参考文献

Discovering new and viable therapies for genetic diseases is a time-consuming and cost-intensive process,especially for rare disorders. In this study,we highlight how a high-throughput drug discovery platform was utilized to uncover drugs at scale that normalized the signature for a rare neurological neurodevelopmental disease,19q12 autism spectrum disorder (ASD) associated with deficiencies in ZNF536 and TSHZ3. We first identified the transcriptomic fingerprint of the disease in an in vitro disease model in the form of dysregulated pathways. Subsequently,we measured the biological impact of small molecule drugs in a relevant wild-type cell line and uncovered an approved drug Entrectinib that induced the opposite effect to that in the disease fingerprint,demonstrating the capability to normalize the disease fingerprint. Entrectinib was further prescribed off-label to the identified patient with 19q12 and drug effect was characterized both from blood collection and neuropsychological assessments. Biomarkers from blood recapitulated Entrectinib’s pharmacodynamic effect and normalized the disease signature. We show how generation of transferrable transcriptomics-derived disease signatures allows for measuring drug effects on a signature in related wild-type cell lines,making the screen universally applicable and reducing the need for expensive screens in disease models. View Publication

Medulloblastoma (MB) is the most prevalent malignant brain tumor in children,exhibiting clinical and genomic heterogeneity. Of the four major subgroups,Group 3 tumors (MYC-MB),display high levels of MYC and metastasis rates. Despite treatment with surgery,radiation,and chemotherapy,patients with Group 3 MB are more likely to develop aggressive recurrent tumors with poor survival. To examine resistance mechanisms in this study,we perform single nuclei multiome analysis of matched primary and recurrent tumors. Therapy resistant Medulloblastoma demonstrates an expanded persistent progenitor population. Additionally,distinct chromatin landscapes link to altered transcription and correspond with metabolic reprogramming. In vivo modeling of radiation resistance exhibits similar chromatin-based metabolic reprogramming focused on wild-type isocitrate dehydrogenase (IDH1) activity. IDH1 inhibition reverses resistance-mediated chromatin changes and enables radiation re-sensitization. Ultimately,these findings demonstrate the efficacy of single-cell multiome analysis in elucidating resistance mechanisms and identifying targetable pathways for MYC-driven medulloblastoma. MYC-driven medulloblastoma is highly aggressive and associated with poor survival. Here,the authors perform single nuclei multi-omic analysis of matched primary and recurrent tumours and identify potential resistance mechanisms and targetable pathways. View Publication

Endometrial receptivity is a critical determinant of embryo implantation and early pregnancy success; however,current methods for assessing endometrial receptivity remain poorly validated and insufficiently reliable for clinical application. Here,we establish a patient-derived vascularised endometrium-on-a-chip (EoC),successfully replicating the dynamic microenvironment and both temporal and spatial architecture of native endometrial tissue. Using our EoC,we develop a clinically relevant endometrial receptivity scoring system,ERS2,which integrates molecular profiling of established receptivity markers with quantitative analyses of angiogenesis. The ERS2 enables personalised assessment of endometrial health and implantation potential,addressing inter-patient variability often overlooked by conventional techniques. By leveraging our EoC to therapeutic monitoring,we observe progressive restoration of the endometrial microenvironment following platelet-rich-plasma treatments,highlighting the translational utility of our model. This study represents the innovative application of a patient-derived EoC and scoring system to assess receptivity,offering personalised infertility management and advancing targeted therapies in reproductive medicine. Accurate assessment of endometrial receptivity remains a challenge in infertility care. Here,authors present a patient-derived vascularised endometrium-on-a-chip and a scoring system for receptivity evaluation. View Publication

Cancer stem cells (CSC) drive therapeutic resistance and recurrence in head and neck squamous cell carcinoma (HNSCC). We and others have shown that treatment with cytotoxic chemotherapy agents (e.g. Cisplatin,Carboplatin) induce Bmi-1 expression and increase the fraction of highly tumorigenic CSC in HNSCC. Notably,Bmi-1 is a master regulator of stem cell self-renewal and DNA repair. The purpose of this work was to test whether therapeutic inhibition of Bmi-1 sensitizes HNSCC cancer stem cells to chemotherapy. HNSCC cells (UM-SCC-1,-22A,-22B) were treated with Cisplatin or Carboplatin and subjected to stemness analyses to evaluate the impact of Bmi-1 on chemoresistance. We observed that both,shRNA-mediated Bmi-1 silencing or pharmacologic inhibition of Bmi-1 with the small molecule inhibitor PTC596,blocked chemotherapy-induced cancer stemness (i.e. increase in the fraction of ALDHhighCD44high cells),CSC self-renewal (i.e. orosphere formation) and inhibited protective DNA damage responses in HNSCC. Further,it is known that high IL-6 serum levels correlate with poor HNSCC patient survival,and that platinum-based therapies induce IL-6 signaling. Here,we observed that Bmi-1 silencing (or PTC596 treatment) inhibited the IL-6R/STAT3 signaling pathway even in presence of platinum-based cytotoxic agents (i.e. Cisplatin,Carboplatin). In vivo,Bmi-1 inhibition with PTC596 suppressed Cisplatin-mediated increase in the fraction of ALDHhighCD44high cells (cancer stemness). Collectively,these preclinical results demonstrate that Bmi-1 is a key mediator of head and neck cancer stemness and suggest that HNSCC patients might benefit from treatment with a Bmi-1 inhibitor combined with a conventional chemotherapeutic agent. View Publication

Atovaquone,an FDA-approved oxidative phosphorylation (OXPHOS) inhibitor,has shown promise in the treatment of epithelial ovarian cancer (EOC),the deadliest gynecologic malignancy. However,the precise mechanisms underlying its antitumorigenic effects remain unclear. We employed a longitudinal transcriptomic approach to characterize the molecular effects of atovaquone on EOC cells. Our findings demonstrate that atovaquone disrupts cellular homeostasis and metabolism,activates stress responses,and primes immune recognition. We observed temporal downregulation of genes and pathways involved in key cellular processes,such as the cell cycle and DNA replication,which correlated with reduced proliferative capacity. Atovaquone also downregulated both OXPHOS and glycolysis while upregulating the pentose phosphate pathway,suggesting a metabolic shift toward redox homeostasis restoration in response to severe oxidative stress. Consistent with oxidative stress,we found that atovaquone activated endoplasmic reticulum (ER) stress,which is linked to immunogenic cell death. During ER stress,calreticulin,a damage-associated molecular pattern (DAMP),translocates to the plasma membrane,where it promotes immune recognition. We observed that calreticulin was upregulated on the plasma membrane of atovaquone-treated EOC cells. Additionally,we detected increased levels of other DAMPs,such as high mobility group box 1 (HMGB1) and mitochondrial transcription factor A (TFAM),in the supernatants of atovaquone-treated cells,indicating the release of immunogenic molecules. Moreover,increased expression of ligands for activating receptors of NK cells was observed,and coculture experiments revealed enhanced NK cell activity toward atovaquone-treated cells. These results highlight atovaquone’s potential to activate immune responses,offering a new avenue for combination therapies in EOC treatment. View Publication

Influenza infection predisposes individuals to secondary pneumonia caused by a range of pathogens,including both bacterial and fungal organisms. Neutrophils are critical effector cells during infection. In this study,we analyzed the transcriptional pathways of lung neutrophils isolated from mouse models of influenza-associated pulmonary aspergillosis (IAPA) and post-influenza methicillin-resistant Staphylococcus aureus (MRSA) pneumonia to examine the immunopathological mechanisms underlying post-influenza super-infection. Pathways associated with neutrophil chemotaxis and degranulation were inhibited in IAPA compared to singular A. fumigatus infection and pathways associated with neutrophil recruitment and phagocytosis were inhibited in IAPA compared to singular influenza infection. Pathways associated with neutrophil recruitment and degranulation were inhibited in post-influenza MRSA pneumonia compared to singular MRSA infection and pathways associated with cytokine signaling were inhibited in post-influenza MRSA pneumonia compared to singular influenza infection. When the 2 types of super-infection were directly compared,pathways related to cytokine induction and neutrophil function were inhibited in IAPA neutrophils compared to post-influenza MRSA pneumonia. These data demonstrate that influenza causes neutrophil dysfunction,predisposing to secondary fungal and bacterial infections. View Publication

Gastrointestinal (GI) dysfunction,characterized by severe diarrhea and dehydration,is a central contributor to morbidity and mortality in filovirus disease in patients,yet the role of the epithelium in this clinical outcome remains poorly defined. Here,we employ induced pluripotent stem cell (iPSC)-derived human intestinal (HIOs) and colonic organoids (HCOs) to model Ebola virus (EBOV) and Marburg virus (MARV) infection. These organoids are permissive to filovirus infection and support viral replication. Bulk RNA sequencing revealed distinct intestinal and colonic epithelial responses,including apical and junctional disruption and a delayed virus-specific induction of interferon-stimulated genes. Moreover,infection impaired adenylate cyclase signaling and CFTR-mediated ion transport,providing mechanistic insight into virus-induced secretory diarrhea. This platform recapitulates key features of human GI pathology in filoviral disease and serves as a powerful system to dissect host-pathogen interactions and identify therapeutic targets. Author summaryEbola virus (EBOV) and Marburg virus (MARV) are among the most lethal viruses known. Infection with these viruses leads to severe disease and death. One of their most harmful effects is damage to the gastrointestinal tract,causing intense diarrhea and life-threatening dehydration. Yet,how these viruses affect the gut remains poorly understood. In this study,we used human mini-guts—small,three-dimensional tissues grown from stem cells that mimic the human intestinal and colonic epithelium—to investigate how these viruses interact with gut epithelial cells. We found that both EBOV and MARV infect and replicate in these tissues,disrupt key barrier structures,and interfere with the cells’ ability to regulate fluid secretion. These effects mirror the severe symptoms seen in patients. Our study provides new insight into how EBOV and MARV damage the gut and identifies specific cellular pathways that may be targeted for treatment. This research not only improves our understanding of EBOV and MARV infections but also offers new infection platforms for testing therapies aimed at protecting the gastrointestinal system during filovirus outbreaks. View Publication

Retinal neurogenesis is mediated by the coordinated activities of a complex gene regulatory network (GRN) of transcription factors (TFs) in multipotent retinal progenitor cells (RPCs). How this GRN mechanistically guides neural competence remains poorly understood. In this study,we present integrated transcriptional,genetic and genomic analyses to uncover the regulatory mechanisms of SOX2,a key factor in establishing neural identity in RPCs. We show that SOX2 is preferentially enriched in the RPC-specific enhancer landscape associated with essential regulators of retinogenesis. Disruption of SOX2 expression impairs retinogenesis,marked by a selective loss of enhancer activity near genes essential for RPC proliferation and lineage specification. We identified the RPC transcription factor VSX2 as a binding partner for SOX2 and,together,SOX2 and VSX2 co-target a core,retina-specific chromatin repertoire characterized by enhanced TF binding and robust chromatin accessibility. This cooperative binding establishes a shared SOX2-VSX2 transcriptional code that promotes the expression of crucial regulators of neurogenesis while repressing the acquisition of alternative lineage cell fate. Our data illuminate fundamental biological insights on how transcription factors act in concert to drive chromatin-based genetic programs underlying retinal neural identity. View Publication

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population,causing infectious mononucleosis,susceptibility to autoimmune diseases and multiple malignancies of epithelial or B cell-origin. EBV infects epithelial cells and B cells through interaction between viral glycoproteins and different host receptors,but it has remained unknown whether a common receptor mediates infection of its two major host cell targets. Here,we establish R9AP as a crucial EBV receptor for entry into epithelial and B cells. R9AP silencing or knockout,R9AP-derived peptide and R9AP monoclonal antibody each significantly inhibit,whereas R9AP overexpression promotes,EBV uptake into both cell types. R9AP binds directly to the EBV glycoprotein gH/gL complex to initiate gH/gL-gB-mediated membrane fusion. Notably,the interaction of R9AP with gH/gL is inhibited by the highly competitive gH/gL-neutralizing antibody AMMO1,which blocks EBV epithelial and B cell entry. Moreover,R9AP mediates viral and cellular membrane fusion in cooperation with EBV gp42-human leukocyte antigen class II or gH/gL-EPHA2 complexes in B cells or epithelial cells,respectively. We propose R9AP as the crucial common receptor of B cells and epithelial cells and a potential prophylactic and vaccine target for EBV. View Publication

Different innate immune cell types are known to release extracellular traps (ETs) in response to invasive pathogens,including parasites. These ETs function to trap,immobilize,and eventually kill pathogens. In line with this,monocytes and macrophages have been shown to release ETs,known as monocyte/macrophage extracellular traps (METs). Toxoplasma gondii (T. gondii) is an apicomplexan zoonotic parasite that infects humans and homeothermic animals. While most studies have focused on prolonged exposure of immune cells to T. gondii,this study characterized the early innate immune reaction of mononuclear phagocytes to vital T. gondii tachyzoites. Methods: Primary human and bovine monocytes,monocytic THP-1 cells,and THP-1 cell-derived macrophages (M0-,M1-,and M2-like) were exposed to T. gondii tachyzoites for 4 h. Scanning electron microscopy (SEM),transmission electron microscopy (TEM),immunofluorescencemicroscopy,and confocal microscopy were used to visualize cell activation and the presence of METs. Additionally,the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-6,and expression of Toll-like receptor (TLR) 2 and TLR4 were analyzed. Results and discussion: Microscopic analysis illustrated the activation of all cell types tested within 4 h of exposure to T. gondii tachyzoites. Numerous tachyzoites were found intracellularly in THP-1 cell-derived M1-like macrophages. Furthermore,the co-localization of extracellular DNA (extDNA) and histones in extracellular web-like fibers proved classical characteristics of extruded T. gondii-induced METs,although this was a rare event. In primary human monocytes,an increased release of IL-1β and IL-6 was observed following exposure to T. gondii tachyzoites. When co-stimulated with lipopolysaccharide (LPS),primary human monocytes showed an enhanced release of IL-1β and IL-6 in response to T. gondii. In contrast to monocytic THP-1 cells,THP-1 cell-derived M1-like macrophages released IL-1β in response to T. gondii tachyzoite exposure. When additionally stimulated by LPS,all THP-1 cell-derived macrophages showed an enhanced release of IL-1β,and monocytic THP-1 cells an increased release of IL-6 in response to T. gondii tachyzoites. This study provides insights into the early innate immune response of human and bovine mononuclear phagocytes to T. gondii. While cytokine secretion was prominent,MET formation was rare in the early response (i.e. < 4 h of exposure) to T. gondii tachyzoites. View Publication