技术资料

Expansion microscopy (ExM) has enabled nanoscale imaging of tissues by physically enlarging biological samples in a swellable hydrogel. However,the increased sample size and water-based environment pose challenges for deep imaging using conventional inverted confocal microscopes,particularly due to the limited working distance of high-numerical-aperture (NA) water immersion objectives. Here,we introduce a practical imaging alternative that utilizes an inverted water-dipping objective and a refractive-index-matched optical path using fluorinated ethylene propylene (FEP) film. Through point spread function (PSF) measurements and simulations,we show that the FEP film introduces predominantly defocus-like wavefront profiles characteristic of high NA systems,which result in an easily correctable axial shift of the focal plane. To ensure stable immersion and refractive index continuity,we use an arrangement relying on an FEP film,Immersol W,water and a FEP-based imaging dish. This configuration achieves sub-micron lateral and axial resolution,supports large tile-scan acquisitions,and maintains image quality across depths exceeding 800 µm. We validate the system by imaging 4×-expanded U2OS cells and human cerebral organoids. Our approach provides a low-cost,plug-and-play solution for high-resolution volumetric imaging of expanded samples using standard inverted microscopes. View Publication

Interleukin-17 (IL-17) plays a central role in the pathogenesis of various autoimmune diseases. Soluble CD14 (sCD14),a marker of innate immune activation,is elevated in several inflammatory conditions. However,its influence on IL-17 production and the differentiation of Th17 cells remains poorly understood. We found that sCD14 enhances Th17-associated cytokine production and up-regulates critical transcription factors such as STAT3 and RORC. Notably,sCD14's effect on Th17 polarization was mediated indirectly through autologous sCD14-treated peripheral blood mononuclear cell (PBMC) supernatant (sCD14-PBMC-Sup). Additionally,we identified a distinct cytokine profile enriched for pro-inflammatory cytokines and chemokines in sCD14-treated T cells,further reinforcing the Th17-promoting role of sCD14. Interestingly,gamma-aminobutyric acid (GABA),a metabolite elevated in sCD14-treated monocytes,was identified as a potential contributor to Th17 polarization. GABA supplementation in T-cell cultures enhanced IL-17A secretion,indicating its role as a signaling molecule in T-cell differentiation. Our findings also revealed the expansion of innate lymphoid cell (ILC)2/3-like cells in T-cell cultures exposed to sCD14-PBMC-Sup and GABA,highlighting the potential role of monocytes in Th17-mediated immunity. Furthermore,while sCD14 promoted Th17 polarization,it simultaneously impaired T-cell activation and proliferation,suggesting an immunosuppressive effect mediated by soluble factors released from monocytes. These results underscore the dual role of sCD14 in modulating T-cell responses,promoting Th17 differentiation while suppressing T-cell effector functions. This study identifies a previously unrecognized role for sCD14 in promoting Th17 induction,highlighting its contribution to immune regulation and its potential as a therapeutic target in Th17-driven autoimmune conditions. View Publication

HIV-associated distal sensory polyneuropathy (HIV-DSP) remains prevalent even in the antiretroviral therapy (ART) era. Previously,we identified the upregulation of nociceptive ion channels transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in the dorsal root ganglia (DRG) of simian immunodeficiency virus (SIV)-infected ART-treated macaques. To investigate upstream mechanisms,we performed bulk RNA-seq and pathway analysis on DRGs from uninfected,SIV-infected,and SIV-infected/ART macaques. SIV infection drove strong activation of upstream regulators of interferon γ (IFNγ) and lipopolysaccharide (LPS). Although ART reduced overall IFNγ and LPS pathway activity,the IFNγ-inducible chemokines C-X-C motif chemokine ligand (CXCL)9 and CXCL10 remained significantly upregulated. To determine whether these chemokines influence TRPV1/TRPA1 expression,we treated induced pluripotent stem cell-derived peripheral sensory neurons (iPSC-PSNs) with CXCL9 and CXCL10,which induced a significant increase in TRPV1 but not TRPA1 expression. In parallel experiments,IFNγ but not LPS stimulated monocyte-derived macrophages (MDMs) to release CXCL9 and CXCL10. Conditioned media from IFNγ-treated MDMs modestly increased TRPV1 expression in iPSC-PSNs,and pharmacological inhibition of CXCR3,the receptor of CXCL9/10,did not reduce this effect. Together,these data indicate that persistent IFNγ-driven CXCL9/10 signaling may be one contributor to nociceptor sensitization underlying HIV-DSP,even in the presence of ART. View Publication

The Mas-related G protein-coupled receptor (MRGPR) X2 is expressed on skin mast cells and can be stimulated by an unusually broad spectrum of ligands,including specific drugs and even endogenous peptides. MRGPRX2 activation can induce mast cell degranulation and consequently mediator release,leading to inflammatory and hypersensitivity reactions. In addition,MRGPRX2 mediates pain and itching sensations,leading to increased efforts to identify MRGPRX2 inhibitors,including plant-derived compounds. Components within oat extracts have been shown to mediate anti-inflammatory and itch-relieving properties,but a possible inhibitory effect on MRGPRX2 activation has not yet been investigated. We aimed to fill this gap and explored whether an oat kernel extract can modulate MRGPRX2 activation. For this purpose,we established a mast cell model with the human LAD2 cell line and used it to investigate the consequences of exposure to oat extract. While we did not observe any influence on cell viability,we analyzed the impact of oat extract on MRGPRX2-mediated mast cell activation and degranulation initiated by the three confirmed MRGPRX2 ligands c48/80,substance P,and cortistatin 14. Exposure to oat extract resulted in a significant reduction in mast cell degranulation for all three ligands,as assessed by the release of β-hexosaminidase,tryptase,cell surface expression of CD63 and CD107a,and phosphorylation of ERK. All results were confirmed with primary human mast cells. Thus,we demonstrated for the first time that oat extract leads to a significant reduction in MRGPRX2 activation,pointing to a previously unrecognized capacity of natural compounds to modulate this pathway. View Publication

The immunoregulatory effects of probiotics have been widely studied,particularly in maintaining immune balance. Conventional in vitro functional screening of probiotics relies on fresh donor-derived primary immune cells,which often exhibit significant inter-individual and temporal variability,limiting reproducibility and interpretation. As an alternative,human-induced pluripotent stem cell (iPSC)-derived dendritic cells were co-cultured with five probiotic strains in the current study to evaluate their immunomodulatory interactions. To assess whether cytokines produced by probiotic-stimulated dendritic cells can influence T cell differentiation,human CD4+ T cells were exposed to the conditioned medium derived from co-cultures. Enzyme-linked immunosorbent assay results demonstrated that iPSC-derived dendritic cells secreted cytokines at distinct concentrations in response to different probiotic strains,suggesting that these cells can distinguish between different microbial stimuli,and supporting their use in functional probiotic screening. Among the five strains tested,Lactiplantibacillus plantarum LPA-56,Limosilactobacillus reuteri RU-23,and Lactobacillus fermentum Fem-99 induced cytokine production levels that promoted the differentiation of the human CD4+ T cells into regulatory T cells. These findings demonstrate that iPSC-derived dendritic cells have immunomodulatory potential,are reliable for in vitro screening of probiotics,and offer a promising strategy for selecting potent immunoregulatory probiotic candidates. View Publication

Antibiotic-resistant infections remain a major challenge in cystic fibrosis (CF),where chronic Pseudomonas aeruginosa colonization drives lung infection. The overexpression of adhesion-related proteins and extracellular matrix components,including fibronectin (Fn),facilitates bacterial colonization. Recent evidence identifies the RNA-binding protein Human Antigen R (HuR) as a key regulator of this process,as it stabilizes Vav3 mRNA,promoting Fn deposition and the formation of bacterial docking platforms. Here,we report the synthesis,optimization,and functional evaluation of the HuR-targeted small-molecule (2S,3S)-BOPC1. Functional assays in CF human airway epithelial cells demonstrated that (2S,3S)-BOPC1 significantly reduced P. aeruginosa adhesion in a dose-dependent manner without detectable cytotoxic effects. These findings provide the first evidence that targeting HuR can disrupt the HuR–Vav3–Fn axis,reducing bacterial attachment. This host-directed approach represents a promising strategy to prevent chronic infections in CF without promoting antibiotic resistance. View Publication

Triple Negative Breast Cancers (TNBCs) are heterogeneous and aggressive tumors with a median overall survival of less than two years. Despite the availability of new drugs,the prognosis remains poor,implicating a more aggressive clinical course in the metastatic setting. This study investigated the effects of metronomic treatment (mCHT) with 5-fluorouracil (5-FU) plus vinorelbine (VNR) on spheroids derived from two different TNBC cell lines (BT-549 and MDA-MB-231) and a patient-derived primary cell line (MS-186). mCHT significantly reduced spheroid growth and altered spheroid architecture,with a pronounced effect in second-generation spheroids,enriched in self-renewing cancer stem cells (CSCs). Expression of CSC-related markers (CD44,CD133,NOTCH-1,and MYC) was more significantly altered—both at the mRNA and protein levels—by mCHT than by standard treatment (STD). In MS-186-derived spheroids,mCHT downregulated EZH2 and STAT3,key regulators of CSC maintenance,and reduced H3K27ac,suggesting a global epigenetic reprogramming. Unlike STD,which partially and transiently reduced stemness markers,mCHT achieved sustained suppression,indicating preferential targeting of therapy-resistant CSCs. These results indicate mCHT as a promising strategy for specifically aiming at the CSC-like compartment in TNBC,underscoring a therapeutic approach that reprograms key epigenetic networks and overcomes resistance to treatment. View Publication

Background/objectives: Interactions between colorectal tumours and their immune microenvironment critically influence disease progression and response to immunotherapy. However,most organoid systems fail to preserve the complex architecture and immune composition of the original tissue. Here,we applied the air-liquid interface (ALI) organoid model to paired tumour and perilesional colon tissues from colorectal cancer patients to evaluate its ability to retain immune and genetic features and to reproduce responses to chemotherapy and immune checkpoint blockade. Methods: Fresh human tumour and matched healthy colon tissues were processed to generate ALI organoids. Their histological organization,immune cell composition (including CD45+ subsets),and genomic profiles were compared with those of the parental tissues and with conventional Matrigel organoids,either alone or co-cultured with peripheral blood mononuclear cells (PBMCs). Organoids were exposed to 5-FU and nivolumab (anti-PD-1) to assess local immune modulation. Results: ALI organoids faithfully preserved the three-dimensional architecture,native immune infiltrates,and somatic mutational landscape of the source tissues. Importantly,upon PD-1 blockade with nivolumab,ALI organoids consistently exhibited a local expansion of regulatory T cells (Tregs),a phenomenon that could contribute to adaptive immune resistance. This response was not reproduced in PBMC-Matrigel co-culture systems,highlighting the importance of preserving endogenous tumour-immune interactions. Conclusions: Patient-derived ALI organoids represent a physiologically relevant platform that conserves key structural,immunological,and genomic hallmarks of colorectal cancer. By capturing clinically relevant immune remodeling events,such as Treg expansion following PD-1 blockade,this model provides a powerful tool for dissecting tumour-immune interactions. View Publication

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi sarcoma,primary effusion lymphoma and multicentric Castleman disease. A vaccine that prevents KSHV infection or serves in the treatment of KSHV-related diseases represents a critical unmet need,however,the types of immune responses a vaccine should elicit have not been well defined. The gH/gL glycoprotein complex is an important target of KSHV-neutralizing antibodies,but the epitope specificities targeted by these antibodies remain unknown. Here,we isolated 12 gH/gL-specific monoclonal antibodies (mAbs) from KSHV-infected donors and performed structure/function analyses. These mAbs bind recombinant gH/gL with nanomolar affinities and epitope binning analyses revealed that the mAbs bind to 5 epitope clusters on gH/gL. Seven mAbs were able to neutralize KSHV infection of epithelial cell lines. Two potent neutralizing mAbs mapped to the EphA2 binding site as determined by inhibition of the receptor-ligand interaction and negative stain electron microscopy (nsEM) of the mAb/gH/gL complex. The epitopes of other neutralizing mAbs targeting novel sites of vulnerability were determined by a combination of cryogenic electron microscopy and nsEM. Together,these mAbs help to define the relevant epitope targets for KSHV vaccine design,have utility in understanding the role of antibodies in preventing KSHV infection,enable the development of immunotherapy approaches,and provide valuable tools to understand the molecular details of the KSHV entry process. Author summaryKSHV is an oncogenic virus that can cause cancer in infected individuals. The virus is most prevalent in sub-Saharan Africa and in men who have sex with men. It is possible this virus could be prevented with an effective vaccine,however,the immune response to this virus has not been well defined. gH/gL,a protein essential for viral fusion,plays an important role in infection and could be a possible vaccine target. To better understand the antibody response to this protein,we sought to isolate and characterize monoclonal antibodies that can bind gH/gL and neutralize viral infection. In this study,we isolate and characterize twelve monoclonal antibodies that could bind to five different regions of the gH/gL protein. Seven of these antibodies can neutralize infection,with two being able to block the gH/gL EphA2 receptor-ligand interaction. View Publication

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with poor overall survival (OS). Resistance to chemotherapeutic drugs such as idarubicin (IDA) remains a major cause of treatment failure. This study investigated the anti-leukemic activity of halofuginone (HF) a synthetic derivative of the natural compound from hydrangea Dichroa febrifuge and its potential to overcome IDA resistance in AML cells. Methods: Apoptosis,proliferation,cell cycle,and colony formation were assessed in AML cells treated with HF. RNA sequencing (RNA-seq) was performed to identify the potential molecular targets of HF. The anti-leukemic efficacy of HF was further assessed in NOD/SCID-IL2Rγ (NSG) mice xenografted with human relapsed/refractory (R/R) AML samples. Results: HF treatment significantly inhibited cell proliferation,reduced colony formation,and induced apoptosis in AML cells. By RNA-seq analysis,S100A8 and S100A9 (S100A8/A9) were identified as potential targets of HF,and HF treatment markedly suppressed their expression. Overexpression of S100A8/A9 abrogated the anti-leukemic effects of HF,indicating that S100A8/A9 are critical mediators of HF activity. Mechanistically,HF activated the amino acid starvation response (AAR),leading to phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α),subsequent downregulation of S100A8/A9,and elevation of cytoplasmic Ca2⁺ levels. Knockdown of eIF2α prevented HF-induced downregulation of S100A8/A9,confirming that HF regulates S100A8/A9 expression via the eIF2α pathway. Furthermore,HF treatment inhibited global protein synthesis,enhanced the cytotoxicity of chemotherapeutic drugs,and reversed IDA resistance by suppressing S100A8/A9 expression. Finally,HF inhibits leukemic infiltration and extended OS in MLL-AF9-transduced AML mice and enhanced IDA-induced anti-leukemic effects in R/R AML-xenografted NSG mice model. Conclusions: These findings reveal that HF exerts anti-leukemic effects by modulating the p-eIF2α–S100A8/A9–Ca2⁺ signaling axis in AML cells. HF represents a promising therapeutic candidate for AML,particularly for patients with IDA-resistant disease. View Publication

Aberrant deposition of β‐amyloid (Aβ) and hyperphosphorylated tau,along with neuroinflammation,are key drivers of Alzheimer's disease (AD) pathology. Here,we identify ramalin,a natural antioxidant,as a promising therapeutic agent that alleviates AD pathology by modulating β‐site APP cleaving enzyme 1 (BACE1),histone deacetylase 6 (HDAC6),and the mitogen‐activated protein kinases (MAPK) pathway. Ramalin reduced BACE1 protein levels,independently of its transcription,translation,or enzymatic activity,an effect mediated by inhibition of HDAC6. Consistently,HDAC6 knockout similarly decreased BACE1 levels,highlighting HDAC6 as a key regulator of BACE1. Ramalin further suppressed neuroinflammatory responses by downregulating inducible nitric oxide synthase (iNOS) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. In AD mouse models,ramalin treatment significantly attenuated neuroinflammation,Aβ plaque burden,and tau hyperphosphorylation,while improving cognitive performance. Notably,ramalin reversed Aβ oligomer‐induced synaptic transmission impairment and restored synaptic vesicle recycling in hippocampal neurons. Transcriptomic analysis identified modulation of the MAPK pathway,with reduced phosphorylation of c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK) implicated in tau pathology. These findings establish ramalin as a disease‐modifying intervention that provides neuroprotection through concurrent regulation of BACE1,HDAC6,and MAPK signaling pathway. Collectively,our findings highlight ramalin as a compelling disease‐modifying candidate with the potential to drive a breakthrough approach targeting AD pathology. Ramalin alleviates Alzheimer's disease pathology by selectively inhibiting HDAC6,reducing BACE1 levels,and suppressing neuroinflammation through downregulation of the NLRP3 inflammasome and iNOS. It restores synaptic function impaired by Aβ toxicity and improves cognitive performance in AD mouse models,APP/PS1 and 3xTg‐AD. Additionally,ramalin modulates the MAPK signaling pathway,reducing tau phosphorylation by inhibiting JNK and ERK activation. View Publication