Gut microbiota has emerged as a key regulator of immune, metabolic, and neuroinflammatory processes, exerting significant influence on central nervous system (CNS) function via the gut-brain axis. Growing evidence suggests that gut dysbiosis not only precedes and worsens stroke severity but is also induced by stroke itself, establishing a bidirectional and self-reinforcing pathological loop. Microbiota-derived metabolites, including short-chain fatty acids and tryptophan derivatives, modulate the activation states of microglial and border-associated macrophage (BAMs), thereby shaping neuroinflammatory responses and tissue repair mechanisms. Although microglia have been extensively studied in this context, the role of BAMs-particularly perivascular-macrophages remains comparatively underexplored, despite their critical involvement in maintaining blood-brain barrier (BBB) integrity and immune surveillance. In addition, dietary patterns strongly influence microbiota composition and, consequently, immune responses within the CNS. Collectively, these findings position gut microbiota as a dynamic regulator of brain-resident immune cells in stroke and highlight diet- and microbiota-targeted interventions as promising therapeutic strategies.

Allogeneic cell-based immunotherapies generated from pluripotent stem cells show considerable promise for the treatment of oncological, autoimmune, and viral diseases, however discovery platforms for induced pluripotent stem cell (iPSC)-derived cell therapies do not translate well to scalable manufacturing platforms. We applied a high-throughput combinatorial screening platform (CombiCult®) to identify novel, manufacturing-ready, feeder-free protocols for the generation of mature, functional NK cells from human iPSCs. We validated seven CombiCult®-derived differentiation protocols for the production of highly cytotoxic, phenotypically mature iPSC-derived NK (iNK) cells, which are comparable to donor-derived NK cells. Translation to a Stirred Tank Bioreactor (STR) system resulted in a 10x increase in productivity, from ∼20 to ∼190 iNK cells per starting iPSC. iNK cells demonstrate mature transcriptomic signatures, retained after translation to bioreactor-based production. The three-dimensional, bead-based screening approach enables seamless translation to bioreactor-based production of iNK cells exhibiting high cytotoxic activity against a range of cancer cell types.

BackgroundGlaucoma is a leading cause of irreversible blindness and is increasingly understood as a chronic neurodegenerative disorder rather than a disease explained solely by elevated intraocular pressure (IOP). Although IOP lowering remains the cornerstone of treatment, many patients continue to progress despite apparently adequate pressure control, indicating that additional mechanisms shape retinal ganglion cell (RGC) vulnerability and disease course. Among these, autophagy and mitophagy have emerged as central regulators of cellular stress adaptation in both anterior and posterior ocular tissues.Main BodyThis review argues that glaucoma can be more coherently interpreted through a stage- and compartment-specific framework of autophagy and selective mitophagy. In the conventional outflow pathway, autophagy contributes to mechanoadaptation, proteostasis, and extracellular matrix homeostasis, whereas chronic oxidative and biomechanical stress may impair lysosomal function and autophagic flux, thereby promoting outflow dysfunction and ocular hypertension. In the posterior segment, RGCs and their axons are highly dependent on autophagy for proteostasis and mitochondrial quality control because of their polarized morphology and substantial metabolic demand. Experimental work suggests that autophagy may be protective during early or acute stress but become insufficient, stalled, or maladaptive during chronic injury. Recent human stem cell and animal studies further implicate optineurin-linked autophagic-lysosomal dysfunction, AMPK–mTORC1 imbalance, and reduced PINK1/Parkin-associated mitophagy as mechanistic nodes linking mitochondrial stress to RGC degeneration. These observations support a model in which glaucoma progression reflects not simply more or less autophagy, but failure to maintain effective quality control across distinct ocular compartments and disease stages.ConclusionA compartment-aware and time-resolved view of autophagy and mitophagy offers a more nuanced framework for glaucoma pathogenesis and therapy. Future progress will likely depend less on indiscriminate pathway modulation than on restoring selective, flux-competent quality control, particularly mitochondrial turnover, in the appropriate tissue and at the appropriate stage of disease.

BackgroundPalmitoylacyltransferase DHHC7 (DHHC7) plays a critical role in various biological processes and diseases. However, its expression, localization, and regulatory mechanisms in sperm function remain largely unknown.MethodsImmunofluorescent staining was used to localize DHHC7 in sperm and examine its colocalization with the estrogen receptor (ER), progesterone receptor (PR), and caveolin-1 (CAV1). To explore DHHC7’s role in sperm function, sperm were treated with a specific DHHC7 antibody. Subsequently, sperm motility and motion parameters were analyzed using a computer-assisted sperm analyzer, and the acrosome reaction (an indicator of sperm capacitation) was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin. To explore the possible regulatory mechanisms of DHHC7 on sperm function, protein palmitoylation was assessed using the acyl-biotin exchange method. Intracellular calcium levels and reactive oxygen species (ROS) production were monitored using fluorescent probes Fluo-4 acetoxymethyl ester (Fluo-4 AM) and 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), respectively, and protein tyrosine phosphorylation levels and DHHC7 protein in sperm were analyzed using Western blotting.ResultsWe found that DHHC7 was localized in the neck, principal piece, midpiece, and end piece of sperm in both mice and humans. Treatment with DHHC7 antibodies significantly impaired sperm motility, progressive movement, hyperactivation, and the acrosome reaction under capacitated conditions. Furthermore, DHHC7 colocalized with the ER, PR, and CAV1 in mouse sperm. Additionally, DHHC7 antibodies reduced protein palmitoylation and tyrosine phosphorylation levels, and reduced intracellular calcium elevation and ROS generation during mouse sperm capacitation.ConclusionThis study identifies DHHC7 as a regulator of sperm motility and capacitation and suggests that its effects involve palmitoylation-dependent modulation of calcium signaling, tyrosine phosphorylation, and ROS signaling by the spatial association with ER, PR, and CAV1. These findings provide novel insights into the biological functions and regulatory mechanisms of DHHC7 in sperm, highlighting its potential significance in reproductive biology.

Background/ObjectiveEarly-onset fetal growth restriction (FGR) is a severe pregnancy complication caused by placental dysfunction. Although superficial trophoblast invasion and sterile inflammation are recognized as characteristics, the specific cellular driving factors and molecular mechanisms that lead to the dysregulation of the immune-trophoblast microenvironment remain unclear.MethodsWe constructed a high-resolution single-nucleus transcriptomic (snRNA-seq) atlas of placental tissues from patients with early-onset FGR and their matched normal controls. By integrating bioinformatics methods, such as pseudotime trajectory inference, gene regulatory network analysis (SCENIC), and intercellular communication modeling (CellChat), along with in vitro validation approaches, such as hypoxia-induced macrophage models, recombinant protein stimulation, and RT-qPCR, we identified specific changes associated with this disease.ResultsOur research revealed impaired differentiation trajectories of trophoblast cells, characterized by the absence of a critical intermediate state essential for acquiring invasiveness. At the same time, we identified a pathogenic Hofbauer cell subset (HBC5), which increases in number in FGR and exhibits high regulon activity of FOS and NFKB1. In vitro experiments have confirmed that hypoxia triggers the polarization of HBC5-like macrophages, leading to the upregulation of pro-inflammatory factors such as CCL4. Furthermore, the CellChat analysis combined with functional validation indicates that the CCL4 produced by HBC5 significantly induces mitochondrial stress markers GDF15 and APP in trophoblast cells, resulting in dysregulation of the placental microenvironment. Mechanistically, unlike physiological HBC that provide trophic support, HBC5 shows impaired secretion of IGF1, and implements dual blockade of inflammation and metabolism on Extravillous Trophoblasts (EVT) and the vascular system through CCL chemokines and NAMPT signaling pathways. Moreover, we have elucidated a vicious cycle: trophoblast cells under stress conditions release danger signals such as GDF15 and APP, which may continuously maintain the pathogenic immune polarization state of HBC5, and polarized HBC5 in turn stimulates trophoblast cells.ConclusionOur study reveals that the HBC5 subset with high FOS/NFKB1 regulon activity acts as a key mediator of placental niche dysregulation. The identified HBC5-CCL4-Trophoblast stress axis provides a potential therapeutic target for early-onset FGR.

Hair follicle miniaturization is a quantifiable histopathological endpoint shared by multiple forms of alopecia. The conventional “stem cell–centric” view often attributes regenerative failure to depletion or intrinsic dysfunction of hair follicle stem cells (HFSCs). However, in canonical trajectories such as human androgenetic alopecia, HFSC-related populations may remain detectable by marker-based analyses, whereas progenitor output is reduced. This pattern suggests that impaired conversion from quiescent HFSCs into an expandable progenitor/transit-amplifying compartment may contribute to miniaturization, while not excluding concomitant HFSC functional decline. We therefore propose “niche identity,” which treats the follicular niche as a set of measurable, stratifiable, and intervention-amenable structural–mechanical constraints. We posit that the collagen network may act as an integrative hub that influences regenerative thresholds and the stability of lineage output through interfacial continuity, fibrillar topology, and local mechanical states. Niche identity is defined here by five coupled state variables: basement membrane boundary integrity, adhesion/anchoring apparatuses, fibrillar topological organization, mechanical set-points, and hair cycle–scaled dynamic remodeling windows. We propose that these elements may drift coordinately under androgen-biased profibrotic remodeling, chronic low-grade inflammation with MMP-mediated matrix degradation, and aging/glycation-associated crosslinking and stiffening, thereby locking follicles into a low-output steady state. Finally, we discuss “signal–structure mismatch” as a plausible basis for unstable therapeutic responses and relapse and propose a niche identity–oriented translational framework intended to guide future experimental testing and endpoint selection.

BackgroundImmune checkpoint inhibitors (ICIs) have transformed cancer therapy but frequently induce immune-related adverse events (irAEs), which can disrupt treatment and worsen outcomes. The mechanisms predisposing certain individuals to irAEs remain unclear, and reliable strategies for early prediction and prevention are urgently needed.MethodsWe analyzed pre-treatment peripheral blood mononuclear cell (PBMC) transcriptomic data from 88 patients receiving ICIs, including 22 who subsequently developed irAEs. Immune infiltration signatures were used to build machine learning models with SHAP-based interpretability. Immune-related differentially expressed genes were then incorporated into the Chemical-Induced Gene Signature (CIGS) framework to predict candidate reversal compounds. Selected compounds were further evaluated in Jurkat T cells to experimentally validate their effects on interferon-γ signaling and underlying mechanisms.ResultsBaseline immune infiltration patterns showed strong predictive value for subsequent irAE development, with the Random Forest model achieving the best performance (AUC = 0.97). SHAP analysis revealed that activated T-cell and NK-cell signatures were dominant predictors, indicating a pre-existing immune-primed state in irAE-prone individuals. Single-cell analysis identified two irAE-enriched myeloid clusters with lung-associated inflammatory features, suggesting baseline myeloid priming. T cells and NK cells from irAE samples exhibited marked upregulation of interferon-stimulated genes and strong enrichment of type I and type II interferon pathways. Perturbation-based screening identified multiple compounds capable of reversing these interferon-amplified signatures, and in vitro experiments demonstrated that alpinetin and momelotinib suppress interferon-γ signaling through distinct STAT1-and JAK–STAT–dependent mechanisms.ConclusionirAEs may arise from the convergence of pre-existing myeloid inflammation and interferon-driven lymphocyte activation before therapy. Our study provides a predictive framework for identifying high-risk patients and highlights mechanistically grounded compounds for potential irAE mitigation.

IntroductionMicrogravity is one type of external stimulus that affects bone homeostasis and bone development. This study investigates the molecular drivers of these effects in order to more fully understand the cellular communication network between bone cells when bone homeostasis is perturbed.MethodsThe transcriptional responses of bone-related genes in zebrafish larvae (Danio rerio) when exposed to simulated microgravity (SMG) using a Random Positioning Machine were analysed. Larvae were initially analyzed at 6, 12, 18, and 24 h post-exposure via RT-qPCR with a focus on the RANKL/RANK/OPG pathway.ResultsShort exposures (6–12 h) produced minimal changes, whereas 18–24 h SMG triggered a two-phase response: initial suppression of osteoblast markers (bglap, sp7, alpl, collagens) followed by activation of osteoclast-associated genes (tnfsf11/RANKL, tnfrsf11b/OPG, tnfrsf11a/RANK, nfatc1, ctsk) and stress-adaptive pathways (hsp family). We then conducted a transcriptomic analysis at 18 and 24 h. Transcriptomic and gene–protein interaction network analyses revealed distinct regulatory clusters encompassing extracellular matrix and osteoclast signaling genes, highlighting the coordinated modulation of bone formation and resorption. Functional enrichment analyses confirmed the involvement of WNT, BMP, HIPPO, and MAPK signaling pathways in skeletal regulation under SMG, and activated stress-adaptive pathways while concurrently downregulating apoptosis-related genes reflecting a complex interplay among developmental, metabolic, and disease-associated bone processes.DiscussionThis data highlights a developmental stage-specific protective response. Collectively, these results demonstrate that SMG disrupts the balance between osteoblast and osteoclast activity, promoting bone resorption via the RANKL/RANK/OPG pathway while suppressing matrix deposition. These findings lay the groundwork for designing targeted interventions to mitigate bone loss during spaceflight and in osteoporotic conditions.

Exosomes, as key mediators of intercellular communication, play a central regulatory role in cellular physiological and pathological processes through their dynamic interaction with the cytoskeleton. The cytoskeleton is a dynamic network composed of microtubules, microfilaments, and intermediate filaments. Microtubules provide track support for the directional transport of MVBs. Microfilament rearrangement generates contractile forces that promote MVB fusion with the plasma membrane. Therefore, the cytoskeleton directly participates in the biogenesis, intracellular transport, and secretion of exosomes. Moreover, cytoskeletal dynamics, coordinated by molecules such as Rab GTPases, affect exosome secretion efficiency. Conversely, exosomes carry bioactive molecules such as proteins, nucleic acids, and lipids. These molecules can regulate cytoskeletal rearrangement in recipient cells by modulating signaling pathways like the TGF-β/Smad signaling pathway and the RhoA/ROCK signaling pathway. Consequently, they influence target cell functions like morphology maintenance, migration, and proliferation. Dysregulation of this interaction is closely related to the progression of various diseases, including tumors and neurodegenerative diseases. For instance, disrupting the dynamic structure of the cytoskeleton or blocking the cytoskeletal remodeling process can significantly reduce exosome secretion, while abnormal exosome transfer disrupts cytoskeletal homeostasis. Current research still faces challenges, such as unresolved details of the molecular regulatory network and a lack of in-depth mechanistic validation in in vivo models. Future studies need to explore in depth novel regulatory factors and signaling pathways and investigate disease diagnosis and treatment strategies based on this interaction. This will provide a theoretical basis and innovative ideas for the prevention and treatment of related diseases.

[This corrects the article DOI: 10.3389/fcell.2024.1348707.].