技术资料

Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here,we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture. Whole-cell patch-clamp demonstrated subthreshold synaptic inputs and action potential firing of neurons. Spontaneous intracellular calcium signals turned into synchronous large-scale oscillations upon combined disinhibition of NMDA receptors and blocking of GABA A receptors. Brief metabolic inhibition to mimic transient energy restriction in the ischemic brain induced reversible intracellular calcium loading of cBOS. Moreover,metabolic inhibition induced a reversible decline in neuronal ATP as revealed by ATeam1.03 YEMK . Overall,cBOS provide a powerful platform to assess morphological and functional aspects of human neural cells in intact minimal networks and to address the pathways that drive cellular damage during brain ischemia. Subject areas: Neuroscience,Cellular neuroscience,Stem cells research View Publication

Despite extensive research on microbiome alterations in ulcerative colitis (UC),the role of the constituent stable microbiota remains unclear. This study,employing 16S rRNA-gene sequencing,uncovers a persistent microbial imbalance in both active and quiescent UC patients compared to healthy controls. Using co-occurrence and differential abundance analysis,the study highlights microbial constituents,featuring Phocaeicola,Collinsella,Roseburia,Holdemanella,and Bacteroides,that are not affected during the course of UC. Co-cultivation experiments,utilizing commensal Escherichia coli and Phocaeicola vulgatus,were conducted with intestinal epithelial organoids derived from active UC patients and controls. These experiments reveal a tendency for a differential response in tight junction formation and maintenance in colonic epithelial cells,without inducing pathogen recognition and stress responses,offering further insights into the roles of these microorganisms in UC pathogenesis. These experiments also uncover high variation in patients’ response to the same bacteria,which indicate the need for more comprehensive,stratified analyses with an expanded sample size. This study reveals that a substantial part of the gut microbiota remains stable throughout progression of UC. Functional experiments suggest that members of core microbiota – Escherichia coli and Phocaeicola vulgatus – potentially differentially regulate the expression of tight junction gene in the colonic epithelium of UC patients and healthy individuals. The online version contains supplementary material available at 10.1186/s13099-024-00612-0. View Publication

Homology Directed Repair (HDR) enables precise genome editing,but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes,such as Non-Homologous End Joining (NHEJ). In this study,we develop a functional,pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target,53BP1,identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing,synergize with a DNAPK inhibitor molecule,and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes. Subject terms: Targeted gene repair,Homologous recombination,High-throughput screening View Publication

Mutations in mexZ,encoding a negative regulator of the expression of the mexXY efflux pump genes,are frequently acquired by Pseudomonas aeruginosa at early stages of lung infection. Although traditionally related to resistance to the first-line drug tobramycin,mexZ mutations are associated with low-level aminoglycoside resistance when determined in the laboratory,suggesting that their selection during infection may not be necessarily,or only,related to tobramycin therapy. Here,we show that mexZ -mutated bacteria tend to accumulate inside the epithelial barrier of a human airway infection model,thus colonising the epithelium while being protected against diverse antibiotics. This phenotype is mediated by overexpression of lecA,a quorum sensing-controlled gene,encoding a lectin involved in P. aeruginosa tissue invasiveness. We find that lecA overexpression is caused by a disrupted equilibrium between the overproduced MexXY and another efflux pump,MexAB,which extrudes quorum sensing signals. Our results indicate that mexZ mutations affect the expression of quorum sensing-regulated pathways,thus promoting tissue invasiveness and protecting bacteria from the action of antibiotics within patients,something unnoticeable using standard laboratory tests. Subject terms: Antimicrobial resistance,Pathogens,Infection View Publication

Hepatocellular carcinoma (HCC) is widely recognized as a globally prevalent malignancy. Immunotherapy is a promising therapy for HCC patients. Increasing evidence suggests that lncRNAs are involved in HCC progression and immunotherapy. The study reveals the mechanistic role of long non‐coding RNA (lncRNA) FOXD1‐AS1 in regulating migration,invasion,circulating tumor cells (CTCs),epithelial‐mesenchymal transition (EMT),and immune escape in HCC in vitro. This study employed real‐time PCR (RT‐qPCR) to measure FOXD1‐AS1,miR‐615‐3p,and programmed death‐ligand 1 ( PD‐L1 ). The interactions of FOXD1‐AS1,miR‐615‐3p,and PD‐L1 were validated via dual‐luciferase reporter gene and ribonucleoprotein immunoprecipitation (RIP) assay. In vivo experimentation involves BALB/c mice and BALB/c nude mice to investigate the impact of HCC metastasis. The upregulation of lncRNA FOXD1‐AS1 in malignant tissues significantly correlates with poor prognosis. The investigation was implemented on the impact of lncRNA FOXD1‐AS1 on the migratory,invasive,and EMT of HCC cells. It has been observed that the lncRNA FOXD1‐AS1 significantly influences the generation and metastasis of M CTC in vivo analysis. In mechanistic analysis,lncRNA FOXD1‐AS1 enhanced immune escape in HCC via upregulation of PD‐L1,which acted as a ceRNA by sequestering miR‐615‐3p. Additionally,lncRNA FOXD1‐AS1 was found to modulate the EMT of CTCs through the activation of the PI3K/AKT pathway. This study presents compelling evidence supporting the role of lncRNA FOXD1‐AS1 as a miRNA sponge that sequesters miR‐655‐3p and protects PD‐L1 from suppression. View Publication

Neocortex expansion during evolution is linked to higher numbers of neurons,which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here,we show that EREG,encoding the growth factor EPIREGULIN,is expressed in the human developing neocortex and in gorilla cerebral organoids,but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells,whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation,and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally,we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone. View Publication

Picornaviruses are a leading cause of central nervous system (CNS) infections. While genotypes such as parechovirus A3 (PeV-A3) and echovirus 11 (E11) can elicit severe neurological disease,the highly prevalent PeV-A1 is not associated with CNS disease. Here,we expand our current understanding of these differences in PeV-A CNS disease using human brain organoids and clinical isolates of the two PeV-A genotypes. Our data indicate that PeV-A1 and A3 specific differences in neurological disease are not due to infectivity of CNS cells as both viruses productively infect brain organoids with a similar cell tropism. Proteomic analysis shows that PeV-A infection significantly alters the host cell metabolism. The inflammatory response following PeV-A3 (and E11 infection) is significantly more potent than that upon PeV-A1 infection. Collectively,our findings align with clinical observations and suggest a role for neuroinflammation,rather than viral replication,in PeV-A3 (and E11) infection. Subject terms: Infection,Central nervous system infections,Viral host response,Innate immunity View Publication

Dou et al. demonstrate that Parkinson’s disease-associated hyperactive LRRK2 decreases the trafficking of synaptic vesicle proteins within neurons by disrupting the regulation of the synaptic vesicle precursor protein RAB3A. Impaired delivery of synaptic proteins to presynaptic sites could contribute to the progression of debilitating non-motor PD symptoms. View Publication

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis,erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis 1 to define the anatomy of normal and stress haematopoiesis. In the steady state,across the skeleton,single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels,where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells,which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults,as it was maintained after haemorrhage,systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment,and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF,and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis,define the anatomy of normal and stress responses,identify discrete microanatomical production sites that confer plasticity to haematopoiesis,and uncover unprecedented heterogeneity of stress responses across the skeleton. Subject terms: Bone marrow cells,Haematopoietic stem cells,Ageing,Imaging the immune system,Haematopoietic stem cells View Publication

Breast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however,the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues,especially in malignant tissues; however,its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance. Both bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung,breast,gastric,colon,and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next,colony formation assay,MTT assay,EdU assay,transwell assay,wound healing assay,flow cytometric analysis,sphere formation assay,western blotting analysis,mouse xenograft model analysis,RNA-sequencing assay,immunofluorescence assay,and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation,invasion,stemness,chemotherapy sensitivity,and integrin/FAK/TAZ/mTOR activation. Further,liquid chromatography–tandem mass spectrometry analysis,GST pull-down assay,and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B,myosin heavy chain 9 (MYH9),and CUL9. TMEM120B expression was elevated in lung,breast,gastric,colon,and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage,lymph node metastasis,and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation,invasion,and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9,which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore,TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely,overexpression of TMEM120B-∆CCD delayed the formation of FAs,suppressed TAZ-mTOR signaling,and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1–2) than in those with better outcomes (Miller/Payne grades 3–5). Our study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the β1-integrin/FAK-TAZ-mTOR signaling axis,maintaining stemness and accelerating chemotherapy resistance. The online version contains supplementary material available at 10.1186/s13058-024-01802-z. View Publication

Acute myeloid leukaemia (AML) is a haematological malignancy characterised by the accumulation of transformed myeloid progenitors in the bone marrow. Piplartine (PL),also known as piperlongumine,is a pro-oxidant small molecule extracted from peppers that has demonstrated antineoplastic potential in solid tumours and other haematological malignancies. In this work,we explored the potential of PL to treat AML through the use of a combination of cellular and molecular analyses of primary and cultured leukaemia cells in vitro and in vivo. We showed that PL exhibits in vitro cytotoxicity against AML cells,including CD34 + leukaemia-propagating cells,but not healthy haematopoietic progenitors,suggesting anti-leukaemia selectivity. Mechanistically,PL treatment increased reactive oxygen species (ROS) levels and induced ROS-mediated apoptosis in AML cells,which could be prevented by treatment with the antioxidant scavenger N -acetyl-cysteine and the pancaspase inhibitor Z-VAD(OMe)-FMK. PL treatment reduced NFKB1 gene transcription and the level of NF-κB p65 (pS536),which was depleted from the nucleus of AML cells,indicating suppression of NF-κB p65 signalling. Significantly,PL suppressed AML development in a mouse xenograft model,and its combination with current AML treatments (cytarabine,daunorubicin and azacytidine) had synergistic effects,indicating translational therapeutic potential. Taken together,these data position PL as a novel anti-AML candidate drug that can target leukaemia stem/progenitors and is amenable to combinatorial therapeutic strategies. Subject terms: Acute myeloid leukaemia,Cancer stem cells,Pharmacology View Publication

SorLA,encoded by the gene SORL1,is an intracellular sorting receptor of the VPS10P domain receptor gene family. Although SorLA is best recognized for its ability to shuttle target proteins between intracellular compartments in neurons,recent data suggest that also its microglial expression can be of high relevance for the pathogenesis of brain diseases,including glioblastoma (GBM). Here,we interrogated the impact of SorLA on the functional properties of glioma-associated microglia and macrophages (GAMs). In the GBM microenvironment,GAMs are re-programmed and lose the ability to elicit anti-tumor responses. Instead,they acquire a glioma-supporting phenotype,which is a key mechanism promoting glioma progression. Our re-analysis of published scRNA-seq data from GBM patients revealed that functional phenotypes of GAMs are linked to the level of SORL1 expression,which was further confirmed using in vitro models. Moreover,we demonstrate that SorLA restrains secretion of TNFα from microglia to restrict the inflammatory potential of these cells. Finally,we show that loss of SorLA exacerbates the pro-inflammatory response of microglia in the murine model of glioma and suppresses tumor growth. View Publication