技术资料

Silencing remains a significant challenge for exogenous gene expression,limiting both the penetrance and expressivity of transgenes. In particular,silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here,we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably,the incorporation of an intron into a CRISPRa construct results in reduced silencing,increased expression levels,and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition,we find that introns can work synergistically with chromatin opening elements to further mitigate silencing,suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes. Silencing of transgenes such as Cas9 limits gene editing and CRISPRa applications. Here,the authors show that adding intronic sequences reduces silencing and boosts transgene expression,enabling improved CRISPRa-mediated gene activation and more stable expression of the transgene over time. View Publication

In dynamic light microscopy applications,imaging specimens from multiple angles while maintaining controlled temperature conditions is crucial for comprehensive and accurate analysis. To address these challenges,we present iCAT,an open-source multifunctional accessory designed to enhance light microscopy. It enables specimen rotation along the axial plane,incorporates built-in modules for precise temperature control,features an integrated LED,and includes a camera for real-time specimen monitoring. It can be easily 3D-printed and assembled using readily available electrical components. Combined with any up-right microscope,this versatile device allows researchers to capture detailed images and videos of organoid cultures and live or fixed specimens,such as C. elegans,zebrafish,drosophila,or mouse embryos. The potential applications of iCAT in investigating dynamic cellular processes and complex developmental phenomena are vast,inspiring researchers to explore its possibilities and push the boundaries of biological research. iCAT,an open-source,3D-printable microscopy accessory,enables axial specimen rotation,temperature control,and live monitoring for high-resolution imaging of organoids and diverse model organisms. View Publication

Eosinophils are major effector cells in type 2 immune responses,contributing to host defense and allergic diseases. They also contribute to maintaining tissue homeostasis by regulating various immune cell types,including neutrophils. Here we show that eosinophils directly associate with neutrophils in the lungs of asthma-induced mice. Eosinophil-specific deficiency of the short-chain fatty acid receptor,GPR43,results in hyperactivation of eosinophils and increases the expression of neutrophil chemoattractants and PECAM-1,thereby enhancing the interaction between eosinophils and neutrophils. This interaction exposes neutrophils to eosinophil-derived IL-4 and GM-CSF,which induce the conversion of conventional neutrophils into more pathogenic,Siglec-Fhi neutrophils capable of enhancing Th17 cell differentiation and aggravating asthma symptoms in mouse models. Our results thus implicate GPR43 as a critical regulator of eosinophils,and describe eosinophil-mediated modulation of neutrophil differentiation and function. Eosinophils contribute to type 2 immunity,but their interaction with neutrophils in this context is incompletely understood. Here the authors use mouse asthma models and in vitro culture to show that eosinophil-specific deficiency of GPR43 promotes Siglec-Fhi neutrophil differentiation and downstream induction of Th17 to aggravate lung inflammation and asthma. View Publication

Background: Biliary atresia (BA) is the most prevalent serious neonatal biliary obstructive disorder and is a complex multifactorial liver disorder. Genome-wide association studies have identified Adducin 3 (ADD3) as a BA susceptibility gene but the mechanisms involved in disease causation and progression remain unclear. Methods: ADD3 knockout human pluripotent stem cells were differentiated into cholangiocyte organoids to assess the effect of ADD3 deletion on biliary development in vitro. Add3 deletion in rhesus rotavirus (RRV)-induced experimental BA mice were employed as the in vivo model to address the impact of reduced Add3 expression on BA pathogenesis. Findings: ADD3 knockout organoids displayed defective cholangiocyte differentiation,failure in the recruitment of βII-spectrin to the cell membrane,abnormal primary cilia development,reduced expression of tight junction proteins,lower transepithelial electrical resistance (TEER) and increased paracellular permeability. Statistical significantly reduced tight junction (TJ) proteins expression and lower TEER in Add3+/− and Add3−/− liver tissue-derived cholangiocytes were observed. Reduced number of TJs and enlarged paracellular spaces without any detectable TJ were detected in the intra-hepatic bile ducts of Add3+/− and Add3−/− livers. A statistical significantly higher incidence and a more advanced form of BA with statistical significantly higher serum bilirubin,liver necrosis and fibrosis,and accumulation of macrophages and activated hepatic stellate cells were observed in Add3 knockout BA mice as compared to wild-type BA mice. Interpretation: Dysregulated ADD3 expression caused an abnormal development and impaired barrier function of cholangiocytes,and the resultant increase in bile duct permeability rendered the foetus/neonate susceptible to a more severe injury response to an external insult. The findings support the hypothetical pathogenic model of genetic susceptibility genes being involved in hepatobiliary development/structure,and the perturbed embryogenesis of the biliary tree and its disrupt integrity increase the host susceptibility to biliary injury and BA. View Publication

Mutations in the GBA gene,which reduce β-glucocerebrosidase (GCase) activity,represent the most significant genetic risk factor for Parkinson’s disease (PD). Decreased GCase activity has also been observed in sporadic PD cases,supporting a broader role for GCase in the poorly understood mechanisms underlying PD etiopathogenesis. While most studies on the relationship between GBA mutations and PD have focused on neurons,evidence suggests that PD pathology promoted by GCase deficiency involves other cell types and,in particular,interactions between neuronal and glial cells.Here,we identify microglia as primary players undergoing significant alterations at early stages of the pathological processes triggered by a GCase impairment. Using both pharmacological and genetic mouse models of GCase deficiency,we observed microglial morphological,transcriptional and metabolic changes. Interestingly,these changes were associated with a cell-specific,significant reduction of microglial ATP levels. When microglial ATP depletion was reproduced in an in vitro system of co-cultured microglial and neuronal cells,the neuroprotective properties of microglia were compromised,resulting in reduced cytoprotective and detoxifying pathways in neurons.These findings underscore the role of microglia in PD pathogenesis and point to a pathogenetic mechanism by which microglial metabolic disturbances leading to ATP depletion enhance neuronal vulnerability to injury and neurodegeneration. This mechanism could be targeted for therapeutic intervention aimed at mitigating PD risk and counteracting the development of PD pathology. View Publication

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

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

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

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