技术资料

Although activated inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs) are potent T cell suppressors,nonactivated IMCs and IDCs promote T cell activation and Th1/Th17 cell differentiation. In this study,we investigated how to reduce the proinflammatory properties of IMCs and IDCs and further convert them into immune regulatory dendritic cells (DCs). We found that IL-4 and retinoic acid (RA) cotreatment of GM-CSF-differentiated IDCs synergistically induced the expression of aldehyde dehydrogenase family 1,subfamily A2,a rate-limiting enzyme for RA synthesis in DCs. IL-4 plus RA-treated IDCs upregulated CD103 expression and markedly reduced the production of proinflammatory cytokines upon activation. IL-4 plus RA-treated IDCs strongly induced CD4�?�Foxp3�?� regulatory T cell differentiation and suppressed Th1 and Th17 differentiation. Mechanistically,the transcription factors Stat6 and RA receptor $$ play important roles in aldehyde dehydrogenase family 1,subfamily A2,induction. In addition,IL-4 and RA signaling pathways interact closely to enhance the regulatory function of treated DCs. Adoptive transfer of IL-4 plus RA-treated DCs significantly increased regulatory T cell frequency in vivo. Direct treatment with IL-4 and RA also markedly suppressed actively induced experimental autoimmune encephalomyelitis. Our data demonstrate the synergistic effect of IL-4 and RA in inducing a regulatory phenotype in IDCs,providing a potential treatment strategy for autoimmune diseases. View Publication

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells,but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC₅₀ = ˜15 ng/mL) by inducing apoptosis,and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations,BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes,such as glucose-regulated protein 78 (GRP78),X-box binding protein 1 (XBP1),and C/EBP homologous protein (CHOP). Finally,we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer. View Publication

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs),the molecular mechanism involved remains to be fully elucidated. Here,we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover,BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together,our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy,however,is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs,implying that activation of JNKs is essential for BMP9 osteoinductive activity. View Publication

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor,cyclopamine,and the activators,Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes. View Publication

In cancer microenvironment,aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC,3'-Sialylgalactose,6'-Sialylgalactose and 2'-Fucosylgalactose. The crosslinking,performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne,resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability,biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology,whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation,indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells. View Publication

While readily achieved in cell lines,the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here,we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein. View Publication

The intestinal epithelial regeneration is driven by intestinal stem cells under homeostatic conditions. Differentiated intestinal epithelial cells,such as Paneth cells,are capable of acquiring multipotency and contributing to regeneration upon the loss of intestinal stem cells. Paneth cells also support intestinal stem cell survival and regeneration. We report here that depletion of an RNA-binding protein named polypyrimidine tract binding protein 1 (PTBP1) in mouse intestinal epithelial cells causes intestinal stem cell death and epithelial regeneration failure. Mechanistically,we show that PTBP1 inhibits neuronal-like splicing programs in intestinal crypt cells,which is critical for maintaining intestinal stem cell stemness. This function is achieved at least in part through promoting the non-productive splicing of its paralog PTBP2. Moreover,PTBP1 inhibits the expression of an AKT inhibitor PHLDA3 in Paneth cells and permits AKT activation,which presumably maintains Paneth cell plasticity and function in supporting intestinal stem cell niche. We show that PTBP1 directly binds to a CU-rich region in the 3' UTR of Phlda3,which we demonstrate to be critical for downregulating the mRNA and protein levels of Phlda3. Our results thus reveal the multifaceted in vivo regulation of intestinal epithelial regeneration by PTBP1 at the post-transcriptional level. View Publication

Age-related gastrointestinal decline contributes to whole-organism frailty and mortality. Genistein is known to have beneficial effects on age-related diseases,but its precise role in homeostasis of the aging gut remains to be elucidated. Here,wild-type aging mice and Zmpste24-/- progeroid mice were used to investigate the role of genistein in lifespan and homeostasis of the aging gut in mammals. A series of longitudinal,clinically relevant measurements were performed to evaluate the effect of genistein on healthspan. It was found that dietary genistein promoted a healthier and longer life and was associated with a decrease in the levels of systemic inflammatory cytokines in aging mice. Furthermore,dietary genistein ameliorated gut dysfunctions,such as intestinal inflammation,leaky gut,and impaired epithelial regeneration. A distinct genistein-mediated alteration in gut microbiota was observed by increasing Lachnospira abundance and short-chain fatty acid (SCFA) production. Further fecal microbiota transplantation and dirty cage sharing experiments indicated that the gut microbiota from genistein-fed mice rejuvenated the aging gut and extended the lifespan of progeroid mice. It was demonstrated that genistein-associated SCFAs alleviated tumor necrosis factor alpha-induced intestinal organoid damage. Moreover,genistein-associated propionate promoted regulatory T cell-derived interleukin 10 production,which alleviated macrophage-derived inflammation. This study provided the first data,to the authors' knowledge,indicating that dietary genistein modulates homeostasis in the aging gut and extends the healthspan and lifespan of aging mammals. Moreover,the existence of a link between genistein and the gut microbiota provides a rationale for dietary interventions against age-associated frailty. View Publication

Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene's effects on many biological and pathological processes. Here,we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes,organoid cell sorting,and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis. View Publication

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However,it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here,we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons,we observed rapid translocation of a subpopulation of MAP2,present in dendritic shafts,to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently,immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore,LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether,this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks. View Publication

Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses,simulate compound diffusion through complex structures,and assess cellular heterogeneity of tissues,making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell culture that are grown from stem cells,are self-organizing,and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development,model diseases,test drug sensitivity and toxicity,and advance regenerative medicine. However,traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging,phenotypic assessment,and isolation from heterogenous organoid populations. To address these bottlenecks,we have adapted our tissue culture consumable and instrumentation to enable automated imaging,identification,and isolation of individual organoids. Organoids grown on the 3D CytoSort? Array can be reliably tracked,imaged,and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time,then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids,we have demonstrated the use of this technology for single-organoid imaging,clonal organoid generation,parent organoid subcloning,and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR? System to facilitate efficient,user-friendly,and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment,2) establishment of single cell-derived organoids,and 3) isolation and retrieval of single organoids for downstream applications. View Publication

BACKGROUND Circular RNA of vimentin (circ-VIM) is a predictor for poor prognosis of acute myeloid leukemia,but we had little information on its function in esophageal cancer (EC). Here we examined the effects of circ-VIM together with sevoflurane on immune escape and multiple oncogenic activities of EC. METHODS Bioinformatic tools,luciferase assay,and RNA immunoprecipitation were used to examine regulations between circ-VIM,miR-124-3p (miR-124),and PD-L1. CCK-8,wound healing,and Transwell assays were used to measure cell proliferation,migration,and invasion,respectively. The impacts of EC cells on cytotoxicity,proliferation,and apoptosis of CD8+ T cells were examined using LDH assay,CFSE staining,and Annexin V/PI staining,respectively. The in vivo tumorigenesis and lung metastases were assessed using xenograft model and tail vein injection of EC cells. RESULTS Significant upregulation of circ-VIM and PD-L1 and downregulation of miR-124 were detected in EC tissues or cells. Circ-VIM sponged miR-124 and released its suppression on the downstream target PD-L1. Sevoflurane,independent of circ-VIM,also upregulated miR-124 to lower PD-L1 expression. By modulating miR-124/PD-L1 axis,silencing circ-VIM and applying sevoflurane both inhibited immune escape and multiple oncogenic activities of EC in vitro,and suppressed xenograft growth and lung metastases in vivo. The inactivation of Ras/ERK signaling pathway was involved in suppression of malignant phenotypes by silencing circ-VIM and sevoflurane treatment. CONCLUSIONS Silencing circ-VIM and applying sevoflurane,by separately regulating miR-124/PD-L1 axis,presented synergistic effects in inhibiting immune escape and multiple malignant phenotypes of EC cells. View Publication