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The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand,FasL. Although Fas is highly expressed in cancer cells,insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here,we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor,fasudil,that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL),fasudil promoted cancer cell apoptosis,but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis. View Publication

T cells have been reported to play critical roles in preventing of microsporidia dissemination. However,there roles and functions of each subset remain unclear. Here in the study,we performed a thorough analysis of murine splenic T-cell response analysis via single-cell RNA sequencing during microsporidia E. cuniculi infection. We demonstrated that Type I T helper (Th1) cells,T follicular helper (Tfh) cells,effector CD8 + T cells and proliferating CD8 + T cells were activated and expanded after infection. Activated Th1 cells and Tfh cells presented significantly upregulated gene expression of Ifng and Il21,respectively. A subcluster of Th1 cells with high Csf1 expression was detected after infection. Subsets of activated CD8 + T cells were markedly enriched with high expression of cytotoxic-function related genes such as Gzma and Gzmb,whereas some active CD8 T cells were enriched with proliferation-function related genes Mki67 and Stmn1. Other subsets of T cells including NK T cells,Myb+ T cells,γδ T cells and Cxcr6+ T cells,were also analyzed in this study yet no expansion was observed. In summary,our findings provide in-depth and comprehensive insights into T-cell responses during microsporidia infection,which will be valuable for further investigations. This study provides a comprehensive landscape of mouse T cells responses during microsporidia infection at a single-cell resolution reporting that Th1,Tfh,effector and proliferating CD8 + T cell subsets were activated and expanded upon infection. View Publication

BACKGROUND In colorectal carcinoma (CRC),activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition,the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore,we hypothesized that an ATP-competitive pan-Raf inhibitor,Raf265,is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition,anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally,the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC,providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC. View Publication

BACKGROUND Self-renewing,chemoresistant breast cancer stem cells are believed to contribute significantly to cancer invasion,migration and patient relapse. Therefore,the identification of signaling pathways that regulate the acquisition of stem-like qualities is an important step towards understanding why patients relapse and towards development of novel therapeutics that specifically target cancer stem cell vulnerabilities. Recent studies identified a role for the aryl hydrocarbon receptor (AHR),an environmental carcinogen receptor implicated in cancer initiation,in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in the acquisition of cancer stem cell-like qualities. RESULTS To test this hypothesis,AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells were modulated with AHR ligands,shRNA or AHR-specific inhibitors,and phenotypic,genomic and functional stem cell-associated characteristics were evaluated. The data demonstrate that (1) ALDH(high) cells express elevated levels of Ahr and Cyp1b1 and Cyp1a1,AHR-driven genes,(2) AHR knockdown reduces ALDH activity by 80%,(3) AHR hyper-activation with several ligands,including environmental ligands,significantly increases ALDH1 activity,expression of stem cell- and invasion/migration-associated genes,and accelerates cell migration,(4) a significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of stem cell- and invasion/migration-associated gene sets is seen with genomic data obtained from 79 human breast cancer cell lines and over 1,850 primary human breast cancers,(5) the AHR interacts directly with Sox2,a master regulator of self-renewal; AHR ligands increase this interaction and nuclear SOX2 translocation,(6) AHR knockdown inhibits tumorsphere formation in low adherence conditions,(7) AHR inhibition blocks the rapid migration of ALDH(high) cells and reduces ALDH(high) cell chemoresistance,(8) ALDH(high) cells are highly efficient at initiating tumors in orthotopic xenografts,and (9) AHR knockdown inhibits tumor initiation and reduces tumor Aldh1a1,Sox2,and Cyp1b1 expression in vivo. CONCLUSIONS These data suggest that the AHR plays an important role in development of cells with cancer stem cell-like qualities and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of these properties. View Publication

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene,whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly,the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations),though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover,gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence,our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future,once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved,hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease. View Publication

INTRODUCTION Bone marrow mesenchymal stem cells (BMSCs) have been extensively investigated from a perspective on cardiac regeneration therapy. The current study aimed to investigate the protective effect conferred by BMSCs in subacute myocardial injury,and to identify an appropriate BMSC reinfusion time. METHODS BMSCs were isolated from human bone marrow blood. Daunorubicin (DNR)-induced subacute myocardial models were subsequently established. The rats with DNR-induced subacute myocardial injury were injected with dexrazoxane (DZR) and/or BMSCs at varying time points,after which cardiac function was evaluated by assessing left ventricular ejection fraction (LVEF) and fraction shortening (FS). The myocardial structural changes were analyzed,after which the levels of CD3 and human leukocyte antigen DR (HLA-DR) were examined to further validate the mechanism by which BMSCs could influence subacute myocardial injury. RESULTS BMSCs combined with DZR treatment enhanced the cardiac function of rats with DNR-induced myocardial injury,as reflected by increased LVEF and FS. DNR-induced myocardial injuries were mitigated via the application of BMSCs combined with treatment of DZR,accompanied by diminished infiltration or vacuolization. Moreover,BMSCs were observed to alleviate infiltration of T lymphocyte and antigen-presenting cells,as evidenced by reduced expression of CD3 and HLA-DR. CONCLUSION Taken together,this study demonstrates that BMSCs could protect against DNR-induced myocardial injury,especially in the first three days of DNR administration. BMSCs combined with DZR exert a better therapeutic effect,but there are individual differences. 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

We studied the actions of geldanamycin (GA) and herbimycin A (HMA),inhibitors of the chaperone proteins Hsp90 and GRP94,on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses,T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA,but not HMA,showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase,a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL. View Publication

PURPOSE Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device. MATERIALS AND METHODS Rat CF were cultured at high-density (˜1.6×10(5)/cm(2)) for 10-14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry,immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later,mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer. RESULTS CF-ECM scaffolds are composed of fibronectin (82%),collagens type I (13%),type III (3.4%),type V (0.2%),type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium. CONCLUSIONS High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium. View Publication

Because neurons are difficult to obtain from humans,generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells,we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation,glutamate receptor function,and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2,resulting in high responsiveness of neural cells to glutamate,N-methyl-d-aspartate (NMDA),α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA),and (S)-3,5-d... View Publication

Femtosecond coherent anti-Stokes Raman scattering (CARS) spectroscopy offers several advantages over spontaneous Raman spectroscopy due to the inherently high sensitivity and low average power deposition in the sample. Femtosecond CARS can be implemented in a collinear pump/probe beam configuration for microspectroscopy applications and has emerged as a powerful technique for chemical imaging of biological specimens. However,one serious limitation of this approach is the presence of a high nonresonant background component that often obscures the resonant signals of interest. We report here an innovative pulse-shaping method based on Lorentzian amplitude and phase spectral modulation of a broadband femtosecond probe pulse that yields spectra with both high spectral resolution and no nonresonant background. No further mathematical analysis is needed to extract Raman spectra. The utility of the proposed method for CARS microscopy is demonstrated using a mixture of polystyrene and latex beads,as well as dry-fixed embryonic stem cells. View Publication