技术资料

Pericytes play a crucial role in controlling inflammation and vascular functions in the central nervous system,which are disrupted in Parkinson’s disease (PD). Still,there is a lack of studies on the impact of pericytes on neurodegenerative diseases,and their involvement in the pathology of PD is unclear. Our objective was to investigate the molecular and functional differences between healthy pericytes and pericytes with the LRRK2 G2019S mutation,which is one of the most common mutations associated with PD. Our study employed pericyte-like cells obtained from induced pluripotent stem cells produced from PD patients with the LRRK2 G2019S mutation as well as from healthy individuals. We examined the gene expression profiles of the cells and analyzed how the alterations reflect on their functionality. We have shown differences in the expression of genes related to inflammation and angiogenesis. Furthermore,we observe modified migration speed in PD pericyte-like cells as well as enhanced secretion of inflammatory mediators,such as soluble VCAM-1 and MCP-1,in these pericyte-like cells following exposure to proinflammatory stimuli. In summary,our findings support the notion that pericytes play a role in the inflammatory and vascular changes observed in PD. Further investigation of pericytes could provide valuable insight into understanding the pathogenesis of PD. The online version contains supplementary material available at 10.1186/s12987-024-00592-y. View Publication

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here,we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks,enhancing the reconstitution capacity of edited HSPCs. However,this results in lower HDR efficiency,rendering ex vivo culture necessary yet detrimental. Mechanistically,ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation,which generates mitogenic reactive oxygen species (ROS),promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus,p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs,ultimately endowing edited cells with superior multi-lineage differentiation,persistence throughout serial transplantation,enhanced polyclonal repertoire,and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications. View Publication

Global dissemination of SARS-CoV-2 Omicron sublineages has provided a sufficient opportunity for natural selection,thus enabling beneficial mutations to emerge. Characterisation of these mutations uncovers the underlying machinery responsible for the fast transmission of Omicron variants and guides vaccine development for combating the COVID-19 pandemic. Through systematic bioinformatics analysis of 496,606 sequences of Omicron variants,we obtained 40 amino acid substitutions that occurred with high frequency in the S protein. Utilising pseudoviruses and a trans -complementation system of SARS-CoV-2,we identified the effect of high-frequency mutations on viral infectivity and elucidated the molecular mechanisms. Finally,we evaluated the impact of a key emerging mutation on the immune protection induced by the SARS-CoV-2 VLP mRNA vaccine in a murine model. We identified a proline-to-leucine substitution at the 1263rd residue of the Spike protein,and upon investigating the relative frequencies across multiple Omicron sublineages,we found a trend of increasing frequency for P1263L. The substitution significantly enhances the capacity for S-mediated viral entry and improves the immunogenicity of a virus-like particle mRNA vaccine. Mechanistic studies showed that this mutation is located in the FERM binding motif of the cytoplasmic tail and impairs the interaction between the S protein and the Ezrin/Radixin/Moesin proteins. Additionally,this mutation facilitates the incorporation of S proteins into SARS-CoV-2 virions. This study offers mechanistic insight into the constantly increasing transmissibility of SARS-CoV-2 Omicron variants and provides a meaningful optimisation strategy for vaccine development against SARS-CoV-2. This study was supported by grants from the National Key Research and Development Plan of China (2021YFC2302405,2022YFC2303200,2021YFC2300200 and 2022YFC2303400),the National Natural Science Foundation of China (32188101,32200772,82422049,82241082,32270182,82372254,82271872,82341046,32100755 and 82102389),Shenzhen Medical Research Fund (B2404002,A2303036),the Shenzhen Bay Laboratory Startup Fund (21330111),Shenzhen San-Ming Project for Prevention and Research on Vector-borne Diseases (SZSM202211023),Yunnan Provincial Science and Technology Project at Southwest United Graduate School (202302AO370010). The New Cornerstone Science Foundation through the New Cornerstone Investigator Program,and the Xplorer Prize from Tencent Foundation. View Publication

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Induction of fetal hemoglobin (HbF) has been shown to be a viable therapeutic approach to treating sickle cell disease and potentially other β-hemoglobinopathies. To identify targets and target-modulating small molecules that enhance HbF expression,we engineered a human umbilical-derived erythroid progenitor reporter cell line (HUDEP2_HBG1_HiBiT) by genetically tagging a HiBiT peptide to the carboxyl (C)-terminus of the endogenous HBG1 gene locus,which codes for γ-globin protein,a component of HbF. Employing this reporter cell line,we performed a chemogenomic screen of approximately 5000 compounds annotated with known targets or mechanisms that have achieved clinical stage or approval by the US Food and Drug Administration (FDA). Among them,10 compounds were confirmed for their ability to induce HbF in the HUDEP2 cell line. These include several known HbF inducers,such as pomalidomide,lenalidomide,decitabine,idoxuridine,and azacytidine,which validate the translational nature of this screening platform. We identified avadomide,autophinib,triciribine,and R574 as novel HbF inducers from these screens. We orthogonally confirmed HbF induction activities of the top hits in both parental HUDEP2 cells as well as in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Further,we demonstrated that pomalidomide and avadomide,but not idoxuridine,induced HbF expression through downregulation of several transcriptional repressors such as BCL11A,ZBTB7A,and IKZF1. These studies demonstrate a robust phenotypic screening workflow that can be applied to large-scale small molecule profiling campaigns for the discovery of targets and pathways,as well as novel therapeutics for sickle cell disease and other β-hemoglobinopathies. View Publication

The lack of a permissive cell culture system has limited high-resolution structures of parvovirus B19 (B19V) to virus-like particles (VLPs). In this study,we present the atomic resolution structure (2.2 Å) of authentic B19V purified from a patient blood sample. There are significant differences compared to non-infectious VLPs. Most strikingly,two host protease inhibitors (PIs),inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and serpinA3,were identified in complex with the capsids in all patient samples tested. The ITIH4 binds specifically to the icosahedral fivefold axis and serpinA3 occupies the twofold axis. The protein-coated virions remain infectious,and the capsid-associated PIs retain activity; however,upon virion interaction with target cells,the PIs dissociate from the capsid prior to viral entry. Our finding of an infectious virion shielded by bound host serum proteins suggests an evolutionarily favored phenomenon to evade immune surveillance and escape host protease activity. Subject terms: Cryoelectron microscopy,Virology View Publication

The application of organoids has been limited by the lack of methods for producing uniformly mature organoids at scale. This study introduces an organoid culture platform,called UniMat,which addresses the challenges of uniformity and maturity simultaneously. UniMat is designed to not only ensure consistent organoid growth but also facilitate an unrestricted supply of soluble factors by a 3D geometrically-engineered,permeable membrane-based platform. Using UniMat,we demonstrate the scalable generation of kidney organoids with enhanced uniformity in both structure and function compared to conventional methods. Notably,kidney organoids within UniMat show improved maturation,showing increased expression of nephron transcripts,more in vivo-like cell-type balance,enhanced vascularization,and better long-term stability. Moreover,UniMat’s design offers a more standardized organoid model for disease modeling and drug testing,as demonstrated by polycystic-kidney disease and acute kidney injury modeling. In essence,UniMat presents a valuable platform for organoid technology,with potential applications in organ development,disease modeling,and drug screening. Subject terms: Nanofabrication and nanopatterning,Biomaterials,Stem-cell biotechnology View Publication

Cancer cell plasticity contributes significantly to the failure of chemo- and targeted therapies in triple-negative breast cancer (TNBC). Molecular mechanisms of therapy-induced tumor cell plasticity and associated resistance are largely unknown. Using a genome-wide CRISPR-Cas9 screen,we investigated escape mechanisms of NOTCH-driven TNBC treated with a gamma-secretase inhibitor (GSI) and identified SOX2 as a target of resistance to Notch inhibition. We describe a novel reciprocal inhibitory feedback mechanism between Notch signaling and SOX2. Specifically,Notch signaling inhibits SOX2 expression through its target genes of the HEY family,and SOX2 inhibits Notch signaling through direct interaction with RBPJ. This mechanism shapes divergent cell states with NOTCH positive TNBC being more epithelial-like,while SOX2 expression correlates with epithelial-mesenchymal transition,induces cancer stem cell features and GSI resistance. To counteract monotherapy-induced tumor relapse,we assessed GSI-paclitaxel and dasatinib-paclitaxel combination treatments in NOTCH inhibitor-sensitive and -resistant TNBC xenotransplants,respectively. These distinct preventive combinations and second-line treatment option dependent on NOTCH1 and SOX2 expression in TNBC are able to induce tumor growth control and reduce metastatic burden. View Publication

To enable transmission of information in the brain,synaptic vesicles fuse to presynaptic membranes,liberating their content and exposing transiently a myriad of vesicular transmembrane proteins. However,versatile methods for quantifying the synaptic translocation of endogenous proteins during neuronal activity remain unavailable,as the fast dynamics of synaptic vesicle cycling difficult specific isolation of trafficking proteins during such a transient surface exposure. Here,we developed a novel approach using synaptic cleft proximity labeling to capture and quantify activity-driven trafficking of endogenous synaptic proteins at the synapse. We show that accelerating cleft biotinylation times to match the fast dynamics of vesicle exocytosis allows capturing endogenous proteins transiently exposed at the synaptic surface during neural activity,enabling for the first time the study of the translocation of nearly every endogenous synaptic protein. As proof-of-concept,we further applied this technology to obtain direct evidence of the surface translocation of noncanonical trafficking proteins,such as ATG9A and NPTX1,which had been proposed to traffic during activity but for which direct proof had not yet been shown. The technological advancement presented here will facilitate future studies dissecting the molecular identity of proteins exocytosed at the synapse during activity,helping to define the molecular machinery that sustains neurotransmission in the mammalian brain. View Publication

Patients with chronic myeloid leukemia (CML) respond to tyrosine kinase inhibitors (TKIs); however,CML leukemic stem cells (LSCs) exhibit BCR::ABL kinase-independent growth and are insensitive to TKIs,leading to disease relapse. To prevent this,new therapies targeting CML-LSCs are needed. Rates of mitochondria-mediated oxidative phosphorylation (OXPHOS) in CD34 + CML cells within the primitive CML cell population are higher than those in normal undifferentiated hematopoietic cells; therefore,the inhibition of OXPHOS in CML-LSCs may be a potential cure for CML. NK-128 (C 33 H 61 NO 5 S) is a structurally simplified analog of JCI-20679,the design of which was based on annonaceous acetogenins. NK-128 exhibits antitumor activity against glioblastoma and human colon cancer cells by inhibiting OXPHOS and activating AMP-activated protein kinase (AMPK). Here,we demonstrate that NK-128 effectively suppresses the growth of CML cell lines and that the combination of imatinib and NK-128 is more potent than either alone in a CML xenograft mouse model. We also found that NK-128 inhibits colony formation by CD34 + CML cells isolated from the bone marrow of untreated CML patients. Taken together,these findings suggest that targeting OXPHOS is a beneficial approach to eliminating CML-LSCs,and may improve the treatment of CML. View Publication

Acute myeloid leukemia (AML) is a deadly hematopoietic malignancy. Although many patients achieve complete remission with standard induction therapy,a combination of cytarabine and anthracycline,~40% of patients have induction failure. These refractory patients pose a treatment challenge,as they do not respond to salvage therapy or allogeneic stem cell transplant. Herein,we show that AML patients who experience induction failure have elevated expression of the NF-κB target gene tumor necrosis factor alpha-induced protein-3 (TNFAIP3/A20) and impaired necroptotic cell death. A20 High AML are resistant to anthracyclines,while A20 Low AML are sensitive. Loss of A20 in AML restores sensitivity to anthracycline treatment by inducing necroptosis. Moreover,A20 prevents necroptosis in AML by targeting the necroptosis effector RIPK1,and anthracycline-induced necroptosis is abrogated in A20 High AML. These findings suggest that NF-κB-driven A20 overexpression plays a role in failed chemotherapy induction and highlights the potential of targeting an alternative cell death pathway in AML. Subject terms: Acute myeloid leukaemia,Cancer therapeutic resistance View Publication

The ability to generate natural killer (NK) cells from induced pluripotent stem cells (iPSCs) has given rise to new possibilities for the large-scale production of homogeneous immunotherapeutic cellular products and opened new avenues towards the creation of “off-the-shelf” cancer immunotherapies. However,the differentiation of NK cells from iPSCs remains poorly understood,particularly regarding the ontogenic landscape of iPSC-derived NK (iNK) cells produced in vitro and the influence that the differentiation strategy employed may have on the iNK profile. To investigate this question,we conducted a comparative analysis of two sets of iNK cells generated from the same iPSC line using two different protocols: (i) a short-term,clinically compatible feeder-free protocol corresponding to primitive hematopoiesis,and (ii) a lymphoid-based protocol representing the definitive hematopoietic step. Our work demonstrated that both protocols are capable of producing functional iNK cells. However,the two sets of resulting iNKs exhibited distinct phenotypes and transcriptomic profiles. The lymphoid-based differentiation approach generated iNKs with a more mature and activated profile,which demonstrated higher cytotoxicity against cancer cell lines compared to iNK cells produced under short-term feeder-free conditions suggesting that the differentiation strategy must be considered when designing iNK cell–based adoptive immunotherapies. View Publication