Characterization of merozoite-specific thrombospondin-related anonymous protein (MTRAP) in Plasmodium vivax and P. knowlesi parasites.

Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.

Nobiletin Inhibits Non-Small-Cell Lung Cancer by Inactivating WNT/ß-Catenin Signaling through Downregulating miR-15-5p.

BACKGROUND: Nobiletin is a natural compound with anticancer activity; however, the mechanism is not clear. METHODS: The inhibitory effect of nobiletin on non-small-cell lung cancer (NSCLC) cells was examined using soft agar, Transwell, and apoptosis analyses. Cancer stemness was measured by sphere assay. Genes and miRNAs regulated by nobiletin were identified by whole-genome sequencing. Protein levels were detected by western blot and immunofluorescence assays. RESULTS: Nobiletin significantly inhibited NSCLC cell colony formation and sphere formation and induced apoptosis. Nobiletin upregulated negative regulators of WNT/ß-catenin signaling, including NKD1, AXIN2, and WIF1, while it inhibited the expression of ß-catenin and its downstream genes, including c-Myc, c-Jun, and cyclin D1. Furthermore, we identified that GN inhibits miR-15-5p expression in NSCLC cells and that NKD1, AXIN2, and WIF1 are the target genes of miR-15-5p. CONCLUSIONS: Nobiletin has a strong inhibitory effect on NSCLC, and nobiletin plays an anticancer role by inhibiting miR-15-5p/ß-catenin signaling in NSCLC.

The C-reactive protein to albumin ratio predicts postoperative complication in patients who undergo gastrectomy for gastric cancer.

The aim of this study was to evaluate the preoperative C-reactive protein (CRP) to albumin ratio (CAR) of patients with gastric cancer and to investigate the factors correlated with perioperative complications. From March 2016 to December 2019, 128 patients who underwent curative gastrectomy for gastric cancer were enrolled in a retrospective study. The preoperative cutoff value of the CAR for predicting postoperative complications was 0.265 on receiver operating characteristic (ROC) curve analysis. Clinical characteristics were compared between patients with complications (Clavien-Dindo grade ≥2, n = 20) and without complications (Clavien-Dindo grade <2, n = 108). On univariate and multivariate analyses, estimated blood loss (EBL) during the operation (HR 1.003, p = 0.039) and CAR (HR 2.832, p = 0.045) were independent predictors of postoperative complications. In conclusion, preoperative CAR appears to be a predictor of postoperative complications in the patients undergoing surgical treatment of gastric cancer.

miR-200b/200a/429 Cluster Stimulates Ovarian Cancer Development by Targeting ING5.

Ovarian cancer is the second most common gynaecological malignancy, and microRNAs (miRNAs) play important role in the cancer development. Here, we found that the level of miR-200b/200a/429 was significantly increased in serum and tumor tissues of patients with stage-I ovarian cancer. Consistent with these results, we detected increased expression levels of miR-200b/200a/429 in ovarian cancer cell lines compared with the human nontumorigenic ovarian epithelial cell line T80. The overexpression of miR-200b/200a/429 in T80 cells stimulated proliferation and caused their growth in soft agar and tumor formation in nude mice. Furthermore, we determined that miR-200b/200a/429 targets inhibitor of growth family 5 (ING5) and that the overexpression of ING5 can block miR-200b/200a/429-induced T80 cell transformation and tumorigenesis. Our findings suggest that miR-200b/200a/429 may be a useful biomarker for the early detection of ovarian cancer and that miR-200b/200a/429 significantly contributes to ovarian cancer development through ING5.

Expression of Heat Shock Proteins and Cytokines in Response to Ethanol Induced Damage in the Small Intestine of ICR Mice.

BACKGROUND/AIMS: Ethanol administration causes intestinal epithelial cell damage by increasing intestinal permeability and the translocation of endotoxins from intestinal bacterial flora. Heat shock proteins (HSPs) are associated with recovery and protection from cell damage. The aim of the current study was to investigate differences in the expression of HSPs in the small intestine and the biochemical changes attributable to ethanol-induced intestinal damage. METHODS: Ethanol (20%) was injected intraperitoneally (2.75 g/kg, 5.5 g/kg, 8.25 g/kg) in ICR mice and the same volume of saline was administered to controls. After 1 hour, the proximal, middle, and distal segments were taken from the small intestine and the degree of damage was analyzed. In each segment, the expression of HSPs was analyzed by western blotting. The expression of inflammatory mediators including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and antioxidant enzyme such as glutathione-S-transferase were compared using real-time polymerase chain reaction assays. RESULTS: In the control group, HSP70 increased in all segments of small intestine. Additionally, increases in the expression of HSP40 and HSP90 in the distal regions and an increase in HSP32 in the middle regions were observed. After ethanol treatment, greater histological damage was observed in the distal small intestine and significant decreases in HSPs were observed generally. Increased expression of IL-1ß, TNF-α, and COX-2 was observed in small intestinal tissues exposed to ethanol-induced damage. However, there was no significant difference in the expression of an antioxidant enzyme. CONCLUSIONS: Significant differences in the expression of HSPs in different intestinal regions were observed. These differences may have been attributable to the distribution of intestinal bacteria.

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase mediate double-strand RNA-induced inducible nitric oxide synthase expression in microglial cells.

Double-stranded RNA (dsRNA) has been implicated as a potential immune stimulant in activating microglia, which can cause chronic neurodegeneration. In this study, we examined the involvement of different types of mitogen-activated protein kinases (MAPKs) in the induction of inducible nitric oxide synthase (iNOS) by dsRNA in microglial cells. Nitric oxide production was increased after exposure of microglia to 50mug/mL dsRNA. Levels of dsRNA-induced nitrite production in a line of immortalized murine microglia (BV2) and in primary cultures of murine microglia were decreased by inhibition of JNK or p38 MAPK, but were increased by inhibition of extracellular signal-regulated kinase. Similar results were shown in the levels of dsRNA-induced iNOS gene expression in BV2 cells. Phosphorylation levels of p38 MAPK were increased, depending on p38 MAPK inhibitor concentrations, while activation levels of MAPKAPK2, a known p38 substrate, were inhibited. Thus, it is likely that SB203580 inhibited the kinase activity of p38 MAPK, resulting in the loss of a feedback inhibition regulatory loop of p38 MAPK in BV2 cells. These findings suggest that dsRNA stimulated iNOS expression via MAPK signaling pathways, including JNK and p38 MAPK.

Differences between lipopolysaccharide and double-stranded RNA in innate immune responses of BV2 microglial cells.

Microglial cells are thought to be major inflammatory cells in the central nervous system; however, sufficient information about the effects of double-stranded RNA (dsRNA) in microglial cells is lacking. The present study compared the innate immune responses of the murine microglial cell line BV2 to dsRNA and lipopolysaccharide (LPS). It showed that the effect of dsRNA was similar to that of LPS treatment. The dsRNA induced several pro-inflammatory factors such as TNF-alpha, IL-6, IL-1beta, and IL-1Ra. Furthermore, the expression level of COX-2 was increased after treatment with dsRNA. However, the induction level of IL-1beta by dsRNA was less than those of the other cytokines that were measured. These results suggest that, although both dsRNA and LPS trigger pro-inflammatory responses, the intracellular signaling pathway and inflammation pattern of dsRNA and LPS may be different. Therefore, dsRNA produced during viral infection could precipitate neurological abnormalities through chronic inflammation.

Celecoxib attenuates 5-fluorouracil-induced apoptosis in HCT-15 and HT-29 human colon cancer cells.

AIM: To investigate the combined chemotherapeutic effects of celecoxib when used with 5-FU in vitro. METHODS: Two human colon cancer cell lines (HCT-15 and HT-29) were treated with 5-FU and celecoxib, alone and in combination. The effects of each drug were evaluated using the MTT [3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay, flow cytometry, and western blotting. RESULTS: 5-FU and celecoxib showed a dose-dependent cytotoxic effect. When treated with 10(-3) mol/L 5-FU (IC(50)) and celecoxib with its concentration ranging from 10(-8) mol/L to 10(-4) mol/L of celecoxib, cells showed reduced cytotoxic effect than 5-FU (10(-3) mol/L) alone. Flow cytometry showed that celecoxib attenuated 5-FU induced accumulation of cells at subG1 phase. Western blot analyses for caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage showed that celecoxib attenuated 5-FU induced apoptosis. Western blot analyses for cell cycle molecules showed that G2/M arrest might be possible cause of 5-FU induced apoptosis and celecoxib attenuated 5-FU induced apoptosis via blocking of cell cycle progression to the G2/M phase, causing an accumulation of cells at the G1/S phase. CONCLUSION: We found that celecoxib attenuated cytotoxic effect of 5-FU. Celecoxib might act via inhibition of cell cycle progression, thus preventing apoptosis induced by 5-FU.