参考文献

Endogenous long double-stranded RNAs (dsRNAs),which are not edited by the RNA editing enzyme ADAR1,may activate the antiviral dsRNA receptor MDA5 to trigger interferon-mediated immune responses. Among the large number of endogenous long dsRNAs,the key substrates that activate MDA5—termed as immunogenic dsRNAs—remain largely unidentified. Here we reveal that human immunogenic dsRNAs constitute a surprisingly small fraction of all cellular dsRNAs. We found that these immunogenic dsRNAs were highly enriched in mRNAs and depleted of introns,consistent with their role as cytosolic MDA5 substrates. We validated the MDA5-dependent immunogenicity of these dsRNAs,which was dampened following ADAR1-mediated RNA editing. Notably,immunogenic dsRNAs were enriched at genetic susceptibility loci associated with common inflammatory diseases,implying their functional importance. We anticipate that a focused analysis of immunogenic dsRNAs will enhance our understanding and treatment of cancer and inflammatory diseases,where the roles of dsRNA editing and sensing are increasingly recognized. The authors show that only a small subset of cytosolic double-stranded RNAs (dsRNAs) requires ADAR1-mediated RNA editing to evade an MDA5-dependent immune response. These immunogenic dsRNAs are enriched in mRNAs and overlap with GWAS signals for common inflammatory diseases. View Publication

Autophagy-based targeted degradation offers a powerful complement to proteasomal degradation leveraging the capacity and versatility of lysosomes to degrade complex cargo. However,it remains unclear which components of the autophagy-lysosomal pathway are most effective for targeted degradation. Here,we describe two orthogonal induced-proximity strategies to identify autophagy effectors capable of degrading organelles and soluble targets. Recruitment of autophagy cargo receptors,ATG8-like proteins,or the kinases ULK1 and TBK1 is sufficient to trigger mitophagy,while only autophagy cargo receptors capable of self-oligomerization degrade soluble cytosolic proteins. We further report a single-domain antibody against p62 and its use as a heterobifunctional degrader to clear mitochondria. Fusing the p62 single-domain antibody to PINK1 enables selective targeting of damaged mitochondria. Our study highlights the importance of avidity for targeted autophagy and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for organelles or protein aggregates. Using proximity-based screening,protein engineering,and structural analysis,this study describes the development of a p62-based biodegrader for the clearance of organelles and aggregated proteins by autophagy-targeted degradation. View Publication

Purpose: Noninfectious uveitis is a sight-threatening autoimmune eye disease lacking effective targeted therapies. Dipyridamole (DIP),a phosphodiesterase (PDE) inhibitor,has demonstrated anti-inflammatory properties in inflammatory diseases. However,its application in uveitis remains unexplored. Methods: We used single-cell RNA sequencing (scRNA-seq) data from experimental autoimmune uveitis (EAU) mice and uveitis patients to assess the potential association of PDE gene expression with disease development. Subsequently,EAU mice received oral DIP (300 mg/kg/day),starting at different time points (preventative,early-therapeutic,or late-therapeutic),and treatment efficacy was assessed. To explore immune components and signaling changes,we profiled cervical draining lymph nodes (CDLNs) from control,EAU,and DIP-treated mice by scRNA-seq and validated key findings with additional experiments. Mechanistically,pharmacologic interventions (an adenylyl cyclase inhibitor and the STAT3 agonist) were used in vitro. Results: Expression of several PDE genes correlated with uveitis severity in both human and mouse. Preventative DIP treatment most effectively reduced fundus inflammation in EAU and modulated the Teff/Treg ratio in the CDLNs and spleens. In vitro,DIP suppressed CD4+ T cell proliferation,and inhibited pathogenic Teff. scRNA-seq analysis revealed that DIP partially reversed EAU-induced transcriptional alterations,with notable changes in immune cell composition and pathway activity. Mechanistically,DIP downregulated STAT3 activity and PIM1 expression in Th17 cells via cAMP,suggesting the involvement of the cAMP-STAT3-PIM1 axis in modulating immune homeostasis. Conclusions: DIP ameliorated intraocular inflammation,modulated Th17/Treg balance,and reduced Th17 pathogenicity in EAU,potentially via cAMP-STAT3-PIM1 signaling. These findings highlight DIP as a promising therapeutic candidate for autoimmune uveitis. View Publication

Cutaneous tuberculosis (CTB) is an infectious disease highly associated with extracellular matrix remodeling and granuloma-driven fibrosis. Fibroblasts play crucial roles in this fibrotic process,but their specific roles in Mycobacterium tuberculosis (Mtb) skin infections remain unclear due to the lack of proper in vitro models. Here,we demonstrate that skin organoids (SKOs) derived from human induced pluripotent stem cells can model CTB infected by Mtb. Single-cell RNA analyses reveal an increase in fibroblasts,upregulation of genes involved in collagen synthesis,and enhanced collagen degradation induced by MMP2 and MMP14 in Mtb-infected SKOs. This is accompanied by the destruction of nerve cells and adipocytes. Importantly,the onset of fibrosis in Mtb-infected SKOs is dependent on the activation of the PI3K-AKT signaling pathway and transcription factor AP1 in fibroblasts. Pharmacological inhibition of PI3K-AKT and AP1 alleviates fibrosis and collagen deposition. Our findings have uncovered distinct alterations in cell populations during Mtb-induced skin fibrosis,highlighting the crucial roles of PI3K-AKT and AP1. The study demonstrates the utility of SKOs for investigating CTB pathogenesis and evaluating potential antifibrotic treatments. Cutaneous tuberculosis is an infectious disease associated with extracellular matrix remodeling and granuloma-driven fibrosis. Here,the authors present an in vitro model of this disease using skin organoids infected with Mycobacterium tuberculosis,and describe infection-induced alterations in specific pathways and cell populations. View Publication

Acute myeloid leukemia (AML) is primarily driven by leukemic stem cells (LSCs),the main cause of relapse and therapy resistance. Here,we discover that LSCs are predominantly small and mechanically soft. These mechanical properties enable their selective isolation using microfluidic chips. Single-cell RNA-sequencing of primary human AML bone marrow identifies enrichment of LSCs within the FSClow ALDH1A1+ subpopulation,which exhibits long-term stemness in functional assays. Notably,inhibiting ALDH1A1 in these cells promotes F-actin polymerization and increases cellular stiffness,reducing their stemness while enhancing their susceptibility to natural killer (NK) cell-mediated cytotoxicity. In AML patient-derived xenograft models,the combination of ALDH1A1 inhibition with NK cell therapy markedly suppresses leukemia progression. These findings suggest that targeting the mechanical properties of LSC offers a promising strategy to overcome AML treatment resistance,providing insights into stem cell mechanobiology and paving the way for combining targeted therapies with immunotherapy to improve clinical outcomes. Leukemic stem cells (LSCs) drive relapse and therapy resistance in acute myeloid leukemia (AML). Here,the authors show that increasing the stiffness of LSCs reduces their stemness and enhances their susceptibility to natural killer cell-mediated immunotherapy in AML. View Publication

Genetic variants in the X‐chromosomal gene coding for the calcium−/calmodulin‐dependent serine protein kinase (CASK) are associated with a neurodevelopmental disorder. CASK is a member of the membrane‐associated guanylate kinase (MAGUK) family of proteins. It acts as a scaffold at presynaptic sites,as a regulator of the transport of glutamate receptors,and as a transcriptional regulator. The PDZ domain of CASK has been reported to bind to presynaptic cell adhesion molecules such as Neurexin1‐3,CNTNAP2,SynCAM and SALM1. Structural analyses of related MAGUKs indicate that the canonical SH3 and GK domains combine with the PDZ domain to form the so‐called PSG supramodule. Conserved aromatic residues (Y723 and W914) flanking the GK domain contribute to the formation of a dimeric structure of two PSG modules,which is required for high‐affinity binding to the type 2 PDZ ligand motif of,for example,Neurexin. Here we identify previously uncharacterized patient variants in the SH3 domain of CASK (I672V; P673L),which alter the intermolecular binding pocket for Y723. Both variants interfere with the binding of Neurexin‐1β,in a manner similar to the previously reported Y723C variant. Intriguingly,binding to the type 1 PDZ ligand of the cell adhesion molecule SALM1 is not altered. Using a set of highly selective patient variants,we show that the binding of SALM1 to CASK is actually not mediated by the CASK PDZ domain or the PSG supramodule,but depends on other type 1 PDZ domain‐containing proteins such as SAP97 and Veli,which associate with CASK through its L27 domains. Our data underline the relevance of an intact PSG tandem of CASK for human health. View Publication

Background: Acute Lung Injury (ALI) and its most severe form,Acute Respiratory Distress Syndrome (ARDS),are critical pulmonary conditions characterized by life-threatening acute hypoxic respiratory failure,affecting over three million individuals globally each year. ALI involves alveolar inflammation and disruption of the alveolar-capillary barrier,primarily driven by neutrophil infiltration and the release of inflammatory mediators. In our previous study using a lipopolysaccharide (LPS)-induced mouse model of ALI,we demonstrated that C6,a peptide inhibitor of voltage-gated proton channels (Hv1),ameliorates lung injury,identifying Hv1 as a potential therapeutic target. However,(i) whether the anti-inflammatory effects of C6 are translatable to a clinically relevant live bacterial infection model,and (ii) the molecular mechanisms underlying these anti-inflammatory effects,remain unknown,and are a crucial next step towards targeted rational drug development. Methods: To induce ALI,we used an intratracheal Pseudomonas aeruginosa infection model,a gram-negative bacterium relevant in ventilated and immunocompromised patients. A separate group of infected mice also received intravenous treatment with C6 (4 mg/kg). Lung injury severity was evaluated using histopathological analysis. Bronchoalveolar lavage (BAL) fluid was collected to quantify neutrophil infiltration and proinflammatory cytokines concentrations. In addition,reactive oxygen species (ROS) production and intracellular calcium levels in BAL neutrophils were measured. RNA sequencing of BAL neutrophils was conducted to assess C6-induced transcriptional changes. Key findings were validated in vitro using human neutrophils. Results: C6 mitigates P. aeruginosa-induced ALI in mice by reducing neutrophil infiltration into the alveolar space by ~ 86%,improving lung injury scores,decreasing BAL fluid proinflammatory cytokine levels,and suppressing neutrophil ROS production and intracellular calcium levels. RNA sequencing of BAL neutrophils revealed 51 downregulated genes,including key regulators of neutrophil migration,cytokine release,and ROS production; only three genes were upregulated and they also have roles in neutrophil immune defense. In human neutrophils,C6 similarly inhibited chemotaxis and reduced ROS and cytokine release,and calcium influx. Conclusions: Targeting Hv1 with C6 effectively protects against P. aeruginosa-induced ALI by limiting neutrophil recruitment and activation. These findings establish C6 as a promising therapeutic candidate against infectious ALI and provide important mechanistic insights into its immunomodulatory effects on neutrophils. View Publication

How the neuroectoderm-derived eye field breaks symmetry to specify iris muscle is not well understood. Recent studies have begun to transcriptionally characterize mouse iris muscle; however,little is known about the transcriptional foundation of human iris development. Human pluripotent stem cells (hPSCs) enable the study of iris muscle specification. Here we compare iris smooth muscle from native adult iris tissues to evaluate successful specification of iris muscle from hPSC lines. We utilize a previously published eye-like organoid protocol that specified cells of the eye field to also generate iris muscle. We describe a population transcriptionally similar to native iris and describe an iris muscle gene signature. Human iris muscle not only contains pigment,but also expresses pigment synthesis genes and is responsive to acetylcholine. Integration of single-cell RNA-seq datasets confirm the similarity between the iris muscle to the adult iris,establishing the usefulness of the model in studying neuroectoderm-derived iris muscle specification,and related diseases. Single-cell RNA sequencing reveals that iris muscle,derived from neuroectoderm,can form in stem cell–derived eye organoids – enabling the modelling of iris muscle pathologies like aniridia and proliferative vitreoretinopathy. View Publication

Silencing remains a significant challenge for exogenous gene expression,limiting both the penetrance and expressivity of transgenes. In particular,silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here,we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably,the incorporation of an intron into a CRISPRa construct results in reduced silencing,increased expression levels,and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition,we find that introns can work synergistically with chromatin opening elements to further mitigate silencing,suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes. Silencing of transgenes such as Cas9 limits gene editing and CRISPRa applications. Here,the authors show that adding intronic sequences reduces silencing and boosts transgene expression,enabling improved CRISPRa-mediated gene activation and more stable expression of the transgene over time. View Publication

USP30,a ubiquitin‐specific protease,primarily characterized as a mitochondrial deubiquitinase regulating mitophagy,has not been previously reported to have nuclear functions. In this study,we demonstrate that USP30 is present in both mitochondrial and nuclear compartments. Nutrient deprivation triggers USP30 nuclear translocation via an N‐terminal nuclear localization signal (NLS),mediated through suppression of mTORC1‐dependent phosphorylation at serine 104,a modification constraining nuclear entry. Nuclear USP30 acts as a tumor suppressor by inhibiting cancer stemness and chemoresistance in triple‐negative breast cancer (TNBC) cells. Mechanistically,USP30 directly interacts with and deubiquitinates the transcription factor TCF/LEF1 at K379 and K382 residues,disrupting recruitment of CBP/P300 co‐activators to the β‐catenin/LEF1 complex. This abolishes β‐catenin/LEF1 transactivation and suppresses WNT signaling. Clinically,USP30 is downregulated in TNBC and cancer stem cells (CSCs),with notably reduced nuclear levels in cancer tissues. Overexpression of nuclear USP30 markedly reduces lung metastatic burden in TNBC mouse models. These findings uncover a novel role for nuclear USP30 in regulating cancer stemness and suggest that targeting the dynamic relocalization of USP30 from mitochondria to the nucleus could offer new therapeutic strategies for breast cancer metastasis. View Publication