技术资料

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

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

Fetch Error (529 Server Error: unknown for url: https://pmc.ncbi.nlm.nih.gov/articles/11628400) View Publication

The COVID-19 pandemic has created a global health crisis,with challenges arising from the ongoing evolution of the SARS-CoV-2 virus,the emergence of new strains,and the long-term effects of COVID-19. Aiming to overcome the development of viral resistance,our study here focused on developing broad-spectrum pan-coronavirus antiviral therapies by targeting host protein quality control mechanisms essential for viral replication. Screening an in-house compound library led to the discovery of three candidate compounds targeting cellular proteostasis. The three compounds are (1) the nucleotide analog cordycepin,(2) a benzothiozole analog,and (3) an acyldepsipeptide analog initially developed as part of a campaign to target the mitochondrial ClpP protease. These compounds demonstrated dose-dependent efficacy against multiple coronaviruses,including SARS-CoV-2,effectively inhibiting viral replication in vitro as well as in lung organoids. Notably,the compounds also showed efficacy against SARS-CoV-2 delta and omicron strains. As part of this work,we developed a BSL2-level cell-integrated SARS-CoV-2 replicon,which could serve as a valuable tool for high-throughput screening and studying intracellular viral replication. Our study should aid in the advancement of antiviral drug development efforts. Subject terms: High-throughput screening,Small molecules View Publication

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines,we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers,while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing. View Publication

NAD(P)-dependent steroid dehydrogenase-like protein (NSDHL),which is involved in breast tumor growth and metastasis,has been implicated in the maintenance of cancer stem cells. However,its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line,MCF-7. NSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2,P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids,tumor initiation and growth were strongly suppressed compared with those in the control. BCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls,along with decreased secretion of TGF-β1 and 3,phosphorylation of Smad2/3,and expression of SOX2. In RNA-sequencing data from The (TCGA) database,a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens ( n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids,suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs,thus preventing breast cancer initiation and progression. Our findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors. The online version contains supplementary material available at 10.1186/s12885-024-13143-3. View Publication

A comprehensive understanding of the cardio-spleen-bone marrow immune cell axis is essential for elucidating the alterations occurring during the pathogenesis of diabetes mellitus (DM). This study investigates the dynamics of immune cell kinetics in DM after myocardial infarction (MI) over time. MI was induced in diabetic and healthy control groups using C57BL/N6 mice,with sacrifices occurring at days 1,3,7,and 28 post-MI to collect heart,peripheral blood (PB),spleen,and bone marrow (BM) samples. Cell suspensions from each organ were isolated and analyzed via flow cytometry. Additionally,the endothelial progenitor cell-colony-forming assay (EPC-CFA) was performed using mononuclear cells derived from BM,PB,and the spleen. The results indicated that,despite normal production in BM and the spleen,CD45+ cells were lower in the PB of DM mice at days 1 to 3. Further analysis revealed a reduction in total and pro-inflammatory neutrophils (N1s) in PB at days 1 to 3 and in the spleen at days 3 to 7 in DM mice,suggesting that DM-induced alterations in splenic neutrophils fail to meet the demand in PB and ischemic tissues. Infiltrating macrophages (total,M1,M2) were reduced at day 3 in the DM-ischemic heart,with total and M1 (days 1–3) and M2 (days 3–7) macrophages being significantly decreased in DM-PB compared to controls,indicating impaired macrophage recruitment and polarization in DM. Myeloid dendritic cells (mDCs) in the heart were higher from days 1 to 7,which corresponded with the enhanced recruitment of CD8+ cells from days 1 to 28 in the DM-infarcted myocardium. Total CD4+ cells decreased in DM-PB at days 1 to 3,suggesting a delayed adaptive immune response to MI. B cells were reduced in PB at days 1 to 3,in myocardium at day 3,and in the spleen at day 7,indicating compromised mobilization from BM. EPC-CFA results showed a marked decrease in definitive EPC colonies in the spleen and BM from days 1 to 28 in DM mice compared to controls in vitro,highlighting that DM severely impairs EPC colony-forming activity by limiting the differentiation of EPCs from primitive to definitive forms. Taking together,this study underscores significant disruptions in the cardio-spleen-bone marrow immune cell axis following MI in DM,revealing delayed innate and adaptive immune responses along with impaired EPC differentiation. 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

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

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4 + T cell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling,resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML,serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR T cell treatment on LSCs in vitro . Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore,promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML. View Publication

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

Liver cancer stem cells (LCSCs) are thought to drive the metastasis and recurrence,however,the heterogeneity of molecular markers of LCSCs has hindered the development of effective methods to isolate them. This study introduced an effective approach to isolate and culture LCSCs from human primary liver cancer (HPLC),leveraging mouse embryonic fibroblasts (MEFs) as feeder cells in conjunction with using defined medium. Isolated LCSCs were further characterized by multiple approaches. Transcriptome sequencing data analysis was conducted to identify highly expressed genes in LCSCs and classify different subtypes of liver cancers. Total sixteen cell strains were directly isolated from 24 tissues of three types of HPLC without sorting,seven of which could be maintained long-term culture as colony growth on MEFs,which is unique characteristics of stem cells. Even 10 of cloned cells formed the tumors in immunodeficient mice,indicating that those cloned cells were tumorgenic. The histologies and gene expression pattern of human xenografts were very similar to those of HPLC where these cloned cells were isolated. Moreover,putative markers of LCSCs were further verified to all express in cloned cells,confirming that these cells were LCSCs. These cloned LCSCs could be cryopreserved,and still maintained the feature of colony growth on MEFs after the recovery. Compared to suspension culture as conventional approach to culture LCSCs,our approach much better maintained stemness of LCSCs for a long time. To date,these cloned cells could be cultured on MEFs over 12 passages. Moreover,bioinformatics analysis of sequencing data revealed the gene expression profiles in LCSCs,and liver cancers were classified into two subtypes C1 and C2 based on genes associated with the prognosis of LCSCs. Patients of the C2 subtype,which is closely related to the extracellular matrix,were found to be sensitive to treatments such as Cisplatin,Axitinib,JAK1 inhibitors,WNT-c59,Sorafenib,and RO-3306. In summary,this effective approach offers new insights into the molecular landscape of human liver cancers,and the identification of the C2 subtype and its unique response to the treatment pave the way for the creation of more effective,personalized therapeutic strategies. The online version contains supplementary material available at 10.1186/s12967-024-05870-9. View Publication