B7-H3 is a member of the B7-family of co-stimulatory molecules, which has been shown to be broadly expressed in various tumor tissues, and which plays an important role in adaptive immune responses. The role of B7-H3 in osteosarcoma, however, remains unknown. In this study we used immunohistochemistry to analyze B7-H3 expression in 61 primary osteosarcoma tissues with case-matched adjacent normal tissues, and 37 osteochondroma and 20 bone fibrous dysplasia tissues. B7-H3 expression was expressed in 91.8% (56/61) of the osteosarcoma lesions, and the intensity of B7-H3 expression in osteosarcoma was significantly increased compared with adjacent normal tissues, osteochondroma and bone fibrous dysplasia tissues (p<0.001). Patients with high tumor B7-H3 levels had a significantly shorter survival time and recurrence time than patients with low tumor B7-H3 levels (p<0.001). Moreover, tumor B7-H3 expression inversely correlated with the number of tumor-infiltrating CD8+ T cells (p<0.05). In vitro, increasing expression of B7-H3 promotes osteosarcoma cell invasion, at least in part by upregulating matrix metalloproteinase-2 (MMP-2). In conclusion, our study provides the first evidence of B7-H3 expression in osteosarcoma cells as a potential mechanism controlling tumor immunity and invasive malignancy, and which is correlated with patients’ survival and metastasis.

During pollen tube growth, the walls of the tube provide the mechanical strength resisting turgor pressure to protect two sperm cells. Cell wall proteins may play an important role in this process. Pollen tube cell wall proteins known as leucine-rich repeat extensins (LRXs) harbor a leucine-rich repeat domain and an extensin domain. In this study, the functions of four pollen-expressed LRXs, LRX8, LRX9, LRX10, and LRX11 (LRX8-11), were characterized in Arabidopsis (). displayed a consistent expression pattern in mature pollen grains and pollen tubes. In a phenotypic analysis of four single mutants, six double mutants, four triple mutants, and a quadruple mutant, the triple and quadruple mutant plants displayed markedly reduced seed set and decreased male transmission efficiency accompanied by compromised pollen germination and pollen tube growth. GFP-fused LRX8, LRX10, and LRX11 were found to be localized to pollen tube cell walls. An immunohistochemical analysis of pollen tube cell wall polysaccharides showed an increase in the amount of rhamnogalacturonan I in the subapical walls of pollen tubes of the and mutants and a decrease in the content of fucosylated xyloglucans in compared with wild-type plants. Moreover, the callose content in the apical walls of pollen tubes increased in the mutant. In conclusion, we propose that LRX8-11 function synergistically to maintain pollen tube cell wall integrity; thus, they play critical roles in pollen germination and pollen tube growth.

The coinhibitory molecules, B7-H3 and B7-H4, have shown negative regulation in T cell activation and tumor-associated macrophage (TAM) polarization in tumor-specific immunity. Here, we investigated the expression of B7-H3 and B7-H4 in human and murine esophageal squamous cell carcinoma (ESCC) tissues to define their clinical significance and mechanism in a tumor microenvironment. In the present study, B7-H3 and B7-H4 were expressed in 90.6 and 92.7 % samples, respectively. High B7-H3 and B7-H4 expression was associated with advanced TNM stage and lymph node metastasis (p < 0.05, respectively). Patients with both B7-H3 and B7-H4 high-expressed tumors had the poorest prognosis (26.7 months), whereas those with both low-expressed tumors had the best survival (56.7 months). B7-H3 and B7-H4 expression were inclined to be positively related to the infiltration intensity of Treg cells and TAMs (p < 0.05, respectively), and B7-H3 expression is negatively associated with the intensity of CD8(+) T cells (p < 0.05). In 4-nitroquinoline 1-oxide (4-NQO)-induced murine models, high B7-H3 expression could only be detected at carcinoma stage, but abnormal B7-H4 expression appeared a little earlier at dysplasia stage. In vitro studies revealed that knockdown of B7-H3 on tumor cells suppressed ESCC cell migration and invasion, while knockdown of B7-H4 could inhibit ESCC cell growth. Overall, B7-H3 and B7-H4 are involved in ESCC progression and development and their coexpression could be valuable prognostic indicators. Interference of these negative regulatory molecules might be a new strategy for treating ESCC.

SUMMARY Pollen tube integrity, growth and guidance are crucial factors in plant sexual reproduction. Members of the plant Skewed5 (SKU5) Similar (SKS) family show strong similarity to multicopper oxidases (MCOs), but they lack conserved histidines in MCO active sites. The functions of most SKS family members are unknown. Here, we show that Arabidopsis pollen‐expressed SKS11 and SKS12 play important roles in pollen tube integrity, growth and guidance. The sks11sks12 mutant exhibited significantly reduced male fertility. Most of the pollen from sks11sks12 plants burst when germinated, and the pollen tubes grew slowly and exhibited defective growth along the funiculus and micropyle. SKS11‐GFP and SKS12‐mCherry were detected at the cell wall in pollen tubes. The contents of several cell wall polysaccharides and arabinogalactans were decreased in the pollen tube cell walls of sks11sks12 plants. Staining with a reactive oxygen species (ROS)‐sensitive dye and use of the H2O2 sensor HyPer revealed that the ROS content in the pollen tubes of sks11sks12 plants was remarkably reduced. SKS11444His‐Ala, in which the last conserved histidine was mutated, could restore the mutant phenotypes of sks11sks12. Thus, SKS11/12 are required for pollen tube integrity, growth and guidance possibly by regulating the ROS level and cell wall polysaccharide deposition or remodeling in pollen tubes.

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) have been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like “on-target, off-tumor” toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.Methods: First we fused anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures with intracellular domains, respectively. Using non-viral minicircle DNA (mcDNA) vectors to generate co-specific CAR T cells (CoG133-CAR T cells) against CD133 and GPC3 double-positive HCC cells. We exhibited the transduction efficiency of CoG133-CAR T cells and the antigen expression of tumor cell lines. Finally, the antitumor efficacy of CoG133-CAR T cells both in vitro and in vivo was detected. Results: GPC3-CAR and CD133-CAR were successfully prepared using non-viral mcDNA vectors to generate effector cells. For the GPC3 and CD133 double-positive HCC (Huh7) xenograft mice, co-specific CAR T cells possessed stronger tumor growth suppression compared to single-targeted CAR (GPC3-CAR and CD133-CAR) T cells which induced only one antigen-mediated signal pathway. The same results also occurred on the in vitro experiments including cytokine secretion, cytotoxicity and proliferation ability of CAR T cells. Vital organs from CoG133-CAR T cells and normal T cells respectively treated Huh7 xenograft mice were stained by hematoxylin and eosin (H&E), the images showed no difference. Conclusions: The mcDNA vectors loading CAR structures were transfected into T cells by electroporation without genetic mutation or mismatch. Huh7 is an HCC cell line with two antigens of GPC3 and CD133 highly expressed. The antitumor efficacy of co-specific CAR (CoG133-CAR) T cells against Huh7 cells is significantly enhanced. The joint design of two specific targets and non-viral vectors leads much more safety, also.