参考文献

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

USP30,a ubiquitin‐specific protease,primarily characterized as a mitochondrial deubiquitinase regulating mitophagy,has not been previously reported to have nuclear functions. In this study,we demonstrate that USP30 is present in both mitochondrial and nuclear compartments. Nutrient deprivation triggers USP30 nuclear translocation via an N‐terminal nuclear localization signal (NLS),mediated through suppression of mTORC1‐dependent phosphorylation at serine 104,a modification constraining nuclear entry. Nuclear USP30 acts as a tumor suppressor by inhibiting cancer stemness and chemoresistance in triple‐negative breast cancer (TNBC) cells. Mechanistically,USP30 directly interacts with and deubiquitinates the transcription factor TCF/LEF1 at K379 and K382 residues,disrupting recruitment of CBP/P300 co‐activators to the β‐catenin/LEF1 complex. This abolishes β‐catenin/LEF1 transactivation and suppresses WNT signaling. Clinically,USP30 is downregulated in TNBC and cancer stem cells (CSCs),with notably reduced nuclear levels in cancer tissues. Overexpression of nuclear USP30 markedly reduces lung metastatic burden in TNBC mouse models. These findings uncover a novel role for nuclear USP30 in regulating cancer stemness and suggest that targeting the dynamic relocalization of USP30 from mitochondria to the nucleus could offer new therapeutic strategies for breast cancer metastasis. 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

Alongside their well-established role in hemostasis,platelets are key modulators of immune cell function. This is particularly the case for macrophages,as platelets can either promote or dampen macrophage activation in a context-specific manner. Whilst the role of platelets in modulating classical (M1) macrophage activation following bacterial challenge is relatively well understood,whether platelets control other macrophage responses is less clear. We investigated the role of platelets in type 2 inflammation using a mouse model of chronic schistosomiasis. Schistosome infection caused thrombocytopenia which was not fully reversed after drug-induced parasite death. Reduced platelet levels in infection were coincident with lower levels of systemic TPO and extensive liver damage caused by parasite eggs. Infection also reduced the ploidy and size (but not number) of bone marrow megakaryocytes,which was associated with reduced platelet output. We show schistosome infection accelerated platelet clearance and promoted the formation of platelet-leukocyte aggregates. This was particularly the case for liver macrophages and monocytes. Phenotypic analysis shows that platelet-associated liver macrophages had a distinct activation phenotype that included elevated expression of the alternative (M2) activation marker RELMα. Despite this,in vitro studies indicated that platelets do not directly promote macrophage alternative activation. Similarly,whilst in vivo pharmacological treatment with a TPO mimetic enhanced platelet numbers and platelet-leukocyte aggregates,this did not alter macrophage phenotype. Conversely,antibody-mediated depletion of platelets or use of platelet-deficient mice both led to extensive bleeding following infection which impacted host survival. Together,these data indicate that whilst platelets are essential to prevent excessive disease pathology in schistosomiasis,they have a more nuanced role in myeloid cell activation and type 2 immune responses. Author summaryPlatelets are the second most abundant blood cell and are best known for their role in stopping bleeding after blood vessel damage. More recent studies have revealed another important function of platelets is their ability to control immune cell activation. Here,we investigate the role of platelets in immune responses to schistosomes,parasitic worms that cause the disease schistosomiasis that affects hundreds of millions worldwide. Schistosome worms live in our blood vessels and release large numbers of eggs that must exit the blood and move through our tissues to exit the body for onward transmission. However,a large number of eggs become trapped in different organs causing inflammation and disease pathology. We find that schistosome infection reduces the numbers of platelets in the blood of laboratory mice. Platelets are taken up by liver macrophages,and whilst these macrophages have a distinct activation profile compared to other cells,platelets themselves do not cause these changes. However,platelets are essential to survive schistosomiasis due to excessive bleeding in their absence. Together,this work shows that platelets are key to surviving schistosome infection but this reflects more their role in preventing bleeding rather than controlling immune cell function. 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

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