技术资料

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry,its exact role during the first steps of HIV-1 infection is not well characterized. Here,we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall,our data suggest that dynamin-2,as a tetramer,might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion. View Publication

Fellows et al. report that individual dynein motors can move the entire axon length during retrograde transport. They find factors LIS1 and NDEL1,needed for transport initiation,also move with cargos. In the anterograde direction,dynein and its cofactor dynactin are transported separately,keeping them apart until required. Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein,which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level,it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here,we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction,we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore,LIS1 and NDEL1 also undergo long-distance movement,despite being mainly implicated with the initiation of dynein transport. Intriguingly,in the anterograde direction,dynein/LIS1 moves faster than dynactin/NDEL1,consistent with transport on different cargos. Therefore,neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required. View Publication

During anemia erythropoiesis is bolstered by several factors including KIT ligand,oncostatin-M,glucocorticoids,and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia,however,reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia,both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments,DYRK3-/- progenitors also supported enhanced erythroblast formation,and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo,DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice,in contrast,produced fewer reticulocytes during hemolytic anemia,and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally,as studied in erythroid K562 cells,DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia. View Publication

Low-density neutrophils (LDNs) have been described in tumors and various autoimmune diseases,where they exhibit immune dysfunction and alter disease progression. Nevertheless,LDNs have been rarely reported in sepsis. We studied sepsis patients admitted to the intensive care unit. Wright-Giemsa stain assay and Transmission electron microscopy were performed to detect the morphology of neutrophils. Flow cytometry was used to analyze the number and function of LDNs. Concentration of cytokines was measured using ELISA. Neutrophil chemotaxis was examined using an under-agarose chemotaxis model. We found that LDNs were significantly elevated in patients with sepsis. Phenotypes and morphological characteristics suggest that LDNs may be formed by mixtures of neutrophils at various maturation stages. In vitro experiments showed that LDN formation was closely associated with neutrophil degranulation. We preliminarily discussed changes in immune function in LDNs. Compared with high-density neutrophils,expression levels of CXC chemokine receptor 4 on LDN surfaces were increased,phagocytotic capacity was decreased,and life span was prolonged. The chemotactic ability of LDNs was significantly reduced,possibly related to the increased expression of P2X1. These data suggest that LDNs are essential components of neutrophils in sepsis. To clarify the source and dysfunction mechanism of LDN in sepsis may be helpful for the diagnosis and treatment of sepsis in the future. View Publication

Objectives gamma$delta$ T cells,a non-conventional innate lymphocyte subset containing cells that can be activated by lipids and phosphoantigens,are abnormally regulated in systemic sclerosis (SSc). To further evaluate the significance of this dysregulation,we compared how exposure to an autoantigenic lipid,cardiolipin (CL),during co-stimulation with an amino-bisphosphonate (zoledronate,zol),affects the activation and cytokine production of SSc and healthy control (HC) gamma$delta$ T cells. Methods Expression of CD25 on Vgamma$9+,Vdelta$1+,and total CD3+ T cells in cultured peripheral blood mononuclear cells (PBMCs),their binding of CD1d tetramers,and the effect of monoclonal antibody (mAb) blockade of CD1d were monitored by flow cytometry after 4 days of in vitro culture. Intracellular production of IFNgamma$ and IL-4 was assessed after overnight culture. Results Percentages of CD25+ among CD3+ and Vdelta$1+ T cells were elevated significantly in short-term cultured SSc PBMC compared to HC. In SSc but not HC,CL and zol,respectively,suppressed {\%}CD25+ Vgamma$9+ and Vdelta$1+ T cells but,when combined,CL + zol significantly activated both subsets in HC and partially reversed inhibition by the individual reagents in SSc. Importantly,Vdelta$1+ T cells in both SSc and HC were highly reactive with lipid presenting CD1d tetramers,and a CD1d-blocking mAb decreased CL-induced enhancement of {\%}SSc CD25+ Vdelta$1+ T cells in the presence of zol. {\%}IFNgamma$+ cells among Vgamma$9+ T cells of SSc was lower than HC cultured in medium,CL,zol,or CL + zol,whereas {\%}IFNgamma$+ Vdelta$1+ T cells was lower only in the presence of CL or CL + zol. {\%}IL-4+ T cells were similar in SSc and HC in all conditions,with the exception of being increased in SSc Vgamma$9+ T cells in the presence of CL. Conclusion Abnormal functional responses of gamma$delta$ T cell subsets to stimulation by CL and phosphoantigens in SSc may contribute to fibrosis and immunosuppression,characteristics of this disease. View Publication

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However,functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC,a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover,strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα),were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors,and may contribute to the block in definitive hematopoiesis from hESC. View Publication

Solid tumor malignancy (STM) patients experience increased risk of breakthrough SARS-CoV-2 infection owing to reduced COVID-19 vaccine immunogenicity. However,the underlying immunological causes of impaired neutralization remain poorly characterized. Furthermore,non-neutralizing antibody functions can contribute to reduced disease severity but remain understudied within high-risk populations. We dissected polyfunctional antibody responses in STM patients and age-matched controls who received adenoviral vector- or mRNA-based COVID-19 vaccine regimens. Elevated inflammatory biomarkers,including agalactosylated IgG,interleukin (IL)-6,IL-18,and an expanded population of CD11c−CD21− double negative 3 (DN3) B cells were observed in STM patients and were associated with impaired neutralization. In contrast,mRNA vaccination induced Fc effector functions that were comparable in patients and controls and were cross-reactive against SARS-CoV-2 variants. These data highlight the resilience of Fc functional antibodies and identify systemic inflammatory biomarkers that may underpin impaired neutralizing antibody responses,suggesting potential avenues for immunomodulation via rational vaccine design. View Publication

Anti-melanoma differentiation-associated gene 5 (MDA5)-positive dermatomyositis (DM) is often complicated by rapidly progressive interstitial lung disease (RP-ILD),leading to early mortality. Previous studies on the pathogenesis of anti-MDA5-positive DM highlighted type I interferons (IFNs),while recent investigations reported the significance of a type III IFN,IFN-λ3. We investigated a range of cytokines,including type I/II/III IFNs,in serum samples from anti-MDA5-positive DM patients collected at diagnosis before treatment introduction. Elevations of IFN-β and λ3 were identified as the hallmark of anti-MDA5-positive DM,in comparison with other myositis subtypes,systemic lupus erythematosus,and COVID-19 pneumonia. The elevation of IFN-λ3 was associated with decreased CD56dimCD16pos NK cells in circulation. The unique cytokine profile with type I/III IFN upregulation in anti-MDA5-positive DM was replicated in independent validation cohorts. A cluster analysis using serum type I/III IFN levels identified three subgroups in anti-MDA5-positive DM: mild elevations of IFN-α/β and λ3; a marked increase in IFN-λ3 alone; and pronounced elevations of IFN-α/β with mild to moderate increase in IFN-λ3. Patients in the cluster with a marked elevation of IFN-λ3 alone tended to present with RP-ILD and decreased survival. The combination of serum type I/III IFN levels could serve as a prognostic biomarker in anti-MDA5-positive DM. View Publication

Dysregulation of mitochondrial function has been implicated in Parkinson’s disease (PD),but the role of mitochondrial metabolism in disease pathogenesis remains to be elucidated. Using an unbiased metabolomic analysis of purified mitochondria,we identified alterations in ?-ketoglutarate dehydrogenase (KGDH) pathway upon loss of PD-linked CHCHD2 protein. KGDH,a rate-limiting enzyme complex in the tricarboxylic acid cycle,was decreased in CHCHD2-deficient male mouse brains and human dopaminergic neurons. This deficiency of KGDH led to elevated ?-ketoglutarate and increased lipid peroxidation. Treatment of CHCHD2-deficient dopaminergic neurons with lipoic acid,a KGDH cofactor and antioxidant agent,resulted in decreased levels of lipid peroxidation and phosphorylated ?-synuclein. CHCHD10,a close homolog of CHCHD2 that is primarily linked to amyotrophic lateral sclerosis/frontotemporal dementia,did not affect the KGDH pathway or lipid peroxidation. Together,these results identify KGDH metabolic pathway as a targetable mitochondrial mechanism for correction of increased lipid peroxidation and ?-synuclein in Parkinson’s disease. An unbiased metabolomic analysis identifies ?-ketoglutarate dehydrogenase metabolic pathway as a targetable mitochondrial mechanism for correction of increased lipid peroxidation in CHCHD2-linked Parkinson’s disease models. View Publication

N-terminal acetyltransferases including NAA10 catalyze N-terminal acetylation,an evolutionarily conserved co- and post-translational modification. However,little is known about the role of N-terminal acetylation in cardiac homeostasis. To gain insight into cardiac-dependent NAA10 function,we studied a previously unidentified NAA10 variant p.(Arg4Ser) segregating with QT-prolongation,cardiomyopathy,and developmental delay in a large kindred. Here,we show that the NAA10R4S variant reduced enzymatic activity,decreased NAA10-NAA15 complex formation,and destabilized the enzymatic complex N-terminal acetyltransferase A. In NAA10R4S/Y-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs),dysregulation of the late sodium and slow delayed rectifier potassium currents caused severe repolarization abnormalities,consistent with clinical QT prolongation. Engineered heart tissues generated from NAA10R4S/Y-iPSC-CMs had significantly decreased contractile force and sarcomeric disorganization,consistent with the pedigree’s cardiomyopathic phenotype. Proteomic studies revealed dysregulation of metabolic pathways and cardiac structural proteins. We identified small molecule and genetic therapies that normalized the phenotype of NAA10R4S/Y-iPSC-CMs. Our study defines the roles of N-terminal acetylation in cardiac regulation and delineates mechanisms underlying QT prolongation,arrhythmia,and cardiomyopathy caused by NAA10 dysfunction. N-terminal acetylation dysregulation in the heart causes severe arrhythmia and cardiomyopathy. The authors show that stem cell models demonstrate ion channel trafficking defects and sarcomeric disarray as the underlying mechanisms,with gene therapy reversing both phenotypes View Publication

Increasing shreds of evidence suggest that neurogenic-to-gliogenic shift may be critical to the abnormal neurodevelopment observed in individuals with Down syndrome (DS). REST,the Repressor Element-1 Silencing Transcription factor,regulates the differentiation and development of neural cells. Downregulation of REST may lead to defects in post-differentiation neuronal morphology in the brain of the DS fetal. This study aims to elucidate the role of REST in DS-derived NPCs using bioinformatics analyses and laboratory validations. We identified and validated vital REST-targeted DEGs: CD44,TGFB1,FN1,ITGB1,and COL1A1. Interestingly,these genes are involved in neurogenesis and gliogenesis in DS-derived NPCs. Furthermore,we identified nuclear REST loss and the neuroblast marker,DCX,was downregulated in DS human trisomic induced pluripotent stem cells (hiPSCs)-derived NPCs,whereas the glioblast marker,NFIA,was upregulated. Our findings indicate that the loss of REST is critical in the neurogenic-to-gliogenic shift observed in DS-derived NPCs. REST and its target genes may collectively regulate the NPC phenotype.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-87314-y. View Publication

The importance of cancer metabolism has been appreciated for many years,but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis,we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway,paced by its rate-limiting enzyme,hydroxymethylglutaryl coenzyme A reductase (HMGCR),is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease,the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway,achieved by ectopic expression of either full-length HMGCR or its novel splice variant,promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and,importantly,cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together,our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. View Publication