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The cell cycle (CC) underpins diverse cell processes like cell differentiation,cell expansion,and tumorigenesis but current single-cell (sc) strategies study CC as: coarse phases,rely on transcriptomic signatures,use imaging modalities limited to adherent cells,or lack high-throughput multiplexing. To solve this,we develop an expanded,Mass Cytometry (MC) approach with 48 CC-related molecules that deeply phenotypes the diversity of scCC states. Using Cytometry by Time of Flight,we quantify scCC states across suspension and adherent cell lines,and stimulated primary human T cells. Our approach captures the diversity of scCC states,including atypical CC states beyond canonical definitions. Pharmacologically-induced CC arrest reveals that perturbations exacerbate noncanonical states and induce previously unobserved states. Notably,primary cells escaping CC inhibition demonstrated aberrant CC states compared to untreated cells. Our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems with simultaneous multiplexing and integration with MC platforms. Subject terms: Assay systems,Proteomics,Cell biology,Immunology,Systems biology View Publication

Cancer drug resistance poses a significant challenge in oncology,often driven by intricate cross-talk among membrane-bound receptors that compromise mono-targeted therapies. We develop a dual membrane receptor degradation strategy leveraging Folate Receptor α (FRα) to address this issue. Folate Receptor α Targeting Chimeras-dual (FolTAC-dual) are engineered degraders designed to selectively and simultaneously degrade distinct receptor pairs: (1) EGFR/HER2 and (2) PD-L1/VISTA. Through modular optimization of modality configurations and geometries,we identify the “string” format as the most effective construct. Mechanistic studies demonstrate an ~85% increase in EGFR-binding affinity compared to the conventional knob-into-hole design,likely contributing to the improved efficiency of dual-target degradation. Proof-of-concept studies reveal that EGFR and HER2 FolTAC-dual effectively counteracts resistance in Trastuzumab/Lapatinib-resistant HER2-positive breast cancer models,while PD-L1 and VISTA FolTAC-dual rejuvenates immune responses in PD-L1 antibody-resistant syngeneic mouse models. These findings establish FolTAC-dual as a promising dual-degradation platform for clinical translation. Subject terms: Cancer immunotherapy,Targeted therapies,Protein design,Drug discovery and development View Publication

Extracellular vesicles (EVs) are nano-sized lipid vesicles released into the extracellular space. We investigated the role of mouse brain-derived EVs in α-synuclein (α-syn) degradation and pathology transmission. Using sucrose gradient isolation and biochemical characterization,we found that EVs harbor active proteases that cleave both monomeric α-syn and pre-formed fibrils (PFFs). Protease activity and inhibitor profiling identified cathepsins B and S as key enzymes mediating this cleavage. EV-mediated proteolysis reduced the seeding capacity of α-syn PFFs in vitro and in vivo,whereas protease inhibition enhanced aggregation. Proteomic analysis revealed a restricted protease repertoire within EV cargo. Our findings suggest that EVs regulate extracellular α-syn levels via proteolysis,thereby modulating its prion-like spreading potential. We suggest that EVs represent a novel post-translational mechanism to regulate the levels of extracellular α-syn and may thus affect the spreading of α-syn pathology. Targeting this proteolytic capacity may offer new therapeutic interventions for mitigating synucleinopathies. Subject terms: Biochemistry,Cell biology,Neuroscience,Pathogenesis View Publication

Background: Lipoprotein(a) [Lp(a)]-induced inflammation contributes to coronary artery spasm (CAS) by the contraction of vascular smooth muscle cells. However,the interaction between Lp(a) and soluble CD36 (sCD36)/interleukin (IL)-6/RAS Homolog Family Member A (RhoA)-GTP signaling pathway has not been evaluated. Methods: We investigated the relevance of Lp(a)/CD36 signaling in CAS patient monocyte-derived macrophages (PMDMs) and a human coronary artery smooth muscle cell (HCASMC) line using expression profile correlation analyses,molecular docking,RNA sequencing,flow cytometry,immunoblotting,and quantitative reverse transcription polymerase chain reaction. Results: Plasma Lp(a) and sCD36 levels in 41 CAS patients were significantly higher ( p = 0.001) and positively correlated (r 2 = 0.3145,p < 0.001),a trend not observed in 36 non-CAS controls. RNA sequencing indicated a significant co-overexpression of CD36 and RhoA in Lp(a)-treated CAS PMDMs and HCASMCs,of which the mRNA and protein expression of CD36 and RhoA were significantly enhanced ( p < 0.001) dose-dependently. Lp(a) rather than LDL preferentially induced CD80+ PMDM (M1) polarization. In HCASMCs,the CD36 knockdown using either short hairpin RNA or natural biflavonoid amentoflavone suppressed Lp(a)-upregulated protein expression of CD36,RhoA-GTP,IL-6,tumor necrosis factor (TNF)-α,nuclear factor (NF)-κB,and CD80; however,overexpressed CD36 increased their levels. Lp(a) decreased and amentoflavone increased the epigenetic expression of CD36 inhibitors,miR-335-5p,and miR-448,respectively. Reciprocally,an miRNA inhibitor or mimic could magnify or diminish Lp(a)-induced CD36,TNF-α,NF-κB and IL-6 expressions in HCASMCs,respectively. Conclusions: Elevated Lp(a) levels upregulate the CD36-dependent TNF-α/NF-κB/IL-6/RhoA-GTP signaling pathway in CAS PMDMs and HCASMCs,indicating that Lp(a)/CD36 inflammatory signaling,HCASMC activation,and macrophage M1 polarization mediate CAS development. View Publication

Monoclonal antibody-based immune checkpoint inhibitors,which have brought breakthrough effects in cancer treatments,are expected to assist in the treatment of viral diseases. However,antibody therapies may cause immune-related side effects,such as inflammation and pneumonia,due to cytokine storms. Small-molecule PD-1/PD-L1 inhibitors are an alternative to monoclonal antibody-based therapeutics. We have identified a novel small-molecule PD-1/PD-L1 inhibitor having a functional group (disulfide group),namely compound 2 (molecular weight: 456.6),from our library of sulfur-containing protein–protein interaction inhibitor compounds. Compound 2 selectively bound to PD-L1 over PD-1,with the dissociation rate constant (K D ) of 77.60 ± 4.44 nM (obtained by affinity analysis) and showed promising T cell activation recovery. A molecular docking simulation study between 2 and PD-L1 suggested that 2 binds to PD-L1 in a binding mode different from those of other small-molecule PD-L1/PD-1 inhibitors. Notably,oral administration of 2 to mice pre-infected with influenza A virus (A/NWS/33,H1N1 subtype) caused a significant increase in the neutralizing antibody titers,as well as recovery from influenza-induced pneumonia. Overall,2 provides insight for the development of therapeutic drugs against early viral infections,with both virus titer-reducing and antibody titer-boosting effects. Moreover,2 is widely used as a rubber peptizing agent in the production process of tires and other rubber products. Our findings may provide useful information for investigating its influence on living organisms. The online version contains supplementary material available at 10.1038/s41598-025-17982-3. Subject terms: Drug discovery and development,Pharmacology,Screening,Structure-based drug design View Publication

Tissue remodeling is a key feature of allergic rhinitis (AR),but its underlying molecular mechanisms remain unclear. Lysyl oxidase-like 2 (LOXL2),a regulator of tissue remodeling,has not been studied in AR. Proteomic analysis was performed on nasal mucosal tissues from 8 AR patients and 8 healthy controls (HCs) to identify differentially expressed proteins (DEPs). The top three upregulated DEPs and their association with tissue remodeling markers were validated by immunofluorescence,Western blot,and RT-qPCR in an independent cohort of 30 AR patients and 30 HCs. In vitro,human nasal epithelial cells (HNECs) were treated with IL-4,and the effects of candidate protein inhibitors on remodeling were assessed. An AR mouse model was used to evaluate the impact of these inhibitors on nasal inflammation and remodeling. Proteomic analysis revealed a disease-specific protein expression profile in the nasal mucosa of AR patients,with the top three upregulated proteins being LOXL2,TGF-β1,and TIRAP. Tissue validation showed that LOXL2 was significantly upregulated in the nasal mucosa of AR patients compared to HCs and was significantly correlated with EMT markers (TGF-β1,α-SMA,and E-cadherin). In vitro,IL-4 stimulation significantly upregulated LOXL2,TGF-β1,and α-SMA,while downregulating E-cadherin in a dose-dependent manner in human nasal epithelial cells. These effects were reversed by inhibition of LOXL2. Further investigations demonstrated that LOXL2 promotes tissue remodeling through activation of the TGF-β1/Smad signaling pathway. In the AR mouse model,LOXL2 inhibitors significantly reduced nasal mucosal inflammation and tissue remodeling. Our proteomic analysis suggests that LOXL2 may be involved in the pathological remodeling processes of AR,potentially through modulation of the TGF-β1/Smad signaling pathway. These findings provide preliminary evidence that LOXL2 could serve as a candidate biomarker and a possible therapeutic target in AR,warranting further investigation. View Publication

Despite recent approval of monoclonal antibodies that reduce amyloid (Aβ) accumulation,the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimer's disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target,where its weak inhibition activates neuroprotective signalling,benefiting AD mouse models with Aβ and p-Tau pathologies. Rational design and structure‒activity relationship studies yielded mtCI inhibitors profiled in a drug discovery funnel designed to address safety,selectivity,and efficacy. The lead compound C458 is highly protective against Aβ toxicity,has favourable pharmacokinetics,and minimal off-target effects. C458 exhibited excellent brain penetrance,activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted using functional tests,metabolic assessment,in vivo 31 P-NMR spectroscopy,blood cytokine panels,ex vivo electrophysiology,and Western blotting. Chronic oral administration improved long-term potentiation,reduced oxidative stress and inflammation,and enhanced mitochondrial biogenesis,antioxidant signalling,and cellular energetics. Efficacy against Aβ and p-Tau was confirmed in human organoids. These studies provide further evidence that the restoration of mitochondrial function in response to mild energetic stress represents a promising disease-modifying strategy for AD. This research was supported by grants from NIH AG 5549-06,NS1 07265,AG 062135,UG3/UH3 NS 113776,and ADDF 291204 (all to ET); U19 AG069701 (to TK); the Alzheimer’s Association Research Fellowship grant 23AARF-1027342 (to TKON). View Publication

Cholesterol is an essential plasma membrane component,and altered cholesterol metabolism has been linked to cholesterol accumulation in the airways of COPD and cystic fibrosis patients. However,its role in airway epithelial differentiation is not well understood. Tandem mass spectrometry-based proteomic analysis of differentiating primary human bronchial epithelial cells (phBECs) revealed an overall inhibition of the cholesterol biosynthesis pathway. We hypothesized that excess cholesterol impairs the differentiation of phBECs into a fully functional bronchial epithelium. PhBECs were differentiated in the presence of 80 µM cholesterol for 21 days,the main airway cell type populations monitored using qRT-PCR and immunofluorescent stainings,and epithelial barrier integrity was analyzed via transepithelial electrical resistance measurements. Chronic cholesterol exposure led to a significant increase in CC10 + secretory cells at the expense of ciliated cells. Pathway enrichment analysis suggested the tumor protein p53 as a master regulator of genes during normal differentiation of phBECs. Chronic cholesterol exposure drastically impaired the nuclear translocation of p53. Our findings suggest that this inhibition underlies the cholesterol-induced expansion of CC10 + secretory cell populations at the expense of ciliated cells. In conclusion,we identify cholesterol as an important regulator of normal bronchial epithelial cell differentiation through inhibition of p53 nuclear translocation. View Publication

Cancer research has long focused on mutations in normal body cells,but this approach has not produced major breakthroughs for most cancers. Our study explores a different concept that some aggressive cancers may actually arise from early reproductive cells called primordial germ cells,which normally develop into eggs and sperm. We created a new experimental model showing how a virus can transform human primordial germ cell-like cells into virus-positive Merkel cell carcinoma,a rare but deadly skin cancer. This model shows that cancers can emerge through changes in developmental states rather than relying solely on genetic mutations. By linking cancer development to early germ cells,our findings suggest a unifying explanation for both germ cell cancers and body cancers. This new perspective may guide more effective approaches to study,diagnose,and treat cancer by focusing on early human development rather than only DNA mutations and later developmental stages. View Publication

With the approval of the antibody-drug conjugate enfortumab vedotin (EV),NECTIN4 has emerged as a bona fide therapeutic target in urothelial carcinoma (UC). Here,we report the development of a NECTIN4-directed chimeric antigen receptor (CAR) T cell,which exhibits reactivity across cells expressing a range of endogenous NECTIN4,with enhanced activity in high expressors. We demonstrate that the PPARγ pathway,critical for luminal differentiation,transcriptionally controls NECTIN4,and that the PPARγ agonist rosiglitazone primes and augments NECTIN4 expression,thereby increasing sensitivity to NECTIN4-CAR T cell-mediated killing. NECTIN4-CAR T cells have potent anti-tumor activity even against EV resistant cells,which largely retain NECTIN4 expression,including in a post-EV biopsy cohort. Our results elucidate a therapeutically actionable mechanism that UC cells use to control NECTIN4 expression and suggest therapeutic approaches that leverage PPARγ agonists for rational combinations with NECTIN4-targeting agents in UC,as well as future potential treatment options for EV-refractory patients. Subject terms: Bladder cancer,Cancer immunotherapy,Cancer therapeutic resistance,Oncology,Bladder cancer View Publication

In bone marrow,cell numbers are balanced between production and loss. After chemotherapy,blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand,although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy,RBC concentrations in bone marrow peaked on day 5 posttreatment,coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis,and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture,and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress. View Publication

Particulate matters such as diesel exhaust particles induce oxidative stress in cells and thereby have a negative impact on health. The aim of this study was to test whether the membrane-permeable,anti-inflammatory metabolite 4-Octyl Itaconate can counteract the oxidative stress induced by diesel exhaust particles and to analyze the downstream-regulated pathways both in human nasal epithelial cells and PBMCs. Human nasal epithelial cells were cultured from nasal swabs,and the response of the cells to diesel exhaust particles either alone or in combination with 4-Octyl Itaconatee was investigated using RNA sequencing,qPCR,and cytokine measurement. The presence of reactive oxygen species in the cells was analyzed using CellROX staining and flow cytometric DCFDA assay. Diesel exhaust particles caused an upregulation of CYP1A1 in nasal epithelial cells. The administration of 4-Octyl Itaconate reduced the reactive oxygen species and increased the expression of antioxidant genes regulated by the transcription factor NRF2,which was also confirmed in PBMCs. IL-6 secretion from NEC was elevated by diesel exhaust particles and reduced when 4-Octyl Itaconate was administered. 4-Octyl Itaconate can reduce the diesel-exhaust-particle-induced oxidative damage by the activation of NRF2-regulated antioxidative pathways. View Publication