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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

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis,binding and uptake of exosomes are well characterized4,5,delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology,we show that these two lipid receptors,which interact in the late endosome9,are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway,which Ebola virus exploits to infect cells9,mediates HAV infection by exo-HAV,which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen,but not the endosomal uncoating of HAV particles contained in the exosomes,is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV,infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses,and call for a reassessment of the role of envelope glycoproteins in infection. View Publication

Na{\{i}}ve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions T cell activation and T cell expansion in vivo. Our results showed that na{\"{i}}ve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient na{\"{i}}ve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse and that these conjugates were less stable than their WT counterparts. Finally we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions which promotes the initiation of stable T cell conjugates during APC scanning. Therefore Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo." View Publication

BACKGROUND In kidney transplantation,early allograft inflammation impairs long-term allograft function. However,precise mediators of early kidney allograft inflammation are unclear,making it challenging to design therapeutic interventions. METHODS We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. RESULTS Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period,they were also induced to express a high level of Ccl8,which,in turn,promoted recipient monocyte graft infiltration,their differentiation to resident macrophages,and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed,including CD4,CD8,and ?? T cells. Consequently,blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. CONCLUSIONS Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation. View Publication

Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

Progressive resistance exercise training (PRT) is the most effective known intervention for combating aging skeletal muscle atrophy. However,the hypertrophic response to PRT is variable,and this may be due to muscle inflammation susceptibility. Metformin reduces inflammation,so we hypothesized that metformin would augment the muscle response to PRT in healthy women and men aged 65 and older. In a randomized,double-blind trial,participants received 1,700 mg/day metformin (N = 46) or placebo (N = 48) throughout the study,and all subjects performed 14 weeks of supervised PRT. Although responses to PRT varied,placebo gained more lean body mass (p = .003) and thigh muscle mass (p {\textless} .001) than metformin. CT scan showed that increases in thigh muscle area (p = .005) and density (p = .020) were greater in placebo versus metformin. There was a trend for blunted strength gains in metformin that did not reach statistical significance. Analyses of vastus lateralis muscle biopsies showed that metformin did not affect fiber hypertrophy,or increases in satellite cell or macrophage abundance with PRT. However,placebo had decreased type I fiber percentage while metformin did not (p = .007). Metformin led to an increase in AMPK signaling,and a trend for blunted increases in mTORC1 signaling in response to PRT. These results underscore the benefits of PRT in older adults,but metformin negatively impacts the hypertrophic response to resistance training in healthy older individuals. ClinicalTrials.gov Identifier: NCT02308228. View Publication

Human cortical neural progenitor cell transplantation holds significant potential in cortical stroke treatment by replacing lost cortical neurons and repairing damaged brain circuits. However,commonly utilized human cortical neural progenitors are limited in yield a substantial proportion of diverse cortical neurons and require an extended period to achieve functional maturation and synaptic integration,thereby potentially diminishing the optimal therapeutic benefits of cell transplantation for cortical stroke. Here,we generated forkhead box G1 (FOXG1)-positive forebrain progenitors from human inducible pluripotent stem cells,which can differentiate into diverse and balanced cortical neurons including upper- and deep-layer excitatory and inhibitory neurons,achieving early functional maturation simultaneously in vitro. Furthermore,these FOXG1 forebrain progenitor cells demonstrate robust cortical neuronal differentiation,rapid functional maturation and efficient synaptic integration after transplantation into the sensory cortex of stroke-injured adult rats. Notably,we have successfully utilized the non-invasive 18F-SynVesT-1 PET imaging technique to assess alterations in synapse count before and after transplantation therapy of FOXG1 progenitors in vivo. Moreover,the transplanted FOXG1 progenitors improve sensory and motor function recovery following stroke. These findings provide systematic and compelling evidence for the suitability of these FOXG1 progenitors for neuronal replacement in ischemic cortical stroke. Human NPCs show promise for stroke treatment,but challenges remain in neuron diversity and maturation time. Here,the authors describe the generation of FOXG1 progenitors from iPSCs that quickly mature into functional cortical neurons,enhancing stroke recovery in rats. View Publication

IntroductionCRISPR/Cas9-edited induced pluripotent stem cells (iPSCs) are valuable research models for mechanistic studies. However,gene conversion between a gene-pseudogene pair that share high sequence identity and form direct repeats in proximity on the same chromosome can interfere with the precision of gene editing. Mutations in the human beta-glucocerebrosidase gene (GBA1) are associated with Gaucher disease,Parkinson’s disease,and Lewy body dementia. During the creation of a GBA1 KO iPSC line,we detected about 70% gene conversion from its pseudogene GBAP1. These events maintained the reading frame and resulted from GBA1-specific cleavage by CRISPR/Cas9,without disrupting the GBA1 gene.MethodTo increase the percentage of alleles with out-of-frame indels for triggering nonsense-mediated decay of the GBA1 mRNA,we supplied the cells with two single-stranded oligodeoxynucleotide (ssODN) donors as homology-directed repair (HDR) templates.ResultsWe demonstrate that HDR using the ssODN templates effectively competes with gene conversion and enabled biallelic KO clone isolation,whereas the nonallelic homologous recombination (NAHR)-based deletion rate remained the same.DiscussionHere,we report a generalizable method to direct cellular DNA repair of double strand breaks at a target gene towards the HDR pathway using exogenous ssODN templates,allowing specific editing of one gene in a gene-pseudogene pair without disturbing the other. View Publication

Genomic imprinting controls parental allele-specific gene expression via epigenetic mechanisms. Abnormal imprinting at the GNAS gene causes multiple phenotypes,including pseudohypoparathyroidism type-1B (PHP1B),a disorder of multihormone resistance. Microdeletions affecting the neighboring STX16 gene ablate an imprinting control region (STX16-ICR) of GNAS and lead to PHP1B upon maternal but not paternal inheritance. Mechanisms behind this imprinted inheritance mode remain unknown. Here,we show that the STX16-ICR forms different chromatin conformations with each GNAS parental allele and enhances two GNAS promoters in human embryonic stem cells. When these cells differentiate toward proximal renal tubule cells,STX16-ICR loses its effect,accompanied by a transition to a somatic cell-specific GNAS imprinting status. The activity of STX16-ICR depends on an OCT4 motif,whose disruption impacts transcript levels differentially on each allele. Therefore,a biallelically active embryonic enhancer dictates GNAS imprinting via different chromatin conformations,underlying the allele-specific pathogenicity of STX16-ICR microdeletions. STX16 microdeletions cause pseudohypoparathyroidism type-1B only on the maternal allele. Here,the authors show that the allele-specific pathogenicity reflects differential conformations of a biallelically active enhancer dictating GNAS imprinting. View Publication

Brain tumors are commonly treated with radiotherapy,but the efficacy of the treatment is limited by its toxicity to the normal tissue including post-irradiation contrast enhanced lesions often linked to necrosis. The poorly understood mechanisms behind such brain lesions were studied using cerebral organoids. Here we show that irradiation of such organoids leads to dose-dependent growth retardation and formation of liquid-filled cavities but is not correlated with necrosis. Instead,the radiation-induced changes comprise of an enhancement of cortical hem markers,altered neuroepithelial stem cell differentiation,and an increase of ZO1+/AQP1+/CLDN3+-choroid plexus (CP)-like structures accompanied by an upregulation of IGF2 mRNA,known to be expressed in CP and cerebrospinal fluid. The altered differentiation is attributed to changes in the WNT/BMP signaling pathways. We conclude that aberrant CP formation can be involved in radiation-induced brain lesions providing additional strategies for possible countermeasures. Human cerebral organoids provide insights into mechanisms behind the formation of choroid plexus (CP)-like structures that may contribute to radiation-induced brain image changes. View Publication