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DNA double-strand breaks (DSBs) can be induced by cellular byproducts or genotoxic agents. Improper processing of these lesions leads to increased genome instability, which constitutes a hallmark of pathological conditions and fuels carcinogenesis. DSBs are primarily repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) and the proper balance between these two pathways is finely modulated by specific molecular events. Here, we report that the histone chaperone DAXX plays a fundamental role in the response to DSBs. Indeed, in human cells, DSBs induce ATM/ATR-dependent phosphorylation of DAXX on serine 424 and 712 and promote its binding to chromatin and the deposition of the histone variant H3.3 in proximity to DNA breaks. Enrichment of H3.3 at DSBs promotes 53BP1 recruitment to these lesions and the repair of DNA breaks by HR pathways. Moreover, H3.3-specific post translational modifications, particularly K36 tri-methylation, play a key role in these processes. Altogether, these findings indicate that DAXX and H3.3 mutations may contribute to tumorigenesis-enhancing genome instability.

Adipose tissue thermogenesis is a promising strategy to counter obesity and metabolic disease, but sustained activation of thermogenic adipocytes elevates oxidative and lipid-peroxidation stress, increasing susceptibility to ferroptotic cell death. Existing models often treat redox buffering, hypoxia signaling and ferroptosis as separate processes, which cannot explain why similar interventions-such as antioxidants, β-adrenergic agonists or iron modulators-alternately enhance thermogenesis or precipitate tissue failure. Here, we propose the Redox-Thermogenesis-Ferroptosis Compass (RTF-Compass) as a framework that maps adipose depots within a space defined by ferroptosis resistance capacity (FRC), ferroptosis signaling intensity (FSI) and HIF-1α-dependent hypoxic tone. Within this space, thermogenic output follows a hormetic, inverted-U trajectory, with a Thermogenic Ferroptosis Window (TFW) bounded by two failure states: a Reductive-Blunted state with excessive antioxidant buffering and weak signaling, and a Cytotoxic state with high ferroptotic pressure and inadequate defense. We use this model to reinterpret genetic, nutritional and pharmacological studies as state-dependent vectors that move depots through FRC-FSI-HIF space and to outline principles for precision redox medicine. Although the TFW is represented as coordinates in FRC-FSI-HIF space, we use 'Compass' to denote a coordinate framework in which perturbations act as vectors that orient depots toward thermogenic or cytotoxic outcomes. Finally, we highlight priorities for testing the model in vivo, including defining lipid species that encode ferroptotic tone, resolving spatial heterogeneity within depots and determining how metabolic memory constrains reversibility of pathological states.

Pulmonary hypertension (PH) causes morbidity and mortality in sickle cell disease (SCD). The release of heme during hemolysis triggers endothelial dysfunction and contributes to PH. Long non-coding RNAs (lncRNAs) may play a pivotal role in endothelial dysfunction and PH pathogenesis. This study assessed the regulatory role of the lncRNA-heme oxygenase-1 (HMOX1) axis in SCD-associated PH pathogenesis. Total RNAs were isolated from the lungs of 15-17-week-old sickle cell (SS) mice and littermate controls (AA) mice and subjected to lncRNA expression profiling using the Arrystar™ lncRNA array. Volcano plot filtering was used to screen for differentially expressed lncRNAs and mRNAs with statistical significance (fold change > 1.8, p < 0.05). A total of 3915 lncRNAs were upregulated and a total of 3545 lncRNAs were downregulated in the lungs of SS mice compared to AA mice. To validate differentially expressed lncRNAs, six upregulated lncRNAs and six downregulated lncRNAs were selected for quantitative PCR. MALAT1 expression was significantly upregulated in the lungs of SS mice and in hemin-treated human pulmonary artery endothelial cells (HPAECs), suggesting that hemolysis induces MALAT1. Functional studies revealed that MALAT1 depletion increased, while MALAT1 overexpression decreased, the endothelial dysfunction markers endothelin-1 (ET-1) and vascular cell adhesion molecule-1 (VCAM1), indicating a protective role of MALAT1 in maintaining endothelial homeostasis. In vivo, adenoviral MALAT1 overexpression attenuated PH, right ventricular hypertrophy (RVH), vascular remodeling, and reduced ET-1 and VCAM1 expression in SS mice. Given that HMOX1 protects endothelial cells during hemolysis, we observed that HMOX1 expression and activity were elevated in SS mouse lungs and hemin-treated HPAECs. HMOX1 knockdown enhanced ET-1 and VCAM1 expression, confirming its endothelial-protective function. Importantly, MALAT1 overexpression increased HMOX1 expression and activity, whereas MALAT1 knockdown reduced HMOX1 levels and mRNA stability. Collectively, these findings identify MALAT1 as a protective regulator that mitigates endothelial dysfunction, vascular remodeling, and PH in SCD, at least in part through the induction of HMOX1. These results suggest that SCD modulates the MALAT1-HMOX1 axis, and further characterization of MALAT1 function may provide new insights into SCD-associated endothelial dysfunction and PH pathogenesis, as well as identify novel therapeutic targets.

Malaria remains a major global health burden, and the emergence of resistance to blood stage antimalarials underscores the need for new interventions targeting earlier stages of the parasite's life cycle. The pre-erythrocytic liver stage represents a critical bottleneck and an attractive target for chemotherapeutic and prophylactic interventions. In this study, we functionally characterized the putative E3 ubiquitin ligase Trophozoite Exported Protein 1 (TEX1; PBANKA_0102200) in Plasmodium berghei using gene knockout, tagging, and imaging approaches across the mosquito and liver stages. TEX1 knockout parasites (PbTEX1-KO) showed impaired development during mosquito-stage transitions, with significant reductions in ookinete formation, oocyst numbers, and sporozoites reaching the salivary glands. In hepatic stages, TEX1-KO parasites displayed reduced growth, abnormal nuclear division, and impaired liver stage maturation, ultimately leading to a dramatic decline in detached cell formation and blood stage infectivity. Endogenous C-terminal tagging of TEX1 with GFP and 3×HA revealed a discrete subnuclear localization pattern, indicating a critical role in DNA synthesis and/or mitotic regulation. Our findings reveal that TEX1 is required for nuclear replication and division and successful development in both the mosquito and liver stages of Plasmodium. Given its pivotal role and nuclear localization during hepatic schizogony, TEX1 represents a promising target for the development of liver stage antimalarial interventions.

Overwinter Syndrome (OWS) affects grass carp (Ctenopharyngodon idellus) aquaculture in China, causing high mortality and economic losses under low temperatures. Failure of antibiotic therapies shows limits of the 'low-temperature-pathogen' model and shifts focus to mucosal barrier dysfunction and host-microbiome interactions in OWS. We compared healthy and diseased grass carp collected from the same pond using histopathology, transcriptomics, proteomics, and metagenomics. This integrated approach was used to characterize intestinal structure, microbial composition, and host molecular responses at both taxonomic and functional levels. Results revealed a three-layer barrier failure in OWS fish: the physical barrier was compromised, with structural damage and reduced mucosal index; microbial dysbiosis featured increased richness without changes in diversity or evenness, and expansion of the virobiota, notably uncultured Caudovirales phage; and mucosal immune dysregulation indicated loss of local immune balance. Multi-omics integration identified downregulation of lysosome-related and glycosphingolipid biosynthesis pathways at transcript and protein levels, with disrupted nucleotide metabolism. Overall gut microbial richness, rather than individual taxa abundance, correlated most strongly with host gene changes linked to immunity, metabolism, and epithelial integrity. Although biological replicates were limited by natural outbreak sampling, matched high-depth multi-omics datasets provide exploratory insights into OWS-associated intestinal dysfunction. In summary, OWS entails a cold-triggered breakdown of intestinal barrier integrity and immune homeostasis. This breakdown is driven by a global restructuring of the gut microbiome, which is marked by increased richness, viral expansion, and functional shifts, ultimately resulting in altered host-microbe crosstalk. This ecological perspective informs future mechanistic and applied studies for disease prevention.

Breast cancer is a heterogeneous disease that exists in multiple subtypes, some of which still lack targeted and effective therapy. A major challenge is to unravel their underlying molecular mechanisms and bring to light novel therapeutic targets. In this study, we investigated the role of WNT-inducible signaling pathway protein 1 (WISP1) matricellular protein in the acquirement of an invasive phenotype by breast cancer cells. To this aim, we treated non-invasive MCF7 cells with WISP1 and assessed the expression levels of macrophage migration inhibitory factor (MIF) and its cellular receptor CD74. Next, we examined the expression of epithelial-to-mesenchymal transition (EMT) markers as well as molecular effectors of the tumor microenvironment, such as CD44, the main hyaluronan receptor that also acts as a co-receptor for MIF, the hyaluronan oncogenic network, and specific matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs). The results showed that WISP1 potently induces the expression of MIF cytokine and affects the expression of specific extracellular matrix molecules with established roles in the promotion of malignant properties. Notably, Src kinases and MIF are critically involved in these processes. Collectively, the present study demonstrates for first time a WISP1/Src/MIF axis as well as its ability to induce an invasive phenotype in MCF7 cells and highlights novel cellular and molecular processes involved in the epithelial-to-mesenchymal transition and the development of invasive breast cancer. This suggests that specific cues from the tumor microenvironment can activate a migratory/invasive phenotype in a subpopulation of cells residing within the heterogeneous breast tumor.

The Repulsive Guidance Molecule a (RGMa) is a multifunctional GPI-anchored protein localized in the sarcolemma and sarcoplasm of the adult skeletal muscle cell. Our research group showed that RGMa overexpression can promote myoblast fusion and induce hypertrophic muscle fibers during in vitro differentiation. Here, we report that RGMa is expressed in primary skeletal muscle cells cultured in vitro, showing a nuclear localization, revealed by immunostaining with an antibody targeting its C-terminal region (C-RGMa). While RGMa was detected in the nuclei, its canonical receptor, Neogenin, was predominantly found in the perinuclear region. Nuclear RGMa was absent in Neogenin-knockdown cells, suggesting that Neogenin mediates its nuclear transport. Functional assays suggested that RGMa promotes primary skeletal muscle cell viability and proliferation and supports their myogenic commitment. These findings reveal a previously unrecognized nuclear function of RGMa-Neogenin signaling and provide new insights into the regulation of skeletal muscle cell behavior in vitro.

Activation of JAK/STAT and MAPK/ERK1,2 signalling pathways has been shown to increase the production of reactive oxygen species (ROS) in multiple cell types involved in cardiovascular diseases (CVDs), including vascular smooth muscle cells (VSMCs). However, these have not yet been studied in human saphenous vein SMCs (HSVSMCs) responsible for the maladaptive remodelling leading to saphenous vein graft failure (VGF), to which patients with type 2 diabetes mellitus (T2DM) are more susceptible. Therefore, this study aimed to evaluate the contributions of the JAK/STAT and MAPK/ERK1,2 pathways towards production of mitochondrial ROS (mROS) in HSVSMCs from T2DM patients versus non-diabetic controls. HSVSMCs explanted from surplus HSV tissues from consenting patients undergoing coronary artery bypass graft surgery were stimulated in vitro with mitogenic stimuli known to be involved in neointimal hyperplasia (NIH) and VGF, which are known activators of the JAK/STAT and the MAPK/ERK1,2 signalling pathways. Flow cytometry was then used to analyse the production of mROS (superoxide) in MitoSOX-stained HSVSMCs. Additionally, we examined the effect of ruxolitinib and trametinib, selective inhibitors of JAK1/2 and MEK1/2 signalling pathways, respectively, on mROS levels in these cells. From our findings, mROS production was significantly higher in HSVSMCs from T2DM patients versus non-diabetic controls. Activation of either the JAK/STAT or MAPK/ERK1,2 signalling pathways did not significantly alter the production of mROS in HSVSMCs from both T2DM and non-diabetic patients. However, inhibition of JAK/STAT and MAPK/ERK1,2 signalling pathways with ruxolitinib and trametinib, respectively, resulted in a significant reduction in mROS in HSVSMCs from both T2DM and non-diabetic patients. Our findings demonstrate a JAK/STAT- and MAPK/ERK1,2-mediated production of mROS in HSVSMCs. Hence, they are potential targets for drug development to limit ROS production in ROS-driven proliferation and migration of HSVSMCs responsible for VGF.

CRISPR/Cas systems have transformed molecular medicine, yet the field still lacks principled guidance on when transient editing suffices versus when sustained exposure through in vivo viral delivery is necessary and how to keep prolonged exposure safe. Notably, EDIT-101 was designed for a permanent edit in post-mitotic photoreceptors with lifelong Cas9 persistence. This review addresses this gap by defining the biological and therapeutic conditions that drive benefit from extended Cas activity while minimizing risk. We will (i) examine relationships between expression window and efficacy across Cas9/Cas12/Cas13 modalities, (ii) identify genome-wide off-target liabilities alongside orthogonal assays, and (iii) discuss controllable, self-limiting, and recallable editor platforms. By separating durable edits from persistent nuclease exposure, and by providing validated control levers, this work establishes a generalizable framework for safe, higher-efficacy CRISPR medicines. Furthermore, we highlight key studies in cell lines, murine models, non-human primates, and humans that examine the long-term effects of sustained expression of CRISPR/Cas systems and discuss the safety and efficacy of such approaches. Current evidence demonstrates promising therapeutic outcomes with manageable safety profiles, although there is a need for continued monitoring as CRISPR/Cas therapies are increasingly applied in clinical contexts and therapies are developed for broader clinical applications.

Declining Leydig cell steroidogenesis contributes to late-onset hypogonadism and to age-associated impairment of male reproductive health. Determinants of dysfunction extend beyond chronological aging. This review synthesizes recent experimental and translational evidence on cellular and molecular processes that compromise Leydig cell endocrine output and the interstitial niche that supports spermatogenesis. Evidence spanning environmental endocrine-disrupting chemicals (EDCs), obesity and metabolic dysfunction, and testicular aging is integrated with emphasis on oxidative stress, endoplasmic reticulum stress, mitochondrial dysregulation, apoptosis, disrupted autophagy and mitophagy, and senescence-associated remodeling. Across model systems, toxicant exposure and metabolic stress converge on impaired organelle quality control and altered redox signaling, with downstream loss of steroidogenic capacity and, in some settings, premature senescence within the Leydig compartment. Aging further reshapes the testicular microenvironment through inflammatory shifts and biomechanical remodeling and may erode stem and progenitor Leydig cell homeostasis, thereby constraining regenerative potential. Single-cell transcriptomic atlases advance the field by resolving Leydig cell heterogeneity, nominating subsets that appear more vulnerable to stress and aging, and mapping age-dependent rewiring of interstitial cell-to-cell communication with Sertoli cells, peritubular myoid cells, vascular cells, and immune cells. Many mechanistic insights derive from rodent in vivo studies and in vitro platforms that include immortalized Leydig cell lines, and validation in human tissue and human clinical cohorts remains uneven. Together, these findings frame mechanistically informed opportunities to preserve endogenous androgen production and fertility through exposure mitigation, metabolic optimization, fertility-preserving endocrine stimulation, and strategies that target inflammation, senescence, and regenerative capacity.