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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

Many biological tissues,such as cardiac muscle,tendons,and the cornea,exhibit highly organized microstructural alignment that is critical for mechanical and physiological functions. Disruptions in this structural organization are commonly associated with pathological conditions such as fibrosis,infarction,and cancer. However,conventional histological imaging techniques rely on immunofluorescence or histochemical staining,and they evaluate tissue alignment via non-physical 2D gradient-based calculation,which is labor-intensive,antibody-dependent,and prone to variability. Here,we demonstrate label-free mid-infrared dichroism-sensitive photoacoustic microscopy (MIR-DS-PAM),an analytical imaging system for cardiac tissue assessments. By combining molecular specificity with polarization sensitivity,this method selectively visualizes protein-rich engineered heart tissue (EHT) and quantifies the extracellular matrix (ECM) alignment without any labeling. The extracted dichroism-sensitive parameters,such as the degree of dichroism and the orientation angle,enable histostructural evaluation of tissue integrity and reveal diagnostic cues in fibrotic EHT. This technique offers a label-free analytical tool for fibrosis research and tissue engineering applications. Mid-infrared dichroism-sensitive photoacoustic microscopy enables label-free,quantitative histostructural analysis by combining spectral specificity and polarization sensitivity to visualize protein-rich components and evaluate anisotropic tissue alignment. View Publication

BACKGROUND: The discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). AIM: To evaluate and compare five iMSC differentiation protocols,assessing their efficiency,phenotypic characteristics,and functional properties relative to primary mesenchymal stem cells (MSCs). METHODS: Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1,iMSC3),an iMatrix-free method (iMSC2),growth factor supplementation (iMSC4),and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria,requiring expression of surface markers (CD105,CD73,CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic),proliferation,mitochondrial function,reactive oxygen species,senescence,and migration. RESULTS: All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001,respectively),while adipogenic potential was reduced in iMSC2 (Adipsin; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01,respectively). Proliferation was comparable or superior to bone marrow MSCs,except in iMSC1,with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001),reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001),and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours). CONCLUSION: While all protocols generated functional iMSCs,variations in differentiation,proliferation,and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications. View Publication

Background: DDB1 and CUL4‐associated factor 7 (DCAF7) is a WD‐repeat adaptor that recruits substrates to the CUL4DDB1 ubiquitinligase complex,but its pan‐cancer relevance and mechanistic contribution to tumor progression remain unclear. Methods: Multi‐omics datasets (genomic,transcriptomic,epigenomic,proteomic and single‐cell) from 33 tumor types were integrated to define DCAF7 expression,regulation,and clinical significance. Somatic alterations and copy‐number variation were analysed across cohorts,and promoter methylation and RNA modification signatures were interrogated. Immune associations were assessed by computational deconvolution and checkpoint‐gene profiling. Pathway and network analyses were performed to infer DCAF7‐linked programmes. Mechanistic and functional validation was conducted in hepatocellular carcinoma (LIHC) cell lines (HepG2,Huh7) using DCAF7 perturbation and pharmacologic Wnt inhibition. Results: DCAF7 was overexpressed in most cancers,consistent with copy‐number gain,focal promoter hypomethylation and putative m6A‐linked post‐transcriptional regulation,whereas hypermethylation at two CpG loci predicted poor prognosis in LIHC. DCAF7 alterations,predominantly amplifications,were associated with shorter overall survival in LIHC and positively correlated with DCAF7 mRNA abundance across cohorts. Immunogenomic analyses linked high DCAF7 to CD4+ T‐cell enrichment,broad upregulation of checkpoint genes (PD‐1/PD‐L1,CTLA‐4,TIGIT),and increased tumour mutational burden,microsatellite instability and neoantigen load,suggesting an immune‐evasive phenotype. Network and enrichment analyses converged on canonical Wnt/β‐catenin,Hippo and cell‐cycle programs. In vitro,DCAF7 promoted LIHC cell proliferation and migration by stabilising β‐catenin via increased inhibitory Ser9 phosphorylation of GSK‐3β,thereby inducing c‐Myc and cyclin D1; DCAF7 knockdown or the Wnt inhibitor XAV939 attenuated these effects. Drug‐response modelling further predicted increased sensitivity of DCAF7‐high tumours to 17‐AAG,docetaxel and alsterpaullone. Conclusions: DCAF7 is frequently activated by genetic and epigenetic mechanisms across cancers,associates with an immunotherapy‐relevant tumour immune milieu,and drives Wnt/β‐catenindependent malignant phenotypes in LIHC. These findings support DCAF7 as a prognostic biomarker and a candidate therapeutic target,particularly for stratified intervention in LIHC. Key points: DCAF7 is up‐regulated in various tumours and correlates with poor prognosis,particularly in LIHC. High DCAF7 expression is linked to CD4+ T cell infiltration,up‐regulation of immune checkpoint genes and increased tumour mutational burden,suggesting a role in tumour immune escape. DCAF7 stabilises β‐catenin by enhancing GSK‐3β Ser9 phosphorylation,thereby driving c‐Myc/cyclin D1 expression and contributing to proliferation and migration in LIHC. DCAF7‐high tumours demonstrate therapeutic vulnerability to 17‐AAG,docetaxel and CDK/GSK‐3 inhibitor,revealing potential targeted treatment strategies. View Publication