, mRNA binding, cadherin binding, catalytic activity, and cell adhesion molecule binding. KEGG pathways were mainly enriched in cell aggregation and adhesion, actin cytoskeleton, extracellular matrix-receptor interaction, PI3K-Akt signaling pathway, complement and coagulation cascades, and cholesterol metabolism. Protein-protein interaction network consisted of 86 (71.07%) DEPs, including complement VII (C7), factor IX (F9), SERPINC1, microfibril-associated glycoprotein 4 (MFAP4), complement C1s subcomponent, complement C1q subcomponent subunit A, complement C1q subcomponent subunit B, tissue factor, and von Willebrand factor, which interacting with numerous other proteins. In the expanded validation for different patients, C7, F9, SERPINC1, and MFAP4, were verified by immunohistochemical staining and Western blotting, which were consistent with the proteomics results. Collectively, this study identifies a series of potential diagnostic biomarkers, and explores the underlying mechanisms associated with AVF dysfunction.

Anthracycline-induced cardiotoxicity remains a major limitation of cancer therapy, and effective preventive strategies are lacking. Topoisomerase IIb has been implicated as a central driver of this toxicity, suggesting that epigenetic regulators may interfere with the pathological cardiac response. Here, we show that doxorubicin promotes topoisomerase IIb accumulation at cardiomyocyte-specific gene promoters (e.g., Actc1, Myl2, and Myh7) overlapping myocyte enhancer factor 2 binding sites and enhances myocyte enhancer factor 2 -dependent transcription. This response is attenuated by the pan-histone deacetylase inhibitor suberoylanilide hydroxamic acid. Suberoylanilide hydroxamic acid -mediated cardioprotection requires class IIa histone deacetylases, as genetic loss of HDAC4 abolishes its effect. Mechanistically, suberoylanilide hydroxamic acid induces acetylation of the chaperone 14-3-3, disrupting its interaction with HDAC4/5, promoting their nuclear accumulation, and repressing myocyte enhancer factor 2 - driven transcription. In vivo, suberoylanilide hydroxamic acid mitigates doxorubicin-induced cardiotoxicity. These findings identify histone deacetylase inhibition as a cardioprotective repurposing strategy and reveal a mechanistic link between epigenetic regulation and anthracycline-associated cardiotoxicity.

Patients with left ventricular assist devices (LVADs) represent a high-risk population at risk of both bleeding and thrombotic complications. A review of the available literature on nonfibrinogen factor concentrate use in LVAD patients up to June 1, 2026, was conducted by searching MEDLINE, Embase, and clinical trial and review registries. A total of 2,355 studies were identified, and 14 retrospective studies were included. Seven studies used factors to treat bleeding during LVAD implant or exchange. In this context, 134 patients received prothrombin complex concentrate (PCC), 487 received usual management or frozen plasma, 82 received factor eight inhibitor bypassing activity (FEIBA), 78 received recombinant activated factor VII (rVIIa), and 5 received isolated factor IX complex/3F-PCC. Given the limited and heterogeneous evidence, this review provides a descriptive summary of eligible studies rather than a formal evidence synthesis. When used first-line and protocolized, PCC is likely safe and effective. rFVIIa appears effective, but higher doses are associated with thrombosis and mortality. Evidence for FEIBA is limited to one small nonrandomized study. Seven studies used factors for emergency anticoagulation reversal, with 139 individual PCC exposures, 47 frozen plasma exposures, and 3 rVIIa exposures. PCC was associated with more rapid International Normalized Ratio normalization, no excess harm, and, in one study, improved survival and progression to transplant. In this context, rFVIIa was associated with a dose-dependent risk of thrombosis. Overall, PCC-based hemostatic management in LVAD patients appears reasonable; however, it is not currently possible to judge the safety of low-dose rVIIa or FEIBA.

Photodynamic therapy (PDT) is a clinically established approach that relies on the localized generation of reactive oxygen species (ROS) mediated by a photosensitizer (PS), light, and molecular oxygen. Protoporphyrin IX (PpIX) is an approved PS; however, its intracellular retention is limited, factor that can alter its overall photodynamic efficiency. In this study, we report how the design of PpIX derivatives bearing piperazine (PpIX-Pip) or morpholine (PpIX-Morp) substituents may modulate lipophilicity, cellular uptake, oxidative stress, and phototoxicity. The photodynamic activity of these derivatives was evaluated in the aggressive triple-negative human breast cancer cell line MDA-MB-231. Both derivatives showed greater time-dependent cellular retention and enhanced photodynamic activity compared to PpIX, promoting oxidative stress, and triggering loss of mitochondrial membrane potential and caspase-3 activation. These results highlight the impact of rational structural modification of PpIX on photodynamic efficiency and provide valuable structure-activity relationship insights for the development of improved porphyrin-based PS for PDT.

Background: Prophylaxis in children with hemophilia B (HB) lacks quantitative approaches that integrate both plasma exposure and tissue distribution. This study aimed to develop and validate a physiologically based pharmacokinetic (PBPK) model of factor IX (FIX) for pediatric HB. The model incorporated the binding of FIX to type IV collagen (Col4) to characterize its distribution in both plasma and extravascular tissues. Methods: A total of 20 children with severe HB were included, contributing 219 plasma samples. The base PBPK model was first established and verified using adult and plasma-derived FIX (pdFIX) data. It was subsequently extrapolated to children by integrating FIX-CTBB parameters and pediatric observations for model calibration. The validated model was used to characterize plasma pharmacokinetics, predict tissue distribution and target attainment, and simulate alternative prophylactic dosing regimens. Results: The model adequately described the plasma pharmacokinetics of FIX in children and predicted substantial extravascular distribution. Total extravascular exposure was approximately sixfold higher than plasma exposure. Marked heterogeneity in target attainment was identified across tissues. Lower target attainment was observed in the colon, pancreas, and brain, whereas delayed attainment occurred in bone and muscle. Simulations of prophylactic dosing regimens suggested that 75 IU/kg twice weekly may provide a favorable balance among sustained FIX exposure, tissue-level target attainment, and treatment burden. Conclusions: This PBPK model provides a mechanistic and quantitative framework for characterizing plasma and tissue exposure to FIX in children with HB and may support individualized optimization of FIX prophylactic dosing.

Skeletal muscle development and regeneration rely on coordinated transcriptional programs controlled by muscle stem cells and their progeny. Nuclear Factor I X (NFIX) plays essential roles in fetal myogenesis and adult muscle regeneration. However, the mechanisms regulating its transcription and isoform expression remain unclear. Here, integrating multi-omics analyses with in vitro functional assays, we define the transcriptional and epigenetic landscape controlling Nfix expression during skeletal muscle development and regeneration. We show that Nfix promoter usage is dynamically regulated according to myogenic cell state: promoter 2 is associated with quiescent and stem-like states, whereas promoter 1 is activated during myogenic commitment and regeneration. These programs are accompanied by differential enhancer accessibility and DNA methylation changes within the Nfix locus, indicating coordinated epigenetic regulation. We further demonstrate that alternative promoter usage and exon 7/9 splicing generate distinct NFIX isoforms affecting myoblast proliferation and fusion, while transcriptomic profiling identified NFIX-dependent networks involved in muscle structure and metabolism. Overall, our findings uncover multilayered mechanisms controlling Nfix expression and identify promoter- and isoform-specific NFIX programs associated with stem, regenerative, and differentiated myogenic states. These results provide a molecular framework for future therapeutic strategies targeting NFIX in muscle diseases.

The structural and spectroscopic properties of well-defined NH⋯S hydrogen bonding interactions is scarce in the literature, only few suitable model compounds have been described. Herein, we describe a set of macrocyclic Zn amino-thiophenolato complexes and their deuterated derivatives, in which secondary amino groups are brought into close proximity to Zn-bound S (thiophenolate) atoms, thereby stabilizing intramolecular R2NH⋯S bonding interactions. Metrical data, spectroscopic signatures, strength, type, and dynamic properties of these NH⋯S bonding interactions were determined for the liquid and solution state, and referenced to an integrated, non-H bonded NH donor serving as "internal standard". Structural and spectroscopic properties for the NH⋯S bonds are as follows: (i) N⋯S bond and H⋯S bond distances ∼3.14 Å and ∼2.36 Å, respectively (ii) frequency shift Δν ∼ 130 cm[-1] (for ν(NH)), (iii) no intensity enhancement of the ν(NH) stretching band, (iv) relative line broadening factor of the ν(NH) bands (FWHM(ν(NH⋯S))/FWHM(ν(NH)) ∼ 2.0), (v) wavenumber ratio ν(NH⋯S)/ν(ND⋯S) = 1.34, (vi) downfield shift of 1H NMR signals Δδ ∼ 5 ppm (for δ(NH)), (vii) bond critical point of type (3, -1) (i.e. attractive H⋯S interaction, QTAIM analysis), (viii) dominant electrostatic interaction with a binding energy of ∼ 9 kcal mol[-1] (at B3LYP-D3/def2-TZVP level of theory), (ix) according to NBO analysis, the NH⋯S bond is described by the type lone pair(S) → σ*(N-H) interaction with stabilization energies of ∼ 7.5 kcal mol[-1], and (x) NH/ND exchange rate slower than NMR timescale. These findings offer benchmarks for theoretical explorations and molecular design of specific, intramolecular R2NH⋯S bonding interactions within constrained environments, that may also be relevant for understanding sulfur-containing active sites in biological systems.

Cofactor-independent activity of FIX has previously been achieved in FIX-IDAV and FIX-FIAV variants containing L6F, V181I, E185D, K265A, and I383V substitutions. Cofactor-dependent FIX hyperactivity was attained through the V10K, R338L, and S377W (KLW) modifications. We evaluated whether combining IDAV/FIAV and KLW substitutions could enhance cofactor-independent activity and elucidated the biochemical mechanisms underlying FVIII-independent FIX-FIAV function. Recombinant FIX variants, expressed in HEK293 cells, were analyzed for FIX-specific and FVIII-equivalent clotting activities. Highly purified FIX-FIAV was characterized using purified component assays and FXIa-triggered thrombin generation in FVIII-deficient plasma. FIX-IDAV-KLW and FIX-FIAV-KLW exhibited 20-28-fold higher FIX-specific activity and a modest twofold increase in FVIII-equivalent clotting activity. Owing to thrombotic safety concerns, KLW combinations were not further pursued. FIX-FIAV displayed normal FIX-specific activity and fourfold enhanced FVIII-equivalent clotting activity. The FIAV substitutions did not alter activation by the extrinsic (TF-FVIIa) or intrinsic (FXIa) pathways, nor cofactor-dependent FX activation, but conferred FVIII-independent FX conversion. Kinetic analyses revealed a modified active site conformation with a trend toward reduced antithrombin inhibition. In FVIII-deficient plasma, FIX-FIAV dose-dependently increased FXIa-triggered thrombin generation by shortening the lag time and time to peak and shifted coagulation phenotype categories based on FVIII-equivalent thrombin generation (TG) activity from severe toward moderate/mild ranges in plasma-based models, without exceeding normal FVIII-equivalent TG activity. FIX-FIAV represents a FIX variant with FVIII-independent activity, normal FIX-specific activity, and reduced inactivation by antithrombin. Targeted modification of regions surrounding the FIXa active site enables allosteric activation, providing a potential foundation for novel bypassing strategies in hemophilia A therapy.