Inhibition of Melanoma Growth by Subcutaneous Administration of hTERTC27 Viral Cocktail in C57BL/6 Mice

hTERTC27 is a 27 kDa C-terminal polypeptide of human telomerase reverse transcriptase that has previously been shown to reduce tumorigenicity of HeLa cells and suppress growth of xenografted glioblastoma in nude mice. Although ectopic expression of hTERTC27 upregulated genes that are involved in apoptosis, cell cycle, and immune response, the mechanism for hTERTC27-induced tumor suppression has not been completely elucidated. Since hTERT was identified as a universal tumor-associated antigen, we hypothesize that hTERTC27 inhibits tumor growth in vivo through activation of anti-tumor immune response. Immunocompetent C57BL/6 mice were used for mouse B16 melanoma model. Mice bearing B16 melanoma were administered rAAV-/rAdv viral cocktail expressing hTERTC27, and tumor growth was monitored after viral cocktail treatment. Blood and splenocytes were used to determine the level of cytokines and the activity of immune cells, respectively. B16 tumor growth was significantly inhibited by subcutaneous administration of a single dose of 1.5×10(11) vg rAAV-hTERTC27 and 2.5×10(9) pfu rAdv-hTERTC27 viral cocktail (rAAV-/rAdv-hTERTC27). The population and cytotoxicity of NK cells in the mice were significantly augmented by rAAV-/rAdv-hTERTC27 treatment, and selective depletion of the NK cell population in mice by intraperitoneal injection of anti-GM1 antibody abrogated the growth suppression of melanoma induced by rAAV-/rAdv-hTERTC27 administration. Activation of NK cells by administration of rAAV-/rAdv-hTERTC27 is critical for growth suppression of melanoma in mouse model.published_or_final_versio

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors

Molecular cloning and functional studies of cyprinid calmodulin

tocabstractpublished_or_final_versionZoologyDoctoralDoctor of Philosoph

Platform pricing and blockchain adoption for capacity sharing with cross-network externality and supply risk

Platform-based capacity sharing is a new business format of sharing economy to reallocate spare production resources via industrial internet, generating collaborative manufacturing related platform operations issues. This paper examines the impact of cross-network externality and supply risk of the two-sided market on the pricing and blockchain adopting decisions of capacity sharing platforms. The mean–variance model is employed to depict the impact of purchasers’ or suppliers’ risk-sensitive types on platform pricing, where purchasers’ or suppliers’ risk-sensitive types can be identified by blockchain technology and be intertwined by the cross-network externality. The obtained platform’s optimal pricing under two scenarios with risk-sensitive capacity purchasers (D) and risk-sensitive suppliers (S) shows that the platform charges risk-seeking, risk-neutral and risk-averse users in descending order for the platform side containing risk-sensitive users, regardless of the comparison of bilateral cross-network externalities. However, for risk-neutral side users, the price ranking under three risk-sensitive types depends on the relative size of bilateral cross-network externalities. In addition, blockchain technology always decreases the surplus of risk-sensitive side users, whereas it increases the surplus of risk-insensitive side users. Further, we find that platform prefers to introduce blockchain technology when the fixed cost of introducing it is less than the profit increment it brings. In addition, by comparing platform profits and blockchain value in the two scenarios, we argue that platforms need to comprehensively balance the comparison of fixed costs of introducing blockchain on both sides and the importance of risk-preferring and risk-averse users to improve their profitability. Introducing blockchain technology to the side where the cost of blockchain technology is higher may result in higher blockchain value for the platform. Finally, we examine the platform with two-sided risk-sensitive users and the results are robust. This study proposes theoretical explanation for platform pricing considering the cross-network externality and risk-sensitive users, as well as some management insights for the application of blockchain technology in platform operations

Predicting the comprehensive geospatial pattern of two ephedrine-type alkaloids for Ephedra sinica in Inner Mongolia.

Ephedra sinica Stapf. is a shrubby plant widely used in traditional Chinese medicine due to its high level of medicinal value, thus, it is in high demand. Ephedrine (E) and pseudoephedrine (PE) are key medicinal components and quality indicators for E. sinica. These two ephedrine-type alkaloids are basic elements that exert the medicinal effect of E. sinica. Recently, indiscriminate destruction and grassland desertification have caused the quantity and quality of these pharmacological plants to degenerate. Predicting potentially suitable habitat for high-quality E. sinica is essential for its future conservation and domestication. In this study, MaxEnt software was utilized to map suitable habitats for E. sinica in Inner Mongolia based on occurrence data and a set of variables related to climate, soil, topography and human impact. The model parametrization was optimized by evaluating alternative combinations of feature classes and values of the regularization multiplier. Second, a geospatial quality model was fitted to relate E and PE contents to the same environmental variables and to predict their spatial patterns across the study area. Outputs from the two models were finally coupled to map areas predicted to have both suitable conditions for E. sinica and high alkaloid content. Our results indicate that E. sinica with high-quality E content was mainly distributed in the Horqin, Ulan Butong and Wulanchabu grasslands. E. sinica with high-quality PE content was primarily found in the Ordos, Wulanchabu and Ulan Butong grasslands. This study provides scientific information for the protection and sustainable utilization of E. sinica. It can also help to control and prevent desertification in Inner Mongolia

rAAV-/rAdv-hTERTC27 treatment increases plasma levels of Th1 cytokines.

<p>Plasma from sacrificed mice on day 20 post tumor cell inoculation were analyzed to determine cytokine levels using mouse Th1/Th2 FlowCytomix assay kit. The numerical values presented with the bar graph indicate the fold differences of cytokine levels between mice treated with hTERTC27 and EGFP.</p

rAAV-/rAdv-hTERTC27 treatment increases the population and cytotoxicity of NK cells.

<p>(A) & (B) Flow cytometric analysis of PMBC from tumor-bearing mice treated with subcutaneous administration of rAAV-/rAdv-hTERTC27, rAAV-/rAdv-EGFP, or PBS. Data shown here represent one of several mice analyzed with similar results. (C) Lymphocyte profiles from A & B (data pooled from 3 mice/group treated with indicated viruses or PBS). *<i>P<0.05</i>, compared with EGFP group. (D) and (E): Cytotoxic activity of spleen NK cells isolated from mice treated with rAAV-/rAdv-hTERTC27, rAAV-/rAdv-EGFP or PBS was analyzed against YAC-1 (D) or B16 (E) cells. Lymphocytes isolated from each mouse were cultured with YAC-1 or B16 cells at various effector to target ratios (E/T). *: <i>P<0.05</i>, compared with EGFP group. (F): Profiles of NKT (NK1.1⁺/CD3⁺), NK (NK1.1⁺/CD3⁻) and T (NK1.1⁻/CD3⁺) cells in PMBC from tumor-bearing mice treated with rAAV-/rAdv-hTERTC27 or rAAV-/rAdv-EGFP. The numerical value presented with the bar graph indicates the percentage of the cell population in PMBC. The cell population without any marker (i.e. NK1.1⁻/CD3⁻) is not shown. Data pooled from 3 mice/group treated with indicated virus through flow cytometry analysis. *: <i>P<0.05</i>, compared with EGFP group.</p

rAAV-/rAdv-hTERTC27 treatment has different effects on the activities of T cells and B cells.

<p>(A) Proliferation activity of T cells and B cells isolated from mice treated with hTERTC27, EGFP or PBS. Data are expressed as means ± SD (n = 3). *: <i>P</i><0.05, compared with EGFP group. (B) Cytotoxicity of T cells isolated from mice treated with hTERTC27, EGFP, or PBS. *: <i>P</i><0.05, compared with EGFP group.</p