Age-related bone defects remain challenging due to the reduced osteogenic capacity of aged bone marrow mesenchymal stem cells (A-BMSCs). An injectable hydrogel incorporating extracellular vesicles (EVs) from young BMSCs (Y-EVs) was designed to enhance bone regeneration. The hydrogel, composed of star-PEG-KA7 and polyphosphate-gelatin (P-GA, generated by conjugating imidazole-modified polyphosphate (PolyP-Im) with gelatin (GA), encapsulates Y-EVs to form the P-GA/KA7@Y-EVs system. In vitro, Y-EVs promoted osteogenic differentiation and alleviated senescence in A-BMSCs, while the hydrogel provided injectability, biocompatibility, and sustained release. In vivo, P-GA/KA7@Y-EVs significantly improved cranial bone regeneration in aged rats, surpassing Y-EVs or hydrogel alone. Transcriptomic and microRNA analyses indicated that Y-EVs rejuvenated A-BMSCs by modulating PI3K-Akt, TGF-β, and MAPK pathways, supported by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment. This EVs-infused bioactive hydrogel offers a minimally invasive and effective strategy for age-related bone regeneration.

Abstract Cell polarity is essential for maintaining intestinal epithelial organization and function. Here we show that combined loss of polarity by epithelial loss of Cdc42 with oncogenic Kras expression in mice causes small intestine failure leading to weight loss, inflammation, epithelial necroptosis, and lethality. These phenotypic defects are characterized by a loss of intestinal stem cells, disrupted epithelial architecture, altered hippo signaling, elevated inflammatory cytokines, and activation of necroptotic cell death, that closely resemble necrotizing enterocolitis (NEC). Single-cell transcriptomic analysis reveals a coordinated dysregulation of polarity machinery, inflammatory pathways, and necroptosis program. Suppression of YAP, IL-1, TNFα signaling or necroptosis rescues the intestinal pathology. Similar NEC-like phenotypes arise when Cdc42 loss and oncogenic Kras activation are initiated from intestinal stem cells. These findings provide mechanism insights involving polarity-YAP-IL1/TNFα signaling induced necroptosis for the synergistic effect of hyperactivation of Kras signaling and loss of polarity in disrupting intestinal epithelia.

Chimeric Antigen Receptor (CAR) T cell therapy is an FDA-approved cancer immunotherapy which currently relies on autologous T cells from patients. Cancer such as lymphoma can accelerate the donor T cell aging, and current ex vivo expansion protocol of CAR-T cells further exacerbates T cell dysfunction by inducing T cell exhaustion and reducing stem-like T cells. CDC42, a cell polarity regulator and cell fate determinant, is an intracellular signal transducer controlling cell cytoskeleton organization, chromatin symmetry, and gene expression. Targeting CDC42 by CASIN, a CDC42 activity-specific inhibitor identified by our lab, is able to functionally rejuvenate hematopoietic stem cells and other stem cells in which CDC42-GTP is elevated upon aging. Here we aim to repress CDC42 activity by CASIN in T cells to maintain T cell stemness and counter T cell aging. In this study, human T cells from healthy donors of varying ages were isolated from the peripheral blood and cultured ex vivo in an exhaustion protocol. CDC42-GTP was found upregulated during T cell culture. Flow cytometry and scRNA-seq analyses showed that CASIN treatment remodeled the T cell composite of aged donors to resemble that of young donors. Specifically, CASIN was able to dose-dependently suppress CDC42-GTP level and increased the TCF1+ stem-like T cells while reducing the PD1+TIM3+ exhausted T cells. CASIN also increased CD4+/CD8+ T cell ratio and decreased the proportion of age-associated T cells (Taa). At the cellular level, similarly to what we reported for aged HSCs, CASIN acted on aged T cells to repolarize the depolarized T cells dose-dependently and realign the mis-localized autophagy flux to the lysosome. Molecularly, CASIN reduced p-Ser45 β-catenin (i.e. increased stability) and increased nuclear β-catenin, and significantly increased TCF1 protein level. CASIN treatment also markedly changed several histone methylation and acetylation markers indictive of epigenetic alterations, and remodeled chromatin accessibility at key immune regulatory loci including TCF7, HAVCR2, IFNG, and IL2, providing strong evidence that CASIN regulates the epigenetic landscape the cultured T cells. RNA-seq analyses saw a drastic reduction by CASIN treatment of gene sets from a series of inflammaging related pathways including that of IL2-STAT5, IL6-JAK-STAT3, TGF-β, and TNF-α. Functionally, we have carried out in vitro and in vivo tumor cell killing assays by the CAR-T cells with or without CASIN treatment and found that CASIN treated anti-CD19 CAR-T cells demonstrated an enhanced killing ability of the B-ALL Nalm1 and Nalm6 cells as well as MV411-CD19 AML cells in culture and significantly increased the activity in suppressing leukemia progression in Nalm6 xenograft NSG mice. Our work identifies CASIN-mediated pharmacological intervention as a unique approach in preventing T cell exhaustion and reverting T cell aging through CDC42-cell polarity mediated transcriptional and epigenetic remodeling, providing a promising strategy to improve CAR-T cell therapy efficacy. Mingjun Cai, Xin Duan, Ryan Jorgensen, Mark Wunderlich, Fukun Guo, Yi Zheng. Targeting CDC42 increases TCF1-mediated “stemness” and rejuvenates human T cells to enhance CAR-T efficacy [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B013.

The second near-infrared (NIR-II, 1000-2000 nm) window, also known as the short-wave infrared (SWIR), represents a transformative frontier for optical bioimaging, offering unprecedented penetration depth and minimal autofluorescence in living tissues. Since 2015, small-molecule fluorophores have been recognized as promising platforms for translating these optical advantages into biological discovery. Yet converting NIR-II fluorophores into reliable, application-ready probes remains challenging because multiple performance requirements must be balanced simultaneously, often under conflicting constraints. Over the past decade, feedback from practical biological applications suggests that the design of an ideal NIR-II molecular probe should be considered across four key dimensions: optical performance (O), multiplexed capability (M), in vivo delivery (D), and biological specificity (S). In this Account, we summarize our efforts to address these challenges through molecular engineering within this OMDS framework. First, we discuss strategies for optimizing optical performance by balancing red shift and brightness through control of electronic structure and aggregation behavior. Second, we describe how spectral engineering of lanthanide complexes expands multiplexed capability, enabling excitation-encoded imaging and ratiometric sensing beyond conventional emission-resolved approaches. Third, we highlight molecular design principles for improving in vivo delivery, including tuning charge distribution, hydrophilicity, and scaffold size to achieve programmable pharmacokinetics and favorable biological transport. Finally, we discuss chemigenetic strategies that integrate the tunable photophysics of small-molecule fluorophores with the precision of genetic targeting to achieve selective labeling and dynamic sensing in living systems. Together, these studies establish a molecular design framework that links fluorophore structure to NIR-II optical behavior and biological performance. We anticipate that this OMDS-guided perspective will support the development of the next generation of reliable, application-ready NIR-II molecular probes for biomedical imaging and chemical biology.

Myeloid skewing is a central and therefore often cited hallmark of hematopoietic aging. Myeloid skewing refers to an elevated myeloid-to-lymphoid cell ratio in aged compared to young mice. Interestingly, whether the extent of myeloid skewing might be in itself a quantitative biological marker of aging has not been addressed yet, nor whether this parameter has also relevance for the extent of aging in humans. Aged mice with high level of myeloid skewing (>50% myeloid cells in blood) showed accelerated hematopoietic aging compared to mice with a low level of myeloid skewing (<30% of myeloid cells in blood), as well as an increased level of inflammatory cytokines and elevated levels of diseases. Hematopoietic stem cells (HSCs) from mice with high myeloid skewing showed an impaired repopulation capacity. Epigenetic clock analyses demonstrated that mice with a high level of myeloid skewing present with a biological age that is older than their chronological age. In humans, a high degree of myeloid skewing was associated with elevated levels of inflammatory markers, reduced mobility, a greater burden of comorbidities, and an increased mortality hazard ratio. The data support that, besides overall myeloid skewing being a central hallmark of aging in mice, the extent of the frequency of myeloid cells in blood might serve as a biological marker of aging and disease in both mice and humans. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, SFB1506 B01/B06/C06, Bausteinprogramm, SFB1074

We have utilized macrophage-dependent and hepatocyte-dependent phagocytosis assays as well as in vivo xenotransfusion experiments to determine the survival and rate of clearance of stored platelets in several conditions that include cold storage and the use of a novel preservative solution. Our novel preservative solution targets RHOA, a key signaling GTPase that controls refrigeration-induced storage damage. Using these approaches, the outcome of fluorescently labeled human platelets can be analyzed for their potential to be cleared by integrin-dependent phagocytes, lectin-binding domain-dependent hepatocytes or in circulation of thrombocytopenic, innate- and adaptive immunity-deficient animals.

Pyroptosis, a lytic and immunogenic form of cell death, holds broad therapeutic potential, yet its selective induction in specific cell populations remains a fundamental challenge. Loss of the NF1 tumor suppressor, one of the most frequent events across pediatric and adult cancers, elevates RAS-GTP and drives tumorigenesis through hyperactivated RAS signaling. Here we demonstrate that protein kinase Cδ (PKCδ) agonism selectively triggers pyroptosis in NF1-deficient cells by exploiting their dependency on KRAS. PKCδ directly phosphorylates KRAS at S39 and S181, inducing KRAS-GDP accumulation and driving endoplasmic reticulum translocation. The dually phosphorylated KRAS-GDP interacts with caspase-8 and competitively displaces inhibitory BCL2, promoting caspase-8/caspase-3/gasdermin-E-mediated pyroptosis. This vulnerability is conserved across multiple NF1-deficient tumor types, and PKC agonism suppresses NF1-deficient neurofibroma and malignant peripheral nerve sheath tumor growth in vivo . These findings establish the inactive KRAS-GDP as a functionally active signaling molecule and PKCδ agonism as a selective therapeutic strategy for NF1-deficient cancers.

Objectives: Iron deficiency impairs intestinal mucosal structure and function, yet its impact on intestinal stem cells (ISCs) remains unclear. This study was therefore designed to examine how iron deficiency affects the proliferation and differentiation of ISCs. Methods: Iron-deficient mouse and enteroid models were established. Expression of key cell markers was analyzed using Western blot, qPCR, and immunofluorescence. Results: Iron deficiency led to structural impairment of the intestinal mucosa, characterized by decreased small intestinal villus height. In iron-deficient mice, expression of ChrA (enteroendocrine cell marker), Lyz (Paneth cell marker), and Muc2 (goblet cell marker) was significantly downregulated across duodenum, jejunum and ileum, whereas Vil1 (enterocyte marker) expression increased. Moreover, both Lgr5 (an ISC marker) expression and the number of Ki67-positive proliferating cells were significantly reduced, along with a decrease in Ki67 transcriptional levels under iron-deficient conditions. Similarly, deferoxamine (DFO)-treated enteroids showed fewer Lgr5-positive ISCs, downregulation of Lgr5, Lyz and Muc2, and upregulation of Vil1. RNA-seq further confirmed that iron deficiency skews ISC differentiation toward absorptive lineage. This shift was associated with modulation of the Notch signaling pathway: upregulation of the ligand Dll1, receptors Notch2 and Notch3, and the protease ADAM10, alongside downregulation of the negative regulator Atoh1. These findings indicate that Notch pathway activation promotes enterocyte differentiation under iron deprivation. Conclusions: Iron deficiency suppressed the proliferation of ISCs and induced their differentiation toward enterocytes, which is associated with the modulation of the Notch signaling pathway, providing a mechanistic insights for impaired intestinal repair and the potential for nutrient-targeted therapies.

Cancer immunotherapy, including immune checkpoint inhibitors (ICIs) and chimera-antigen receptor (CAR)-T cell therapy, has achieved substantial clinical success. However, response rates remain limited in many patients due to tumor-intrinsic immune evasion and immune cell dysfunction within the tumor microenvironment (TME). Rho family small GTPases are key signaling regulators of cytoskeletal dynamics, intracellular trafficking, transcription, and metabolism in cancers. Emerging evidence implicates Rho GTPase signaling in mediating immunotherapy efficiency through its context-dependent functions. Individual Rho GTPases modulate immunotherapy responses in tumor cells and various immune cells through actomyosin-mediated chemotaxis, cell junctions, cell polarity, and gene/epigenetic networks, among other pathways. The present review summarizes both the direct evidence linking Rho GTPases in tumor cells to immunotherapy responses and the indirect role of the selective Rho GTPase signaling network in various immune cells, with a focus on the recent progress in understanding the molecular mechanisms and associated outcomes of the ICIs and CAR-T cell therapies. We highlight current knowledge gaps at the intersection of Rho GTPase biology and cancer immunology and discuss therapeutic implications, proposing that selective modulation of specific Rho GTPase signaling pathways in tumor or TME immune cells represents a promising strategy to improve immunotherapy efficiency.

Homeostasis in the intestinal epithelium depends on intestinal stem cells (ISCs). A reduction in the function of ISCs, caused by a decline of canonical Wnt signaling in ISCs, contributes to a reduced regenerative potential of the aged intestine. The composition of the intestinal microbiota changes upon aging. We report here that aging-associated changes in the composition of the microbiota result in reduced canonical Wnt signaling through Ascl2 in ISCs, which causes a decline in the regenerative potential of aged ISCs in vivo. We demonstrate, using microbiota transfer experiments, that interestingly, elevated levels of Akkermansia muciniphila in the intestine cause a reduction of Ascl2-mediated canonical Wnt signaling in ISCs and thus reduced regeneration of the aged epithelium. The composition of the intestinal microbiota thus plays a critical role in regulating the function of ISCs. Our data imply potential therapeutic approaches via modulation of the composition of microbiota for aging-associated changes in the function of ISCs.

Folic acid, a water-soluble B vitamin, is well known for its critical roles in neural tube development and its contributions to neonatal gut maturation and overall gut health. However, the specific mechanisms by which folic acid influences the intestinal mucosa remain incompletely understood. In this study, we aimed to explore the effects of folic acid on the proliferation and differentiation of intestinal stem cells (ISCs). A mouse model of intestinal mucosal injury was established by intraperitoneal injection of 5-fluorouracil (5-FU) at 50 mg per kg body weight once daily for five consecutive days. Our results demonstrated that folic acid enhanced epithelial barrier integrity and modulated epithelial function through the upregulation expression of tight junction components and nutrient transporters. Additionally, folic acid promoted the differentiation of intestinal epithelial cells and accelerated ISC renewal. Mechanistically, folic acid increased the expression of β-adrenergic receptors, whereas its stimulatory effects on enteroid growth and budding were attenuated by the β-adrenergic receptor antagonist propranolol, suggesting the involvement of β-adrenergic receptor signaling in folic acid-mediated epithelial regeneration. Collectively, these findings indicate that folic acid enhances ISC-driven epithelial regeneration in a β-adrenergic signaling-dependent manner, providing new insights into intestinal homeostasis and potential therapeutic strategies for mucosal injury.

Bone marrow (BM) hematopoietic stem cells (HSCs) are exquisitely sensitive to cues from the BM microenvironment (ME), which is critical for their engraftment and regeneration following myeloablative stress. Retinoic acid signaling, acting on both HSCs and niche cells, has emerged as a central regulator of this process. Among ME components, BM adipocytes (BMAs), which can comprise up to 45% of BM volume and expand dramatically during the pancytopenic phase after myeloablation, play a previously underappreciated role in hematopoietic recovery. Here, we identify retinoid X receptor (RXR) signaling in BMAs as a key regulator of the adipokine Resistin, which promotes HSC self-renewal and functional fitness by activating NF-κB signaling. Conditional loss of RXR in adiponectin-expressing cells suppressed Resistin production, resulting in reduced NF-κB activity in HSCs, impaired self-renewal, and defective multilineage hematopoietic regeneration. Functionally, in vivo Resistin neutralization impaired hematopoietic reconstitution, whereas supplementation with either monomeric or dimeric Resistin enhanced HSC self-renewal and long-term lympho-hematopoietic reconstitution in an NF-κB-dependent manner. Together, these findings establish BMA-derived Resistin as an RXR-dependent, critical extrinsic regulator of HSC self-renewal and regenerative hematopoiesis, underscoring its essential role in lympho-myeloid reconstitution after myeloablation. Disclosures : The authors declare no relevant conflicts of interest.

Biomechanical alterations contribute to the decreased regenerative capacity of hematopoietic stem cells (HSCs) upon aging. RhoA is a key regulator of mechanosignaling, but its role in mechanotransduction in stem cell aging remains unclear. Here we show that murine HSCs respond to increased nuclear envelope (NE) tension by inducing NE translocation of P-cPLA2, which cell-intrinsically activates RhoA. Aged HSCs experience physiologically higher intrinsic NE tension, but reducing RhoA activity lowers NE tension in aged HSCs. Feature image analysis of HSC nuclei reveals that chromatin remodeling is associated with RhoA inhibition, including restoration of youthful levels of the heterochromatin marker H3K9me2 and a decrease in chromatin accessibility and transcription at retrotransposons. Finally, we demonstrate that RhoA inhibition upregulates Klf4 expression and transcriptional activity, improving aged HSC regenerative capacity and lympho/myeloid skewing in vivo. Together, our data outline an intrinsic RhoA-dependent mechanosignaling axis, which can be pharmacologically targeted to restore aged stem cell function. Mejía-Ramírez, Iáñez Picazo, Walter et al. explore how nuclear biomechanical changes limit the regenerative capacity of aged hematopoietic stem cells and show that targeting RhoA rejuvenates aged hematopoietic stem cells by reducing nuclear envelope tension and remodeling nuclear architecture.

Chemotherapy resistance has long stood in the way of therapeutic advancement for lung cancer patients, the malignant tumor with the highest incidence and fatality rate in the world. Patients with lung adenocarcinoma (LUAD) now have a dismal prognosis due to the development of cisplatin (DDP) resistance, forcing them to use more costly second-line therapies. Therefore, overcoming resistance and enhancing patient outcomes can be achieved by comprehending the regulatory mechanisms of DDP resistance in LUAD. WD repeat domain 62 (WDR62) expression in LUAD tissues and in DDP-resistant or sensitive LUAD patients was analyzed bioinformatically, and a K-M plot was utilized to assess survival status. Real-time quantitative PCR was employed for WDR62 expression detection, cell-counting kit-8 assay for half maximal inhibitory concentration determination, flow cytometry for cell apoptosis detection, immunofluorescence for γ-H2AX expression analysis, and western blot for nonhomologous end joining repair and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway-related protein expression analysis. Poor prognosis was linked to WDR62, which was overexpressed in LUAD tissues and cells. Compared to sensitive cells, DDP-resistant cells had increased WDR62 expression. WDR62 knockdown may enhance DDP-induced cell apoptosis while reducing cell proliferation and DNA damage repair. Functional investigations verified that overexpressed WDR62’s encouraging impact on DNA damage repair in A549/DDP cells could be reversed by MAPK inhibitors, increasing the cells’ susceptibility to DDP. LUAD cells became less sensitive to DDP when WDR62 activated the MAPK/ERK pathway, which promoted DNA damage repair, indicating that DDP resistance might be reversed by treating LUAD with inhibitors of the MAPK pathway.

RAC1A159V is a hotspot mutation associated with poor prognosis in several cancers. By gene editing, we generated endogenous homozygous and heterozygous RAC1A159V mutations, which result in up-regulated RAC1 activity and mammalian target of rapamycin (mTOR) signaling. RAC1A159V tumors grow faster than RAC1WT tumors in immune-proficient mice and are resistant to anti-programmed death protein 1 (PD1). Flow cytometry and scRNA-seq analyses reveal that RAC1A159V cells form "cold" tumors with an immunosuppressive microenvironment and reduced tumor-immune cell interactions. Mechanistically, RAC1A159V up-regulates glycosphingolipid biosynthesis to activate mTORC1 signaling in tumor cells, which in turn increases glycolysis, impairs key chemokine production, and decreases IFNGR1 expression of the tumor cells. mTORC1 inhibition by rapamycin resensitizes the RAC1A159V tumors to anti-PD1 treatment by reversing effects of RAC1A159V mutation. These results demonstrate a mechanism of RAC1A159V-driven immune evasion and suggest an approach of combining the targeting of RAC1-mTOR signaling with immune checkpoint inhibitor for the treatment of a type of immune-cold tumors.

Chromodomain Helicase DNA-binding (CHD) proteins are crucial regulators of chromatin remodeling, gene transcription and expression. CHD8 of the CHD proteins is involved in many important signaling pathways, such as p53 and Wnt-β-catenin pathways. It remains unclear whether CHD8 plays a role in immune regulatory T cells (Tregs). In this study, Treg-specific conditional CHD8-deficient mice were generated. We found that CHD8 deficiency in Treg led to early, fatal inflammation, due to increased CD4+ and CD8+ effector T cells. CHD8 deletion did not alter Treg homeostasis but increased their activation and promoted functional plasticity with elevated expression of effector T cell cytokines, such as IFN-g, IL-4 and IL-17. CHIP-seq of Tregs uncovered that CHD8 binding genes were enriched in PI3K-Akt-mTOR signaling and several other pathways. RNA-seq and ATAC-seq revealed that CHD8 deletion upregulated multiple pathways, notably mTORC1 signaling and its mediated glycolysis that have been reported to promote Treg plasticity. Integrating RNA-seq data with ATAC-seq and/or CHIP-seq data indicates that CHD8 regulates gene expression in either chromatin remodeling- and/or CHD8 binding-dependent or -independent manner. Our findings suggest that autism-related CHD8 is important for maintaining Treg fitness, via genetic and epigenetic mechanisms, to control autoimmunity. More in-depth studies are ongoing to further elucidate the underlying molecular mechanisms. This work was supported by NCI (R01CA234038-01A1 and R01CA278756-01A1) awards to F.G. and Y.Z. Immune Response Regulation: Molecular Mechanisms (IRM)

Base editing technology is a novel gene-editing approach derived from the CRISPR/Cas9 system, enabling precise and efficient base conversion. Due to operational simplicity, strong target specificity, high editing efficiency, and minimal editing byproducts, base editing has been widely applied in gene therapy, crop breeding, construction of model organisms, and microbial metabolic engineering. In this review, we systematically summarize the development history and recent advancements of several major base editors, including cytosine base editors (CBEs), adenine base editors (ABEs), CRISPR-free base editors, C-to-G base editors (CGBEs), glycosylase base editors (GBEs), and IscB-derived base editors. Furthermore, we comprehensively summarize optimization strategies for base editors and its application in constructing efficient industrial microorganism, such as Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yarrowia lipolytica, and Aspergillus niger. This review aims to facilitate the broader application of base editing technologies in synthetic biology and accelerate their translational potential.