技术资料

Cell-autonomous barriers to reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remain poorly understood. Using a focused CRISPR-Cas9 screen,we identified Ubiquitin-specific peptidase 22 (USP22) as a key chromatin-based barrier to human iPSC derivation. Suppression of USP22 significantly enhances reprogramming efficiency. Surprisingly,this effect is likely to be independent of USP22’s deubiquitinase activity or its association with the SAGA complex,as shown through module-specific knockouts,and genetic rescue experiments. USP22 is not required for iPSC derivation or maintenance. Mechanistically,USP22 loss during reprogramming downregulates fibroblast-specific genes while activating pluripotency-associated genes,including DNMT3L,LIN28A,SOX2,and GDF3. Additionally,USP22 loss enhances reprogramming efficiency under naïve stem cell conditions. These findings reveal an unrecognized role for USP22 in maintaining somatic cell identity and repressing pluripotency genes,highlighting its potential as a target to improve reprogramming efficiency. Ubiquitin-specific peptidase 22 (USP22) is identified as a key chromatin-based barrier to human iPSC derivation through a chromatin-focused CRISPR-Cas9 screen. View Publication

Signaling mediates cellular responses to extracellular stimuli. The c-Jun NH(2)-terminal kinase (JNK) pathway exemplifies one subgroup of the mitogen-activated protein (MAP) kinases,which,besides having established functions in stress response,also contribute to development by an unknown mechanism. We show by genome-wide location analysis that JNK binds to a large set of active promoters during the differentiation of stem cells into neurons. JNK-bound promoters are enriched with binding motifs for the transcription factor NF-Y but not for AP-1. NF-Y occupies these predicted sites,and overexpression of dominant-negative NF-YA reduces the JNK presence on chromatin. We find that histone H3 Ser10 (H3S10) is a substrate for JNK,and JNK-bound promoters are enriched for H3S10 phosphorylation. Inhibition of JNK signaling in post-mitotic neurons reduces phosphorylation at H3S10 and the expression of target genes. These results establish MAP kinase binding and function on chromatin at a novel class of target genes during stem cell differentiation. View Publication

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with one of the worst outcomes among all cancers. PDA often recurs after initial treatment to result in patient death despite the use of chemotherapy or radiation therapy. PDA contains a subset of tumor-initiating cells capable of extensive self-renewal known as cancer stem cells (CSC),which may contribute to therapeutic resistance and metastasis. At present,conventional chemotherapy and radiotherapy are largely ineffective in depleting CSC pool,suggesting the need for novel therapies that specifically target the cancer-sustaining stem cells for tumor eradication and to improve the poor prognosis of PDA patients. In this study,we report that death receptor 5 (DR5) is enriched in pancreatic CSCs compared with the bulk of the tumor cells. Treating a collection of freshly generated patient-derived PDA xenografts with gemcitabine,the first-line chemotherapeutic agent for PDA,is initially effective in reducing tumor size,but largely ineffective in diminishing the CSC populations,and eventually culminated in tumor relapse. However,a combination of tigatuzumab,a fully humanized DR5 agonist monoclonal antibody,with gemcitabine proved to be more efficacious by providing a double hit to kill both CSCs and bulk tumor cells. The combination therapy produced remarkable reduction in pancreatic CSCs,tumor remissions,and significant improvements in time to tumor progression in a model that is considered more difficult to treat. These data provide the rationale to explore the DR5-directed therapies in combination with chemotherapy as a therapeutic option to improve the current standard of care for pancreatic cancer patients. View Publication

Type II diabetes mellitus (T2D) is a chronic metabolic disorder that results from defects in both insulin secretion and insulin action. The deficit and dysfunction of insulin secreting $\$-cell are signature symptom for T2D. Additionally,in pancreatic $\$-cell,a small group of genes which are abundantly expressed in most other tissues are highly selectively repressed. Lactate dehydrogenase A (LDHA) is one of such genes. Upregulation of LDHA is found in both human T2D and rodent T2D models. In this study,we identified a LDHA-suppressing microRNA (hsa-miR-590-3p) and used it together with human embryonic stem cell (hESC) derived pancreatic endoderm (PE) transplantation into a high-fat diet induced T2D mouse model. The procedure significantly improved glucose metabolism and other symptoms of T2D. Our findings support the potential T2D treatment using the combination of microRNA and hESC-differentiated PE cells. View Publication

OBJECTIVE To analyze the combination therapy of Sinomenine (SIN) and Methotrexate (MTX) in rheumatoid arthritis (RA),we herein demonstrated the combination effect of SIN and MTX on collagen-induced arthritis (CIA) in rats through their modulation on osteoclast-related cytokines. METHODS CIA was induced by the immunization of type II collagen (CII) in SD rats. SIN and MTX were administrated alone or in combination after the onset of arthritis. Arthritis index and histological analysis were used to evaluate the effect of treatments. Effects of SIN and MTX on expression of receptor activator of NF-κB ligand (RANKL) and osteopontin (OPN) in synovial tissues were assayed by immunohistochemistry. RANKL,osteoprotegerin (OPG),IL-6,IL-17 and matrix metalloproteinases (MMPs) in rat serum were measured by ELISA. The expression of osteoclast-related cytokines in fibroblast-like synoviocytes (FLS) from RA patients was assayed by RT-PCR. RESULTS SIN and MTX combination additively reduced the inflammatory symptoms and joint damage in CIA. Combination of SIN and MTX significantly repressed synovial RANKL and OPN production. SIN and MTX exhibited complementary and synergistic effect upon down-regulating RANKL,IL-6,IL-17 and MMPs in rat serum. SIN and MTX also modulated the expression of RANKL and OPG in RA-FLS. CONCLUSION SIN and MTX have additive effects,decreasing inflammation and joint damage in CIA rats by modulating osteoclast-related cytokines. These results are indicative of the combined effect of SIN and MTX for anti-arthritic treatment in RA. View Publication

View Publication

SummaryGene-switch techniques hold promising applications in contemporary genetics research,particularly in disease treatment and genetic engineering. Here,we developed a compact drug-induced splicing system that maintains low background using a human ubiquitin C (hUBC) promoter and optimized drug (LMI070) binding sequences based on the Xon switch system. To ensure precise subcellular localization of the protein of interest (POI),we inserted a 2A self-cleaving peptide between the extra N-terminal peptide and POI. This streamlined and optimized switch system,named miniXon2G,effectively regulated POIs in different subcellular localizations both in vitro and in vivo. Furthermore,miniXon2G could be integrated into endogenous gene loci,resulting in precise,reversible regulation of target genes by both endogenous regulators and drugs. Overall,these findings highlight the performance of miniXon2G in controlling protein expression with great potential for general applicability to diverse biological scenarios requiring precise and delicate regulation. Graphical abstract Highlights•miniXon2G is a compact and versatile version of the Xon gene-switch system•A P2A peptide eliminates residual peptides from functional proteins•We demonstrate applications on multiple proteins of interest•miniXon2G is a precise and reversible switch system with minimal background expression MotivationThe Xon drug-inducible splice-switch system is a simple and highly adaptable tool for regulated protein expression. We sought to further engineer this system to expand its applications in contemporary genetics research. In particular,we focused on reducing the size of the switch elements,maintaining minimal background expression,introducing a feature to remove extraneous peptide fragments,and demonstrating genomic integration and validation on a range of targets. Chi et al. develop a compact and versatile miniXon2G drug-inducible splice-switch system based on the Xon system. It features a reduced size,minimal background,and the removal of extraneous peptide fragments,enabling application to various biological scenarios that require precise expression control. View Publication

Regulated cell death is a key component of the innate immune response,which provides the first line of defense against infection and homeostatic perturbations. However,cell death can also drive pathogenesis. The most well-defined cell death pathways can be categorized as nonlytic (apoptosis) and lytic (pyroptosis,necroptosis,and PANoptosis). While specific triggers are known to induce each of these cell death pathways,it is unclear whether all cell types express the cell death proteins required to activate these pathways. Here,we assessed the protein expression and compared the responses of immune and non-immune cells of human and mouse origin to canonical pyroptotic (LPS plus ATP),apoptotic (staurosporine),necroptotic (TNF-α plus z-VAD),and PANoptotic (influenza A virus infection) stimuli. When compared to fibroblasts,both mouse and human innate immune cells,macrophages,expressed higher levels of cell death proteins and activated cell death effectors more robustly,including caspase-1,gasdermins,caspase-8,and RIPKs,in response to specific stimuli. Our findings highlight the importance of considering the cell type when examining the mechanisms regulating inflammation and cell death. Improved understanding of the cell types that contain the machinery to execute different forms of cell death and their link to innate immune responses is critical to identify new strategies to target these pathways in specific cellular populations for the treatment of infectious diseases,inflammatory disorders,and cancer. View Publication

AIM OF THE STUDY Human breast milk reduces the risk and severity of necrotizing enterocolitis (NEC). Exosomes are extracellular vesicles (EVs) found in high concentrations in milk,and they mediate intercellular communication and immune responses. The aim of this study is to compare the protective effects of exosomes that are derived from different time periods of breast milk production against intestinal injury using an ex vivo intestinal organoid model. METHODS Colostrum,transitional and mature breast milk samples from healthy lactating mothers were collected. Exosomes were isolated using serial ultracentrifugation and filtration. Exosomes' presence was confirmed using transmission electron microscopy (TEM) and western blot. To form the intestinal organoids,terminal ileum was harvested from neonatal mice pups at postnatal day 9,crypts were isolated and organoids were cultured in matrigel. Organoids were either cultured with exposure to lipopolysaccharide (LPS),or in treatment groups where both LPS and exosomes were added in the culturing medium. Inflammatory markers and organoids viability were evaluated. MAIN RESULTS Human milk-derived exosomes were successfully isolated and characterized. LPS administration reduced the size of intestinal organoids,induced inflammation through increasing TNF$\alpha$ and TLR4 expression,and stimulated intestinal regeneration. Colostrum,transitional and mature human milk-derived exosome treatment all prevented inflammatory injury,while exosomes derived from colostrum were most effective at reducing inflammatory cytokine. CONCLUSIONS Human breast milk-derived exosomes were able to protect intestine organoids against epithelial injury induced by LPS. Colostrum exosomes offer the best protective effect among the breast-milk derived exosomes. Human milk exosomes can be protective against the development of intestinal injury such as that seen in NEC. View Publication

Rapamycin analogs,temsirolimus and everolimus,are approved for the treatment of advance renal cell carcinoma (RCC). Currently approved agents inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). However,the mTOR kinase exists in two distinct multiprotein complexes,mTORC1 and mTORC2,and both complexes may be critical regulators of cell metabolism,growth and proliferation. Furthermore,it has been proposed that drug resistance develops due to compensatory activation of mTORC2 signaling during treatment with temsirolimus or everolimus. We evaluated Ku0063794,which is a small molecule that inhibits both mTOR complexes. Ku0063794 was compared to temsirolimus in preclinical models for renal cell carcinoma. Ku0063794 was effective in inhibiting the phosphorylation of signaling proteins downstream of both mTORC1 and mTORC2,including p70 S6K,4E-BP1 and Akt. Ku0063794 was more effective than temsirolimus in decreasing the viability and growth of RCC cell lines,Caki-1 and 786-O,in vitro by inducing cell cycle arrest and autophagy,but not apoptosis. However,in a xenograft model there was no difference in the inhibition of tumor growth by Ku0063794 or temsirolimus. A potential explanation is that temsirolimus has additional effects on the tumor microenvironment. Consistent with this possibility,temsirolimus,but not Ku0063794,decreased tumor angiogenesis in vivo,and decreased the viability of HUVEC (Human Umbilical Vein Endothelial Cells) cells in vitro at pharmacologically relevant concentrations. Furthermore,expression levels of VEGF and PDGF were lower in Caki-1 and 786-O cells treated with temsirolimus than cells treated with Ku0063794. View Publication

Background: Preclinical cell tracking is enhanced with a multimodal imaging approach. Bioluminescence imaging (BLI) is a highly sensitive optical modality that relies on engineering cells to constitutively express a luciferase gene. Magnetic particle imaging (MPI) is a newer imaging modality that directly detects superparamagnetic iron oxide (SPIO) particles used to label cells. Here,we compare BLI and MPI for imaging cells in vitro and in vivo. Methods: Mouse 4T1 breast carcinoma cells were transduced to express firefly luciferase,labeled with SPIO (ProMag),and imaged as cell samples after subcutaneous injection into mice. Results: For cell samples,the BLI and MPI signals were strongly correlated with cell number. Both modalities presented limitations for imaging cells in vivo. For BLI,weak signal penetration,signal attenuation,and scattering prevented the detection of cells for mice with hair and for cells far from the tissue surface. For MPI,background signals obscured the detection of low cell numbers due to the limited dynamic range,and cell numbers could not be accurately quantified from in vivo images. Conclusions: It is important to understand the shortcomings of these imaging modalities to develop strategies to improve cellular detection sensitivity. View Publication

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein,we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore,the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFN$\gamma$ production,also Ca2+- and CaM-dependent,was instead not reduced in HNSCC T cells,which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity,but not IFN$\gamma$ production,and reduced their chemotaxis in the presence of adenosine,thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally,1-EBIO enhanced INF$\gamma$ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore,they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers. View Publication