技术资料

Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus’ (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus’ oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors,enabling the translation of this approach to clinical trials,are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM,MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect,replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells,but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly,ZIKV rapidly replicated in GBM,MED,and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover,ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether,our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However,such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects,suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV. View Publication

Genome editing is poised to revolutionize treatment of genetic diseases,but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons,which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this,we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage,compared to just days in isogenic iPSCs. Furthermore,Cas9-treated neurons upregulated unexpected DNA repair genes,including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells,we uncovered unforeseen challenges and opportunities for precise therapeutic editing. View Publication

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study,wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials,resulting in a unique band signal around 150 kDa for ADNP,above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting,comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal,indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector,we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore,we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations,while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies,suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However,western blotting of patient-derived hiPSCs,immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition,an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates,whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome. View Publication

Viruses depend on host metabolic pathways and flaviviruses are specifically linked to lipid metabolism. During dengue virus infection lipid droplets are degraded to fuel replication and Zika virus (ZIKV) infection depends on triglyceride biosynthesis. Here,we systematically investigated the neutral lipid–synthesizing enzymes diacylglycerol O-acyltransferases (DGAT) and the sterol O-acyltransferase (SOAT) 1 in orthoflavivirus infection. Downregulation of DGAT1 and SOAT1 compromises ZIKV infection in hepatoma cells but only SOAT1 and not DGAT inhibitor treatment reduces ZIKV infection. DGAT1 interacts with the ZIKV capsid protein,indicating that protein interaction might be required for ZIKV replication. Importantly,inhibition of SOAT1 severely impairs ZIKV infection in neural cell culture models and cerebral organoids. SOAT1 inhibitor treatment decreases extracellular viral RNA and E protein level and lowers the specific infectivity of virions,indicating that ZIKV morphogenesis is compromised,likely due to accumulation of free cholesterol. Our findings provide insights into the importance of cholesterol and cholesterol ester balance for efficient ZIKV replication and implicate SOAT1 as an antiviral target. Exploring the role of neutral lipid-synthesizing enzymes in Zika virus infection using different cell culture models,inhibition of cholesterol esterification is found to impair ZIKV morphogenesis. View Publication

ABSTRACTExtracellular vesicles (EVs) and secretory factors play crucial roles in intercellular communication,but the molecular mechanisms and dynamics governing their interplay in human pluripotent stem cells (hPSCs) are poorly understood. Here,we demonstrate that hPSC?secreted milk fat globule?EGF factor 8 (MFGE?8) is the principal corona protein at the periphery of EVs,playing an essential role in controlling hPSC stemness. MFGE?8 depletion reduced EV?mediated self?renewal and survival in hPSC cultures. MFGE?8 in the EV corona bound to integrin ?v?5 expressed in the peripheral zone of hPSC colonies. It activated cyclin D1 and dynamin?1 via the AKT/GSK3? axis,promoting the growth of hPSCs and facilitating the endocytosis of EVs. Internalization of EVs alleviated oxidative stress and cell death by transporting redox and stress response proteins that increased GSH levels. Our findings demonstrate the critical role of the extracellular association of MFGE?8 and EVs in modulating the self?renewal and survival of hPSCs. View Publication

Monkeypox virus (MPV) is known to inflict injuries and,in some cases,lead to fatalities in humans. However,the underlying mechanisms responsible for its pathogenicity remain poorly understood. We investigated functions of MPV core proteins,H3L,A35R,A29L,and I1L,and discovered that H3L induced transcriptional perturbations and injuries. We substantiated that H3L upregulated IL1A expression. IL1A,in consequence,caused cellular injuries,and this detrimental effect was mitigated when countered with IL1A blockage. We also observed that H3L significantly perturbed the transcriptions of genes in cardiac system. Mechanistically,H3L occupied the promoters of genes governing cellular injury,leading to alterations in the binding patterns of H3K27me3 and H3K4me3 histone marks,ultimately resulting in expression perturbations. In vivo and in vitro models confirmed that H3L induced transcriptional disturbances and cardiac dysfunction,which were ameliorated when IL1A was blocked or repressed. Our study provides valuable insights into comprehensive understanding of MPV pathogenicity,highlights the significant roles of H3L in inducing injuries,and potentially paves the way for the development of therapeutic strategies targeting IL1A. View Publication

SummaryProtein turnover is a critical component of gene expression regulation and cellular homeostasis,yet methods for measuring turnover rates that are scalable and applicable to different models are still needed. We introduce an improved D2O (heavy water) labeling strategy to investigate the landscape of protein turnover in cell culture,with accurate calibration of per-residue deuterium incorporation in multiple cell types. Applying this method,we mapped the proteome-wide turnover landscape of pluripotent and differentiating human induced pluripotent stem cells (hiPSCs). Our analysis highlights the role of APC/C (anaphase-promoting complex/cyclosome) and SPOP (speckle-type POZ protein) degrons in the fast turnover of cell-cycle-related and DNA-binding hiPSC proteins. Upon pluripotency exit,many short-lived hiPSC proteins are depleted,while RNA-binding and -splicing proteins become hyperdynamic. The ability to identify fast-turnover proteins also facilitates secretome profiling,as exemplified in hiPSC-cardiomyocyte and primary human cardiac fibroblast analysis. This method is broadly applicable to protein turnover studies in primary,pluripotent,and transformed cells. Graphical abstract Highlights•D2O labeling measures protein turnover in primary,pluripotent,and transformed cells•D2O incorporates into multiple amino acids in vitro,including Ala,Glu,Asp,and Pro•Protein turnover analysis shows hiPSC differentiation alters fast-turnover proteins•We show application to secretome analysis in human cardiac myocytes and fibroblasts MotivationDynamic stable isotope labeling by amino acids in cell culture coupled with mass spectrometry is commonly used to measure protein turnover in cell culture but requires altering culture medium composition and may not label some peptides. We describe a simple and convenient alternative for measuring protein turnover kinetics in cultured cells by adding low-volume D2O (heavy water) to standard tissue culture media. Addressing a critical gap,we determined the number of deuterium-accessible atoms on all 20 proteinogenic amino acids across multiple cell types. This allows accurate interpretation of D2O-labeled mass spectra to measure protein turnover kinetics and secretome flux on a proteome scale. Alamillo et al. present a D2O labeling mass spectrometry method to measure protein turnover rates that is compatible with multiple cell cultures and medium formulations. The method reveals a parsimonious protein turnover landscape in human induced pluripotent stem cells and identifies hyperdynamic proteins that are unique to self-renewal states. View Publication

To build in vitro tissues for therapeutic applications,it is essential to replicate the spatial distribution of cells that occurs during morphogenesis in vivo. However,it remains technically challenging to simultaneously regulate the geometric alignment and aggregation of cells during tissue fabrication. Here,we introduce the acoustofluidic bioassembly induced morphogenesis,which is the combination of precise arrangement of cells by the mechanical forces produced by acoustofluidic cues,and the morphological and functional changes of cells in the following in vitro and in vivo cultures. The acoustofluidic bioassembly can be used to create tissues with regulated nano-,micro-,and macro-structures. We demonstrate that the neuromuscular tissue fabricated with the acoustofluidic bioassembly exhibits enhanced contraction dynamics,electrophysiology,and therapeutic efficacy. The potential of the acoustofluidic bioassembly as an in situ application is demonstrated by fabricating artificial tissues at the defect sites of living tissues. The acoustofluidic bioassembly induced morphogenesis can provide a pioneering platform to fabricate tissues for biomedical applications. Tissue engineering is essential for drug screening and regenerative medicine. Here,authors developed an acoustofluidic method that can induce morphogenesis of therapeutic tissues at varied dimensions/scales. View Publication

Inflammatory cytokines,including interleukin 6 (IL6),are associated with ion channel remodeling and enhance the propensity to alterations in cardiac rhythm generation and propagation,in which the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role. Hence,we investigated the consequences of exposure to IL6 on HCN channels in cell models and human atrial biopsies. In murine atrial HL1 cells and in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs),IL6 elicited STAT3 phosphorylation,a receptor-mediated downstream signaling. Downregulation of HCN1,2,4 by IL6 was observed after 24–48 h; in hiPS-CMs,this effect was reverted by 24 h of application of tocilizumab,a human IL6 receptor antagonist. In parallel,hiPS-CM action potentials (APs) showed a reduced spontaneous frequency. Moreover,we assessed IL6 and HCN expression in dilated left atrial samples from patients with mitral valve disease,an AF-prone condition. IL6 levels were increased in dilated atria compared to controls and positively correlated with echocardiographic atrial dimensions. Interestingly,the highest IL6 transcript levels and the lowest HCN4 and HCN2 expression were in these samples. In conclusion,our data uncovered a novel link between IL6 and cardiac HCN channels,potentially contributing to atrial electrical disturbances and a higher risk of dysrhythmias in conditions with elevated IL6 levels. View Publication

Age-related macular degeneration (AMD),a multifactorial type of retinal degeneration represents the most common cause for blindness in elderly. Polymorphisms in complement factor-H increase,while absence of factor-H-related protein-1 (FHR1) decreases the AMD risk,currently explained by their opposing relationship. Here we identify a FHR1-driven pathway fostering chronic cellular inflammation. FHR1 accumulates below the retinal pigment epithelium (RPE) in AMD donor tissue and similarly the murine homolog,muFHR1 is abundant in three AMD-relevant mouse models. These mouse models express the muFHR1 receptor EGF-like module-containing mucin-like hormone receptor 1 (Emr1) on the RPE and on invading mononuclear phagocytes (MP),where both cells form clusters via muFHR1/Emr1. FHR1 ignited EMR2-dependent Ca2+-signals and gene expression in both human RPE cell line and in vivo where muFHR1 affects Emr1+ cells (RPE and MP) gene expression shown by RNAseq analysis. As muFHR1 deletion in mice revealed significantly reduced MP invasion and neoangiogenesis in laser-induced choroidal neovascularization,we hypothesize that FHR1 accumulates,stabilizes and activates MP in the stage of RPE degeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03499-z. View Publication

During the first month of pregnancy,the brain and spinal cord are formed through a process called neurulation. However,this process can be altered by low serum levels of folic acid,environmental factors,or genetic predispositions. In 2018,a surveillance study in Botswana,a country with a high incidence of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs,found that newborns whose mothers were taking dolutegravir (DTG) during the first trimester of pregnancy had an increased risk of neural tube defects (NTDs). As a result,the World Health Organization and the U.S. Food and Drug Administration have issued guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs,we sought to assess the potential neurotoxicity of DTG in stem cell-derived brain organoids. The gene expression of brain organoids developed in the presence of DTG was analyzed by RNA sequencing,Optical Coherence Tomography (OCT),Optical Coherence Elastography (OCE),and Brillouin microscopy. The sequencing data shows that DTG induces the expression of the folate receptor (FOLR1) and modifies the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness. In contrast,reverberant OCE measurements indicate decreased organoid volumes and internal stiffness. View Publication

Background/Objectives: High-altitude environments have a significant detrimental impact on the cognitive functions of the brain. Qi Jing Wan (QJW),a traditional herbal formula composed of Angelica sinensis,Astragalus membranaceus,and Rhizoma Polygonati Odorati,has demonstrated potential efficacy in treating cognitive disorders. However,its effects on cognitive dysfunction in plateau hypoxic environments remain unclear. Methods: In this study,acute and chronic plateau cognitive impairment mouse models were constructed to investigate the preventive and therapeutic effects of QJW and its significant active ingredient,diosgenin (Dio). Behavioral experiments were conducted to assess learning and memory in mice. Morphological changes in hippocampal neurons and synapses were assessed,and microglial activation and inflammatory factor levels were measured to evaluate brain damage. Potential active ingredients capable of crossing the blood–brain barrier were identified through chemical composition analysis and network database screening,followed by validation in animal and brain organoid experiments. Transcriptomics analysis,immunofluorescence staining,and molecular docking techniques were employed to explore the underlying mechanisms. Results: QJW significantly enhanced learning and memory abilities in plateau model mice,reduced structural damage to hippocampal neurons,restored NeuN expression,inhibited inflammatory factor levels and microglial activation,and improved hippocampal synaptic damage. Transcriptomics analysis revealed that Dio alleviated hypoxic brain damage and protected cognitive function by regulating the expression of PDE4C. Conclusions: These findings indicate that QJW and its significant active ingredient Dio effectively mitigate hypoxic brain injury and prevent cognitive impairment in high-altitude environments. View Publication