技术资料

Osteoporotic osteoarthritis (OPOA) is a common bone disease mostly in the elderly,but the relationship between Osteoporotic (OP) and osteoarthritis (OA) is complex. It has been shown that knee loading can mitigate OA symptoms. However,its effects on OPOA remain unclear. In this study,we characterized pathological linkage of OP to OA,and evaluated the effect of knee loading on OPOA. We employed two mouse models (OA and OPOA),and conducted histology,cytology,and molecular analyses. In the OA and OPOA groups,articular cartilage was degenerated and Osteoarthritis Research Society International score was increased. Subchondral bone underwent abnormal remodeling,the differentiation of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts and chondrocytes was reduced,and migration and adhesion of pre-osteoclasts were enhanced. Compared to the OA group,the pathological changes of OA in the OPOA group were considerably aggravated. After knee loading,however,cartilage degradation was effectively prevented,and the abnormal remodeling of subchondral bone was significantly inhibited. The differentiation of BMSCs was also improved,and the expression of Wnt/$\beta$-catenin was elevated. Collectively,this study demonstrates that osteoporosis aggravates OA symptoms. Knee loading restores OPOA by regulating subchondral bone remodeling,and may provide an effective method for repairing OPOA. View Publication

Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells,whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues,whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability,DNA binding,and gene transcription. Moreover,NEUROG3 was highly unstable in the intestinal epithelium,explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships. View Publication

Graphene quantum dots (GQDs) have gained significant attention in various biomedical applications. The physicochemical properties of these nanoparticles,including toxic effects,are largely determined by their surface modifications. Previous studies have demonstrated high in vitro cytotoxicity of the hydroxylated GQDs (OH-GQDs). The focus of this study was on the intestinal toxicity of OH-GQDs. Briefly,C57BL/6J mice were given daily oral gavage of 0.05,0.5 or 5 mg/kg OH-GQD for 7 days,and the indices of intestinal damage were evaluated. Higher doses of the OH-GQDs caused significant intestinal injuries,such as enhanced intestinal permeability,shortened villi and crypt loss. The number of Lgr5+ intestinal stem cells also decreased dramatically upon OH-GQDs exposure,which also inhibited the Ki67+ proliferative progenitor cells. In addition,an increased number of crypt cells harboring the oxidized DNA base 8-OHdG and $\gamma$H2AX foci were also detected in the intestines of OH-GQD-treated mice. Mechanistically,the OH-GQDs up-regulated both total and phosphorylated p53. Consistent with this,the average number of TUNEL+ and cleaved caspase-3+ apoptotic intestinal epithelial cells were significantly increased after OH-GQDs treatment. Finally,a 3-dimensional organoid culture was established using isolated crypts,and OH-GQDs treatment significantly reduced the size of the surviving intestinal organoids. Taken together,the intestinal toxicity of the OH-GQDs should be taken into account during biomedical applications. View Publication

Shiga toxigenic Escherichia coli (STEC) are responsible for a worldwide foodborne disease,which is characterized by severe bloody diarrhea and hemolytic uremic syndrome (HUS). Subtilase cytotoxin (SubAB) is a novel AB5 toxin,which is produced by Locus for Enterocyte Effacement (LEE)-negative STEC. Cleavage of the BiP protein by SubAB induces endoplasmic reticulum (ER) stress,followed by induction of cytotoxicity in vitro or lethal severe hemorrhagic inflammation in mice. Here we found that steroids and diacylglycerol (DAG) analogues (e.g.,bryostatin 1,Ingenol-3-angelate) inhibited SubAB cytotoxicity. In addition,steroid-induced Bcl-xL expression was a key step in the inhibition of SubAB cytotoxicity. Bcl-xL knockdown increased SubAB-induced apoptosis in steroid-treated HeLa cells,whereas SubAB-induced cytotoxicity was suppressed in Bcl-xL overexpressing cells. In contrast,DAG analogues suppressed SubAB activity independent of Bcl-xL expression at early time points. Addition of Shiga toxin 2 (Stx2) with SubAB to cells enhanced cytotoxicity even in the presence of steroids. In contrast,DAG analogues suppressed cytotoxicity seen in the presence of both toxins. Here,we show the mechanism by which steroids and DAG analogues protect cells against SubAB toxin produced by LEE-negative STEC. View Publication

Despite implementation of virtual crossmatches,flow cytometry crossmatches (FCXM) are still used by many transplant centers to determine immunological risk before kidney transplantation. To determine if common profiles of patients prone to false positive FCXM exist,we examined the demographics and native diseases of kidney patients tested with autologous FCXM (n = 480). Improvements to FCXM and cell isolation methods significantly reduced the positive rate from 15.1{\%} to 5.3{\%}. Patients with native diseases considered 'immunological' (vasculitis,lupus,IgA nephropathy) had more positive autologous FCXM (OR = 3.36,p = 0.003) vs. patients with all other diseases. Patients who were tested using our updated method (n = 321) still showed that these immunological diseases were a significant predictor for positive autologous FCXM (OR = 4.79,p = 0.006). Interestingly,patients on peritoneal dialysis (PD) also had significantly more positive autologous FCXM than patients on hemodialysis or waiting for pre-emptive kidney transplants (OR = 3.27,p = 0.02). These findings were confirmed in patients who had false positive allogeneic FCXM. Twenty of 24 (83.3{\%}) patients with false positive allogeneic FCXM tested with updated method either had immunological diseases originally or were on PD. Our findings are helpful when interpreting an unexpected positive FCXM,especially for transplantation from deceased donors. 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

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 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

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

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

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