技术资料

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response,a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses,and to interact with both non-structural (NS) and structural flavivirus proteins. However,the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein–protein interactions were determined by a co-immunoprecipitation assay. From the results,both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably,the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively,the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics. View Publication

Cancer stem cells (CSCs) are a specific subpopulation of cancer cells with the ability of self‐renewal,infinite proliferation,multidifferentiation and tumorigenicity,and play critical roles in cancer progression and treatment resistance. CSCs are tightly regulated by the tumor microenvironment,such as hypoxia; however,how hypoxia regulates CSCs in non‐small cell lung cancer (NSCLC) remains unclear. The proportion of ALDH hi cells was examined using the Aldefluor assay. Tankyrase inhibitor XAV939 and siRNA were used to inhibit β‐catenin while pcDNA3‐β‐catenin (S33Y) plasmid enhanced the expression of β‐catenin. Western blot was administered for protein detection. The mRNA expression was measured by quantitative real‐time PCR. We found that hypoxia led to an increase in the proportion of ALDH hi cells in lung squamous carcinoma (LUSC) H520 cells,while causing a decrease in the ALDH hi cell proportion in lung adenocarcinoma (LUAD) A549 cells. Similarly,β‐catenin expression was upregulated in H520 cells but downregulated in A549 cells upon exposure to hypoxia. Mechanically,the proportion of ALDH hi cells in both cell lines was decreased by β‐catenin inhibitor or siRNA knockdown,whereas increased after β‐catenin overexpression. Furthermore,hypoxia treatment suppressed E‐cadherin expression in H520 cells and enhanced N‐cadherin and β‐catenin expression,while this effect was completely opposite in A549 cells. The hypoxia‐EMT‐β‐catenin axis functions as an important regulator for the proportion of CSCs in NSCLC and could potentially be explored as therapeutic targets in the future. View Publication

T cells protect tissues from cancer. Although investigations in mice showed that amino acids (AA) critically regulate T cell immunity,this remains poorly understood in humans. Here,we describe the AA composition of interstitial fluids in keratinocyte-derived skin cancers (KDSCs) and study the effect of AA on T cells using models of primary human cells and tissues. Gln contributed to ∼15% of interstitial AAs and promoted interferon gamma (IFN-γ),but not granzyme B (GzB) expression,in CD8 + T cells. Furthermore,the Toll-like receptor 7 agonist imiquimod (IMQ),a common treatment for KDSCs,down-regulated the metabolic gatekeepers c-MYC and mTORC1,as well as the AA transporter ASCT2 and intracellular Gln,Asn,Ala,and Asp in T cells. Reduced proliferation and IFN-γ expression,yet increased GzB,paralleled IMQ effects on AA. Finally,Gln was sufficient to promote IFN-γ-production in IMQ-treated T cells. Our findings indicate that Gln metabolism can be harnessed for treating KDSCs. Subject areas: Dermatology,Immunology View Publication

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor,CDC20,preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However,the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression,we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for,gastrointestinal atrophy is detrimental. Remarkably,deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome,while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect,identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast,only overexpression of anti-apoptotic BCL2,but none of the BH3-only protein deficiencies mentioned above,can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo. View Publication

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here,we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro,both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype. In an immunodeficient female mouse model,transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease. Subject terms: Targeted gene repair,Sickle cell disease View Publication

Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery. A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase. Luciferase-expressing hiPSCs were differentiated into islet organoids. The islet organoids were transplanted into the scapular brown adipose tissue (BAT) of nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice as the BAT group and under the left kidney capsule (KC) of NOD/SCID mice as a control group,respectively. Bioluminescence imaging (BLI) of the organoid grafts was performed on days 1,7,14,28,35,42,49,56,and 63 posttransplantation. BLI signals were detected in all recipients,including both the BAT and control groups. The BLI signal gradually decreased in both BAT and KC groups. However,the graft BLI signal intensity under the left KC decreased substantially faster than that of the BAT. Furthermore,our data show that islet organoids transplanted into streptozotocin-induced diabetic mice restored normoglycemia. Positron emission tomography/MRI verified that the islet organoids were transplanted at the intended location in these diabetic mice. Immunofluorescence staining revealed the presence of functional organoid grafts,as confirmed by insulin and glucagon staining. Our results demonstrate that BAT is a potentially desirable site for islet organoid transplantation for T1D therapy. View Publication

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However,the systems-level effects of AS-NMD in this context are poorly understood. We developed an R package,factR2,which offers a comprehensive suite of AS-NMD analysis functions. Using this tool,we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably,this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes. The online version contains supplementary material available at 10.1186/s13059-024-03305-8. View Publication

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy,contingent upon the species is crucial to circumvent transgene silencing,necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly,transgene silencing occurred while using the CAG promoter,contrary to conventional understanding,whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations,confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally,GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats,thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2,mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations,with implications for future research in gene-engineered rat models. The online version contains supplementary material available at 10.1186/s12917-024-04123-7. View Publication

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features,we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT),a specialized DNA polymerase intrinsic to VDJ recombination,broadly expressed within CD34 + progenitors prior to B/T cell emergence. While these TdT + cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype,their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84 lo GMPs demonstrates robust lymphoid potentials ex vivo,while still retaining significant myeloid differentiation capacity,akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors,further defining the lympho-myeloid axis in human hematopoiesis. Subject terms: Lymphopoiesis,Systems analysis,Proteomic analysis,Myelopoiesis View Publication

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality,inter-batch consistency,cryopreservation and scale remain,reducing experimental reproducibility and clinical translation. Here,we report a robust stirred suspension cardiac differentiation protocol,and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines,the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs,bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks,which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible,scalable,and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications,and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols. Subject terms: Cardiovascular biology,Induced pluripotent stem cells,Cardiovascular models View Publication

Numerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally,we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation. Selective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice,as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover,UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro. UiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders. The online version contains supplementary material available at 10.1186/s13578-024-01274-w. View Publication

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease,and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed,and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells,we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions,including irregularly enlarged cell morphology,cell cycle arrest with downregulation of Ki67,and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover,immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly,HPV surrogate marker p16,and stress-associated basal keratinocyte marker KRT17,indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation,and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable. Subject terms: Oral cancer,Mechanisms of disease,Cell death View Publication