技术资料

USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP),a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans,and its earliest pathogenic events remain unknown. Here,we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A−/− induced pluripotent stem cells. Methods: We assessed organoids for cellular,molecular,and morphological defects using serial live imaging and whole organoid and fixed section analyses. Results: Both patient-derived and isogenic-USH2A−/− organoids showed preferential rod photoreceptor loss followed by widespread degeneration,consistent with the clinical phenotype. Additionally,isogenic-USH2A−/− organoids showed early defects in proliferation and structure. Conclusions: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP,and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features,we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies. View Publication

We used high-resolution optical mapping (~50 µm) to investigate potential arrhythmia mechanisms following transplantation of engineered cardiac tissue. We induced myocardial infarction in 6 immunosuppressed pigs and implanted cardiac spheroids into the border zone. One week later,600-µm-thick cardiac slices containing implanted spheroids were harvested and electrical propagation was imaged. Histology showed low connexin-43 expression,scar,and misaligned muscle fibers at the graft-host interface. We observed propagation from host-to-graft in 10 slices from 3 pigs. Host-graft electrical bridges were spaced by millimeters. Propagation was ~4-fold slower in the graft than host. One graft beat spontaneously,but activation did not propagate from graft-to-host in this,or any other slice. We did not observe reentry,but slow in-graft conduction and sparse electrical bridges provided opportunity for reentry induction. These data reveal potential for reentrant or focal arrhythmias 1 week post-implant,which may resolve with maturation of the graft and the graft-host interface. View Publication

DNA repair mechanisms in human primary cells,including error-free repair,and,recurrent nuclease cleavage events,remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR),an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs,small indels,large deletions,and other aberrations in absolute terms in clinically relevant edited cells,including HSPCs,iPSCs,and T-cells. By quantifying up to 90% of loci with unresolved DSBs,CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore,we accurately quantify DNA repair precision,revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics,paving the way for mechanistic studies to advance gene therapy,designer editors,and small molecule discovery. Quantifying genomic aberrations resulting from designer nucleases activity is essential for gene therapy clinical translation. Here,the authors present a modular digital PCR technique that profiles DNA repair precision and cut-repair cycles at the edited loci,exposing current evaluation biases. View Publication

Regulatory T cells are a class of T lymphocytes which respond to activation signals by expanding their cell numbers,and whose culturing and expansion are of significant clinical interest. Cellular activation states are used to inform process control decisions such as restimulation and can be probed with experimental measurements of cell surface markers. However,these measurements are expensive,time-consuming,and invasive,and an urgent need exists for devising a non-invasive method for activation state monitoring that could be deployed on-line. Raman spectroscopy is a label-free and information-rich optical method that,when coupled to data analytical methods,can ameliorate these experimental issues. In this work,we quantitatively estimated experimental measurements of regulatory T cell activation markers with high accuracy. We simulated a clinical manufacturing setting by building an L1-regularized least-squares model with spectroscopic data from six regulatory T cell donors. Then,we validated the constructed model by accurately estimating different experimental measurements of biomarker values from two external donors,unseen by the model. We have devised a robust program to effectively estimate the activation state of regulatory T cells. We anticipate our method to be used with on-line Raman probes integrated into cell manufacturing devices for label-free monitoring of these processes. View Publication

Parechovirus ahumpari 3 (HPeV-3) is among the main agents causing severe neonatal neurological infections such as encephalitis and meningitis. However,the underlying molecular mechanisms and changes to the host cellular landscape leading to neurological disease has been understudied. Through quantitative proteomic analysis of HPeV-3 infected neural organoids,we identified unique metabolic changes following HPeV-3 infection that indicate immunometabolic dysregulation. Protein and pathway analyses showed significant alterations in neurotransmission and potentially,neuronal excitotoxicity. Elevated levels of extracellular glutamate,lactate dehydrogenase (LDH),and neurofilament light (NfL) confirmed glutamate excitotoxicity to be a key mechanism contributing to neuronal toxicity in HPeV-3 infection and can lead to apoptosis induced by caspase signaling. These insights are pivotal in delineating the metabolic landscape following severe HPeV-3 CNS infection and may identify potential host targets for therapeutic interventions. View Publication

Phosphatidylinositol 3-kinase delta (PI3KCD) is a critical signaling enzyme for B cell development,activation,function and immune regulation. Gain-of-function mutations in PI3KCD result in the congenital immunodeficiency known as Activated PI3KCD Syndrome (APDS). APDS patients are prone to repeated infections and other serious clinical manifestations. Here,we determine how B cell-intrinsic expression of the APDS-associated PI3KCDE1021K mutation impacts immune responses to the protozoan parasite Trypanosoma congolense. PI3KCDE1021K/B mice exhibit a significant expansion of IL10-expressing B cells within the spleen and peritoneal cavity,which was associated with impaired control of T. congolense infection. Despite the generation of robust germinal center,plasma cell and antibody responses,PI3KCDE1021K/B mice show elevation in the first wave of parasitemia and increased mortality. We further characterize the phenotype of the expanded IL10-producing B cell population in PI3KCDE1021K/B mice,which show hallmarks of innate-like regulatory B cells (Breg) and expression of multiple inhibitory molecules. This Breg expansion is associated with reduced IFNγ/IL10 ratio,reduced TNFα production and impaired activation of myeloid cells,likely compromising the innate response to infection. These findings highlight the profound impact of dysregulated PI3KCD activity on regulatory B cells that can functionally impair innate immune responses controlling a systemic parasite protozoan disease. Author summaryB cells and antibodies play a critical role in the immune response to Trypanosome parasites. Molecular signaling networks within B cells can control the type of response generated during infection. Here,we studied how a genetic variant in the signaling enzyme PI3KCD,previously linked to human immune deficiencies,impacts B cell responses to Trypanosome infection. We find that mice expressing the PI3KCDE1021K mutation in their B cells show impaired control of Trypanosome infection,and alterations in several aspects of the immune response. Specifically,we noted these mice poorly control parasite growth within the first week of infection,a timeframe where specific antibody responses have not yet been generated. We noted an altered balance between pro-inflammatory and anti-inflammatory cytokine mediators produced within the first week of infection. This was associated with high numbers of regulatory B cells expressing multiple molecules capable of inhibiting other cells of the immune system. We further found that these mice show functional alterations in other critical immune cell types,such as macrophages and T cells. These findings highlight the impact of dysregulated PI3KCD activity on regulatory B cells that can impair immune responses controlling a systemic parasite protozoan disease. View Publication

Conventional human embryonic stem cells (hESCs) are capable of self-renewal and simultaneously poised for differentiation. But the mechanisms underlying this primed pluripotent state,which endows them with elevated responsiveness to differentiation cues,remain largely underexplored. Especially,little is known about the pivotal transcription factors (TFs) that orchestrate hESCs towards primed pluripotency. Here,we report a function of TF ZNF263 in pluripotency priming. Genetic and functional assays reveal that ZNF263 directly initiates the incipient expression of early differentiation genes and concurrently dampens the core pluripotency circuitry in hESCs,greatly tilting the balance from pluripotency maintenance to lineage priming. Importantly,ZNF263 deficiency markedly impairs pluripotency dissolution and multi-lineage differentiation in hESCs,particularly toward ectoderm. Moreover,single-cell transcriptomic profiling reveals that ZNF263 promotes the priming of cell fate commitment in hESCs,suggesting its indispensable requirement for pluripotency priming and lineage commitment continuum. Together,we demonstrate the role of ZNF263 in establishing the primed pluripotent state in hESCs and facilitating their differentiation into primary germ layer lineages. Human embryonic stem cells are simultaneously capable of self-renewal and poised for differentiation. Here,the authors show a role for the ZNF263 transcription factor promotes primed pluripotency and facilitates differentiation into primary germ layer lineages. View Publication

Antibody–drug conjugates (ADCs) represent a promising strategy in cancer therapy,enabling the targeted delivery of cytotoxic agents to tumor cells. In this study,we developed and characterized novel ADCs combining the anti-EGFR monoclonal therapeutic antibody Cetuximab (Cet) with two aminobisphosphonates (N-BPs) analogues of zoledronic acid (ZA): DC310 and the aminothiazole DC315. These conjugates aim to enhance antitumor efficacy of Cet in colorectal cancer (CRC) by both directly inhibiting tumor cell growth and activating Vδ2 T lymphocytes. We optimized the drug-antibody ratio (DAR),achieving significantly higher DARs compared to previously reported Cet-ZA conjugate,particularly with Cet-DC315 (DAR ≈ 23). Both ADCs retained selective EGFR binding in CRC cell lines and patient-derived organoids (PDO). Functionally,Cet-DC315 markedly inhibited proliferation of EGFR⁺ CRC cell lines in conventional cultures and 3D spheroids. Furthermore,Cet-DC-315 uniquely induced expansion and cytotoxic activation of Vδ2 T cells in co-cultures with CRC cell lines,PDO,and primary tumor samples. These findings suggest that ADCs incorporating novel N-BPs such as DC315 represent a potent approach for dual antitumor targeting through direct cytostatic effects and immune activation,offering a potential therapeutic advantage in the treatment of EGFR+ colorectal cancer. View Publication

Since the COVID-19 pandemic,there has been a documented rise in the incidence of neurological manifestations among individuals complicated with encephalitis or myelitis. The spectrum of neurological symptoms associated with HCoVs infections is expanding. However,the infection characteristics and pathogenesis of seasonal HCoVs to the central nervous system remain obscure. No pharmacological agents have demonstrated the capacity to specifically and efficaciously mitigate the neurological symptoms induced by HCoVs infections to date. Methods: We developed human cerebral organoids (HCOs) derived from human induced pluripotent stem cells and established a blood–brain barrier (BBB) HCOs co-culture model. We subjected these models to seasonal human coronavirus (HCoV) infections to investigate the viral characteristics within the central nervous system (CNS). Utilizing RNA sequencing,we conducted a preliminary exploration of the mechanisms underlying virus-induced inflammatory responses in the CNS. Furthermore,we assessed the efficacy of antiviral and anti-inflammatory drugs using the HCO model. Results: Our results showed that among seasonal coronaviruses,HCoV-OC43 replicates efficiently within the organoids,primarily targeting neurons and astrocytes,and disrupts the barrier function of the BBB. RNA sequencing analysis revealed that HCoV-OC43 infection triggers an inflammatory response through the TNF and NF-κB signaling pathways,leading to cell death,impaired neuronal function,and disrupted interneuron signaling. Interestingly,Bardoxolone methyl (CDDO-Me) demonstrated antiviral effects comparable to remdesivir,reducing both inflammation and cell death. Conclusions: Conclusively,HCOs infected with HCoV-OC43 offer valuable insights into the pathogenesis of HCoVs in central nervous system (CNS),and might serve as a tool for developing novel therapeutic strategies for HCoVs infections,including COVID-19,especially on exploring treatment candidates.Graphical abstract View Publication

BackgroundCancer stem cells (CSCs) are considered the ‘seeds’ of recurrence after chemotherapy,but eliminating CSCs remains notoriously challenging. This study aims to examine whether cell cycle checkpoint kinase 1 (CHK1) blockade can abrogate the stemness of ovarian cancer (OC) cells,making them easier targets of anti-tumor immunity. Methods: Prexasertib was used to block CHK1 in OC cell lines and xenografts,and its cytotoxicity was assessed in vitro and in vivo. In vitro tumor-sphere formation assays and stemness markers were used to evaluate cell stemness. PD-L1 expressions were examined via qRT-PCR,Western blot,flow cytometry,and immunohistochemistry. Prexasertib in combination with anti-PD-L1 antibody Atezolizumab was tested in immune-proficient mice bearing OC xenografts in terms of effects on tumor growth,tumor cell stemness,and tumor infiltrating lymphocytes via tumor volume monitoring,immunohistochemistry,and flow cytometry. Results: Prexasertib effectively inhibited CHK1 phosphorylation,exhibited significant anti-tumor effects in vitro and in vivo,accompanied by decreased OC cell stemness. CHK1 was highly expressed in tumor spheres versus tumor cells cultured in 2D system,and Prexasertib treatment suppressed sphere formation and reduced the ALDH+ cell fraction. Unexpectedly,Prexasertib upregulated PD-L1 expression in tumor cells. In vivo,combining Prexasertib with Atezolizumab led to more remarkable remission of tumors,when compared with Prexasertib or Atezolizumab alone. Meanwhile,the tumor-infiltrating CD8+ T cells significantly increased in the combination group,while exhausted T cells decreased; the treatments did not affect CD4+ cell infiltration. Conclusions: Dual targeting of CHK1 and PD-L1 may improve OC treatment by simultaneously suppressing stemness and enhancing anti-tumor immunity. View Publication

In the arms race between a pathogen and the host,the defense mechanisms of the host cell,including the ubiquitin system,are often counteracted by bacteria. Simkania negevensis (Sne),an obligate intracellular Chlamydia-like bacterium connected with respiratory diseases,possesses numerous deubiquitinases,but not much is known about its other ubiquitin-modifying enzymes. Sne infects a wide range of hosts,developing inside a tubular vacuole in close contact with the host endoplasmic reticulum (ER) and mitochondria. Our study describes an uncharacterized Sne ubiquitin E3 RING-ligase (SNE_A12920 or SneRING),which primarily generates K63- and K11-linked ubiquitin chains and preferentially interacts with UbcH5b and UBE2T E2 enzymes. SneRING is expressed upon infection of various human cell lines,as well as amoebae. We show that a portion of the expressed SneRING co-localizes with mitochondria and ER and that the SneRING interactome includes mitochondrial and ER proteins involved in organelle morphology and stress response. Our work offers an initial characterization of a bacterial RING ligase potentially involved in the host cell remodeling to accommodate the unique intracellular lifestyle of Sne. Author summaryUbiquitination is a protein modification system that regulates protein degradation,localization,or interactions. As such,ubiquitination has many important functions in cell signalling,and its dysregulation can lead to cancer and neurodegenerative diseases. Bacteria that live and develop inside human or other eukaryotic cells,such as Chlamydia,often modulate the ubiquitination system to ensure their own survival. Simkania negevensis is a Chlamydia-like bacterium connected to respiratory diseases in humans. We have discovered a novel enzyme expressed by these bacteria that can ubiquitinate other proteins and thus potentially modify host cell processes that would otherwise hinder infection. In this work,we explore the function of this enzyme and determine its possible cellular localization,as well as some of the proteins it interacts with. Our study provides new insights into how bacterial pathogens adapt to and manipulate host cells using one of the major cell function regulatory systems. View Publication

The maintenance of a basal immunoinflammatory signature in hematopoietic stem/progenitor cells (HSPCs) constitutes a fundamental regulatory axis governing hematopoietic competence and immune effector generation. While epigenetic repressors constrain this inflammatory phenotype,the molecular amplifiers that preserve this critical state remain undefined. Through integrated single-cell transcriptomic/epigenomic profiling and functional interrogation,we identify Setd2-mediated H3K36me3 as an indispensable epigenetic amplifier sustaining baseline inflammation in murine HSPCs. Setd2 ablation specifically eliminated interferon (IFN)-enriched HSPC subpopulations and attenuated inflammatory signaling cascades. Functionally,Setd2-deficient HSPCs exhibited impaired IFNγ responsiveness,compromised B-lymphopoiesis,and diminished reconstitution capacity due to Lin−c-Kit+Sca1high cell depletion. Paradoxically,Setd2 loss conferred resistance to IFNγ-induced HSPCs exhaustion,which may contribute to the maintenance of Setd2-deficient HSPCs in our myelodysplastic syndrome (MDS) model under the inflammatory milieu. Mechanistically,Setd2 sustained chromatin accessibility and enhancer (H3K27ac) activity at inflammatory gene loci. This work delineates a critical link between Setd2-mediated chromatin regulation,baseline inflammation,HSPC function,and immune competence,providing insights into inflammatory dysregulation in hematopoietic malignancies like MDS. View Publication