技术资料

Protein disulfide isomerase (PDI,PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies,although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4,CHOP,and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors. View Publication

Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

Although a link between the gut microbiota and alcohol-related liver diseases (ALDs) has previously been suggested,the causative effects of specific taxa and their functions have not been fully investigated to date. Here,we analyze the gut microbiota of 410 fecal samples from 212 Korean twins by using the Alcohol Use Disorders Identification Test (AUDIT) scales to adjust for host genetics. This analysis revealed a strong association between low AUDIT scores and the abundance of the butyrate-producing genus Roseburia. When Roseburia spp. are administered to ALD murine models,both hepatic steatosis and inflammation significantly improve regardless of bacterial viability. Specifically,the flagellin of R. intestinalis,possibly through Toll-like receptor 5 (TLR5) recognition,recovers gut barrier integrity through upregulation of the tight junction protein Occludin and helps to restore the gut microbiota through elevated expression of IL-22 and REG3$\gamma$. Our study demonstrates that Roseburia spp. improve the gut ecosystem and prevent leaky gut,leading to ameliorated ALDs. View Publication

PURPOSE We investigated the effect of the racemic $\beta$-hydroxybutyrate precursor,R,S-1,3-butanediol (BD),on T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells (PBMC) following prolonged,strenuous exercise. METHODS A repeated-measures,randomised,crossover study was conducted in nine healthy,trained male cyclists (age,26.7 ± 5.2 years; VO2peak,63.9 ± 2.5 mL kg-1 min-1). Participants ingested 0.35 g kg-1 of BD or placebo 30 min before and 60 min during 85 min of steady-state (SS) exercise,which preceded a {\~{}} 30 min time-trial (TT) (7 kJ kg-1). Blood samples were collected at pre-supplement,pre-exercise,post-SS,post-TT and 1-h post-TT. Whole blood cultures were stimulated with Staphylococcal enterotoxin B (SEB) for 24 h to determine T-cell-related interleukin (IL)-4,IL-10 and interferon (IFN)-$\gamma$ mRNA expression within isolated PBMCs in vitro. RESULTS Serum cortisol,total circulating leukocyte and lymphocyte,and T-cell subset concentrations were similar between trials during exercise and recovery (all p {\textgreater} 0.05). BD ingestion increased T-cell-related IFN-$\gamma$ mRNA expression compared with placebo throughout exercise and recovery (p = 0.011); however,IL-4 and IL-10 mRNA expression and the IFN-$\gamma$/IL-4 mRNA expression ratio were unaltered (all p {\textgreater} 0.05). CONCLUSION Acute hyperketonaemia appears to transiently amplify the initiation of the pro-inflammatory T-cell-related IFN-$\gamma$ response to an immune challenge in vitro during and following prolonged,strenuous exercise; suggesting enhanced type-1 T-cell immunity at the gene level. View Publication

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication

INTRODUCTION Complexins (CPLXs),initially identified in neuronal presynaptic terminals,are cytoplasmic proteins that interact with the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex to regulate the fusion of vesicles to the plasma membrane. Although much is known about CPLX function in neuronal synaptic vesicle exocytosis,their distribution and role in immune cells are still unclear. In this study,we investigated CPLX2 knockout (KO) mice to reveal the role of CPLXs in exocytosis of lymphocytes. METHODS We examined the expression of CPLXs and SNAREs in lymphocytes. To study the effect of CPLXs on the immune system in vivo,we analyzed the immune phenotype of CPLX2 KO mice. Furthermore,antibodies secretion from the peritoneal cavity,spleen,and bone marrow cells of wild-type (WT) and CPLX2 KO mice were determined. RESULTS CPLX2 was detected in B cells but not in T cells,while other CPLXs and SNAREs were expressed at a similar level in both B and T cells. To clarify the function of CPLX2 in B lymphocytes,serum concentrations of immunoglobulin G (IgG),IgA,IgM,and IgE were measured in WT and CPLX2 KO mice using enzyme-linked immunosorbent assay. The level of IgM,which mainly consists of natural antibodies,was higher in KO mice than that in WT mice,while the levels of other antibodies were similar in both types of mice. Additionally,we found that spontaneous secretion of IgM and IgG1 was enhanced from the splenic antibody-secreting cells (ASCs) of CPLX2 KO mice. CONCLUSION Our data suggest that CPLX2 inhibits spontaneous secretion of IgM and IgG1 from splenic ASCs. This study provides new insight into the mechanism of antibody secretion of ASCs. View Publication

Progressive resistance exercise training (PRT) is the most effective known intervention for combating aging skeletal muscle atrophy. However,the hypertrophic response to PRT is variable,and this may be due to muscle inflammation susceptibility. Metformin reduces inflammation,so we hypothesized that metformin would augment the muscle response to PRT in healthy women and men aged 65 and older. In a randomized,double-blind trial,participants received 1,700 mg/day metformin (N = 46) or placebo (N = 48) throughout the study,and all subjects performed 14 weeks of supervised PRT. Although responses to PRT varied,placebo gained more lean body mass (p = .003) and thigh muscle mass (p {\textless} .001) than metformin. CT scan showed that increases in thigh muscle area (p = .005) and density (p = .020) were greater in placebo versus metformin. There was a trend for blunted strength gains in metformin that did not reach statistical significance. Analyses of vastus lateralis muscle biopsies showed that metformin did not affect fiber hypertrophy,or increases in satellite cell or macrophage abundance with PRT. However,placebo had decreased type I fiber percentage while metformin did not (p = .007). Metformin led to an increase in AMPK signaling,and a trend for blunted increases in mTORC1 signaling in response to PRT. These results underscore the benefits of PRT in older adults,but metformin negatively impacts the hypertrophic response to resistance training in healthy older individuals. ClinicalTrials.gov Identifier: NCT02308228. View Publication

SCOPE Necrotizing enterocolitis (NEC) is a devastating disease that is highly lethal in premature infants. Human milk oligosaccharides (HMOs) efficiently reduce the incidence of NEC. However,the protective mechanism of HMO treatment is unknown. It is hypothesized that HMOs protect against NEC by inhibiting the damage to intestinal epithelial cells. METHODS AND RESULTS C57BL/6 pups are challenged with hypoxia and cold stress to induce NEC. All pups are sacrificed after 72 h. It is found that HMO administration reduces the concentrations of IL-8 in the serum and ileum of all NEC mice. Ileum toll-like receptor 4 (TLR4) protein expression and nuclear factor kappa-B (NF$\kappa$B) pathway activation are inhibited. The proliferative ability of enterocytes in the ileum is restored as determined by labeling with proliferation markers (Ki67,SOX9). In a 3D culture intestinal crypt organoids study,HMO treatment improves the maturation of organoid cells and increases the ratio of proliferative cells under lipopolysaccharides (LPS) treatment. HMO treatment downregulates TLR4 expression in the organoid cells,thus reducing the effect of LPS. CONCLUSION HMOs protect intestinal epithelial cells from injury by accelerating the turnover of crypt cells by reducing the expression of TLR4 on intestinal epithelial cells. View Publication

Myelodysplastic syndromes (MDSs) are a group of clonal disorders of hematopoietic stem cells,resulting in ineffective hematopoiesis. Previous studies have reported that decitabine (DAC) plays an essential role in cell cycle arrest and cell death induction in multiple cell types. Nevertheless,the effect of decitabine on mesenchymal stromal cells derived from bone marrow of patients with MDSs is not completely clarified. Here,we explored the apoptotic and anti-proliferative effect of DAC on MSCs isolated from patients with MDSs. Treatment with DAC inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. We found a positive relationship between cell death triggered by DAC in MSCs and the death receptor family members Fas and FasL mRNA and protein levels (***P {\textless} 0.00085),cleaved caspase (-3,-8,and -9) activity,and mitochondrial membrane potential reduction. Additionally,DAC-induced apoptosis was inhibited by Kp7-6,a FasL/Fas antagonist,indicating a crucial role of FasL/Fas,a cell death receptor,in mediating the apoptotic effect of DAC. DAC also induced reactive oxygen species (ROS) generation in MSCs derived from MDSs patients (*P = 0.038). Furthermore,N-acetyl-L-cysteine (NAC),a widely accepted ROS scavenger,efficiently reversed DAC-induced apoptosis by inhibiting ROS generation (***P {\textless} 0.00051) in mitochondria and restoring mitochondrial membrane potential. Furthermore,ROS production was found to be a consequence of caspase activation via caspases inhibition. Our data imply that DAC triggers ROS production in human MSCs,which serves as a crucial factor for mitochondrial membrane potential reduction,and DAC induces cell death prior to FasL/Fas stimulation. View Publication

The perivascular niche within adipose tissue is known to house multipotent cells,including osteoblast precursors. However,the identity of perivascular subpopulations that may mineralize or ossify most readily is not known. Here,we utilize inducible PDGFR$\alpha$ (platelet-derived growth factor alpha) reporter animals to identify subpopulations of perivascular progenitor cells. Results showed that PDGFR$\alpha$-expressing cells are present in four histologic niches within inguinal fat,including two perivascular locations. PDGFR$\alpha$+ cells are most frequent within the tunica adventitia of arteries and veins,where PDGFR$\alpha$+ cells populate the inner aspects of the adventitial layer. Although both PDGFR$\alpha$+ and PDGFR$\alpha$- fractions are multipotent progenitor cells,adipose tissue-derived PDGFR$\alpha$+ stromal cells proliferate faster and mineralize to a greater degree than their PDGFR$\alpha$- counterparts. Likewise,PDGFR$\alpha$+ ectopic implants reconstitute the perivascular niche and ossify to a greater degree than PDGFR$\alpha$- cell fractions. Adventicytes can be further grouped into three distinct groups based on expression of PDGFR$\alpha$ and/or CD34. When further partitioned,adventicytes co-expressing PDGFR$\alpha$ and CD34 represented a cell fraction with the highest mineralization potential. Long-term tracing studies showed that PDGFR$\alpha$-expressing adventicytes give rise to adipocytes,but not to other cells within the vessel wall under homeostatic conditions. However,upon bone morphogenetic protein 2 (BMP2)-induced ossicle formation,descendants of PDGFR$\alpha$+ cells gave rise to osteoblasts,adipocytes,and pericyte-like" cells within the ossicle. In sum PDGFR$\alpha$ marks distinct perivascular osteoprogenitor cell subpopulations within adipose tissue. The identification of perivascular osteoprogenitors may contribute to our improved understanding of pathologic mineralization/ossification. Stem Cells 2019." View Publication

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies,including liver injury. $\omega$-6 PUFAs exert pro-inflammatory activity,while $\omega$-3 PUFAs promote anti-inflammatory activity that is mediated,in part,through specialized pro-resolving mediators [e.g.,resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the $\omega$-6:$\omega$-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. $\omega$-3 FA desaturase-1 (fat-1) mice,which endogenously increase $\omega$-3 PUFA levels,were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-$\gamma$,Il-6,and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation,stem cell renewal,and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further,gut microbiome and plasma metabolomics revealed several changes in fat-1 versus WT mice that may contribute to a reduced inflammatory response. Finally,these data correlated with a significant reduction in liver injury. Our study suggests that $\omega$-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury. View Publication

The vascular wall is a source of progenitor cells that are able to induce skeletal repair,primarily by paracrine mechanisms. Here,the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First,purified human perivascular stem cells (PSCs) were observed to induce mitogenic,pro-migratory,and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic,pro-migratory,and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins,as demonstrated by EV trypsinization,or neutralizing antibodies for CD9 or CD81. Moreover,shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally,PSC-EVs stimulated bone repair,and did so via stimulation of skeletal cell proliferation,migration,and osteodifferentiation. In sum,PSC-EVs mediate the same tissue repair effects of perivascular stem cells,and represent an 'off-the-shelf' alternative for bone tissue regeneration. View Publication