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Background Osteosarcoma is the most common bone malignancy in children and adolescents, and 20%–30% of the patients suffer from poor prognosis because of individual chemoresistance. The Hippo/yes‐associated protein (YAP) signaling pathway has been shown to play a role in tumor chemoresistance, but no previous report has focused on its involvement in osteosarcoma chemoresistance. This study aimed to investigate the role of the Hippo/YAP signaling pathway in osteosarcoma chemoresistance and to determine potential treatment targets. Methods Using the Cell Titer‐Glo Luminescent cell viability assay and flow cytometry analysis, we determined the proliferation and chemosensitivity of YAP‐overexpressing and YAP‐knockdown osteosarcoma cells. In addition, using western blotting and the real‐time polymerase chain reaction technique, we investigated the alteration of the Hippo/YAP signaling pathway in osteosarcoma cells treated with chemotherapeutic agents. Results Mammalian sterile 20‐like kinase 1 (MST1) degradation was increased, and large tumor suppressor kinase 1/2 (LATS1/2) total protein levels were decreased by methotrexate and doxorubicin, which increased activation and nuclear translocation of YAP. Moreover, YAP increased the proliferation and chemoresistance of MG63 cells. Conclusions The Hippo/YAP signaling pathway plays a role in osteosarcoma chemoresistance, and YAP is a potential target for reducing chemoresistance.

Aim： To investigate the effects and the molecular mechanisms of fucoxanthin, a major carotenoid found in edible seaweed, on HeLa cells. Methods： The cytotoxicity of fucoxanthin was evaluated using MTT assay. Cell cycle and apoptosis were evaluated using flow cytometric analysis. Autophagy was detected with acridine orange staining and transient transfection of the GFP-LC3 plasmid into the cells. Protein expression was detected with Western blotting. Results： Treatment of HeLa cells with fucoxanthin （10-80 μmol/L） for 48 h caused dose-dependent cytotoxicity with an IC50 value of 55.1±7.6 μmol/L. Fucoxanthin （10, 20, and 40 pmol/L） dose-dependently induced Go/G1 arrest, but did not change the apoptosis of HeLa cells. The same concentrations of fucoxanthin dose-dependently increased the protein expression of LC3 II （the autophagosome marker） and Beclin 1 （the initiation factor for autophagosome formation） in HeLa cells. Moreover, fucoxanthin dose-dependently decreased the levels of phosphorylated Akt and its downstream proteins p53, p7OS6K, and mTOR, and increases the expression of PTEN in HeLa cells. Pretreatment of HeLa cells with 3-methyladenine （5 mmol/L） blocked the cytotoxic effect of fucoxanthin as well as fucoxanthin-induced autophagy. Conclusion： Fucoxanthin exerts autophagy-dependent cytotoxic effect in HeLa cells via inhibition of Akt/mTOR signaling pathway.

The molecular pathways contributing to humoral-mediated allograft rejection are poorly defined. In this study, we assessed the role of the herpesvirus entry mediator/B- and T-lymphocyte attenuator （HVEM/BTLA） signalling pathway in the context of antibody-mediated allograft rejection. An experimental setting was designed to elucidate whether the blockade of HVEM/BTLA interactions could modulate de novo induction of host antidonor-specific antibodies during the course of graft rejection. To test this hypothesis, fully allogeneic major histocompatibility complex-mismatched skin grafts were transplanted onto the right flank of recipient mice that were treated with isotype control, anti-CD40L or modulatory antibodies of the HVEM/BTLA signalling pathway. The frequencies of CD4 T follicular helper （Tfh） cells （B220-, CD4＋ CXCR5＋ PD-lhigh）, extrafollicular helper cells （B220-, CD4＋ CXCR5- PD-1＋ and PD-1-） and germinal centre （GC） B cells （B220＋Fas＋ GL7＋） were analysed by flow cytometry in draining and non-draining lymph nodes at day 10 post transplantation during the acute phase of graft rejection. The host antidonor isotype-specific humoral immune response was also assessed. Whereas blockade of the CD40/CD40L pathway was highly effective in preventing the allogeneic humoral immune response, antibody-mediated blockade of the HVEM/BTLA-interacting pathway affected neither the expansion of Tfh cells nor the expansion of GC B cells. Consequently, the course of the host antidonor antibody-mediated response proceeded normally, without detectable evidence of impaired development. In summary, these data indicate that HVEM/BTLA interactions are dispensable for the formation of de novo host antidonor isotype-specific antibodies in transplantation.

Dendritic cells （DCs） and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c~hCD11b＋HLA-DR-hCD86＋ subset in the spleen, along with the previously described subsets of hCDlc＋ DCs, hCD123＋ plasmacytoid DCs （pDCs）, hCD16＋ DCs and hCD141＋ DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c＋HLA-DR＋ and hCD11c＋HLA-DR- cells were produced. The latter resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCDSO and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors.

Advances in fabrication of mesoscopic membrane sensors with unique structures and morphologies inside anodic alumina membrane （AAM） nanochannels have led to the development of various methods for detecting, visualizing, adsorbing, filtering, and recovering ultra-trace concentrations of toxic metal ions, such as Hg^2＋ and Pb^2＋, in water and blood. These often ＂one-pot＂ screening methods offer advantages over conventional methods in that they do not require sophisticated instruments or laborious sample preparation. In the present study, we fabricated two mesoscopic membrane sensors for naked-eye detection, recognition, filtration, and recovery of Hg^2＋ and Pb^2＋ in biological and environmental samples. These sensors were characterized by the dense immobilization of organic colorants on the mesopore surfaces of silica nanotubes that were constructed using the nanochannels of an AAM as a scaffold. We confirmed that the nanotubes were oriented along the long axis of the AAM nanochannels, open at both ends, and completely and uniformly filled with organic colorants; also, the dense immobilization of the organic colorants did not affect the speed of ion-to-ligand binding events. We used simple, desk-top, flow-through assays to assess the suitability of the developed membrane sensors for detection, removal, and filtration of Hg^2＋ and Pb^2＋ with respect to recyclability and continuous monitoring. Removal of the target ions from biological fluids was assessed by means of flow cytometric analysis. Our results demonstrate the potential of our membrane sensors to be used for preventing the health risks associated with exposure to toxic metal ions in the environment and blood.

Aim： To investigate the effects of trans-cinnamaldehyde （TCA） on the human leukemia K562 cell line and the cytotoxicity of cytokineinduced killer （CIK） cells against K562 cells. Methods： Apoptosis, Fas expression, and mitochondrial transmembrane potential in K652 cells were analyzed using flow cytometry. K562 cells were labeled with CFSE. The cytotoxic effect of expanded CtK cells on CFSE-labeled K562 cells was determined by FACS flow cytometry. Results： Treatment with TCA 180 μmol/L for 9 h induced apoptosis in 8.9%±1.23% of K562 cells. Treatment with 120 or 180 pmol/L TCA for 24 h significantly increased the apoptotic cells to 18.63%±1.42 % and 38.98%±2.74%, respectively. TCA significantly upregulates Fas expression and decreases mitochondrial transmembrane potential in K562 cells. TCA treatment at 120 and 180 μmol/L for 9 h enhanced the percentage of lysis of K562 cells by expanded CIK cells from 34.84%±2.13% to 48.21%±2.22 % and 64.81%±3.22% at the E：F ratio of 25：1 and from 49.26%±3.22% to 57.81%±5.13% and 73.36%±5.98% at E：F ratio of 50：1. Conclusion： TCA exerts cytotoxic effects on human leukemia K562 cells by inducing apoptosis and synergizing the cytotoxicity of CIK cells against K562 cells. These properties of TCA are beneficial to the treatment of leukemia, even in the patients who have received hematopoietic stem cells transplantation （HSCT）.

The prevalence of nasopharyngeal cancer （NPC） is high in the southern area of China and some other districts in the world. The pathogenesis of NPC is unclear. It is reported that some microRNAs （miR） are involved in the progression of NPC. This study aims to investigate the role of miR-21 in the induction of immune tolerance of NPC. In this study, NPC tissue was collected from patients with NPC. Assessment of miR was performed with real time quantitative RT-PCR. Western blotting was used to assess proteins of interleukin 10 and nuclear factor I-A （NFI-A）, Immune ceils were analyzed by flow cytometry. The results showed that NPC cell line C666-1 and surgically removed NPC tissue expressed miR-21, which was upregulated by the presence of the Toll-like receptor 3 ligand, Poly h C. Exposure to miR-21 increased the expression of NFI-A and interleukin （IL）-IO in naive B cells. High frequency of IL-10＋ B cells was detected in the NPC tissue. The NPC- or miR-21-primed B ceils suppressed cytotoxic CD8＋ T cell activities. We conclude that NPC-derived miR-21 induces IL-10＋ B ceils; the latter is capable of suppressing CD8＋ T-cell activities, miR-21 may be a potential target in the treatment of NPC.

Aim： Ursolic acid （UA） is a pentacyclic triterpenoid found in most plant species, which has been shown anti-inflammatory and anti-oxidative activities. In this study, we examined the effects of UA on collagen-induced arthritis （CIA） in mice, and to identify the mechanisms underlying the effects. Methods： CIA was induced in mice. Two weeks later, the mice were treated with UA （150 mg/kg, ip, 3 times per week） for 4 weeks. The expression of cytokines and oxidative stress markers in joint tissues was measured with immunohistochemistry. The numbers of CD4~IL-17~, CD4~CD25＊Foxp3~ and pSTAT3 cells in spleens were determined using confocal immunostaining or flowcytometric analyses. Serum antibody levels and B cell-associated marker mRNAs were analyzed with ELISAs and qRT-PCR, respectively. CD4^＋ T cells and CD19^＋ B cells were purified from mice spleens for in vitro studies. Results： UA treatment significantly reduced the incidence and severity of CIA-induced arthritis, accompanied by decreased expression of proinflammatory cytokines （TNF-α, IL-1β, IL-6, IL-21 and IL-17） and oxidative stress markers （nitrotyrosine and iNOS） in arthritic joints. In CIA mice, UA treatment significantly decreased the number of Th17 cells, while increased the number of Treg cells in the spleens, which was consistent with decreased expression of pSTAT3, along with IL-17 and RORyt in the splenocytes. In addition, UA treatment significantly reduced the serum CII-specific IgG levels in CIA mice. The inhibitory effects of UA on Th17 cells were confirmed in an in vitro model of Th17 differentiation. Furthermore, UA dose-dependently suppressed the expression of B cell-associated markers Bcl-6, Blimp1 and AID mRNAs in purified CD19^＋ B cells pretreated with IL-21 or LPS in vitro. Conclusion： UA treatment significantly ameliorates CIA in micevia suppression of Th17 and differentiation. By targeting pathogenic Th17 cells and autoantibody production, UA may be useful for the treatment of autoimmune arthritis and other Th17-related diseases.

The genome size（C-value） of an organism is referring to the DNA content of its non-replicated haploid chromosome complement,generally deduced from measuring somatic diploid nuclei.We presented genome size（C-value） data obtained by flow cytometry for four commercially important crabs（Portunus trituberculatus,Charybdis japonica,Scylla paramamosain,and Eriocheir sinensis） common in the coast of China.Gallus domesticus（2C=2.5 pg） was used as the internal standard.The results showed that the C-value for P.trituberculatus,C.japonica,S.paramamosain,and E.sinensis were（2.31±0.01） pg,（2.33±0.03） pg,（1.64±0.02） pg,and（2.29±0.03） pg,respectively.The C-value of P.trituberculatus,C.japonica and S.paramamosain were reported for the first time.The data represented by the four species indicated that they had lower DNA contents than average DNA values in crustaceans（（4.99±0.48） pg）,and three of the four values were very similar if not identical.The results provide useful data for future studies in the fields of biodiversity,species conservation,and phylogeny of these commercial crabs.They will also be helpful in instructing the hybridization breeding program and estimating the cost of the whole genome sequencing project.