Three GPI-anchored alkaline phosphatases are involved in the intoxication of Cry1Ca toxin to Spodoptera exigua larvae.

Glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (ALP) isoforms are crucial for the intoxication of crystal proteins (Cry) to several insect species. We cloned five SeALPs from the larval midgut of Spodoptera exigua, a major pest of many crops. All five SeALPs contain a signal peptide at the N-terminus, a phosphatase domain, and a GPI-anchor site at the C-terminus. Additionally, the sequences encode two or three potential N-glycosylation sites. The five SeALPs were highly expressed at the larval stage, especially in the larval gut or Malpighian tubules. Ingestion over four consecutive days of double-stranded RNAs (dsRNAs) targeting SeALP1, SeALP2, SeALP3, SeALP4, and SeALP5 significantly reduced the corresponding mRNA levels by 60.0%, 40.0%, 65.6%, 48.1%, and 69.1% respectively, compared with the levels in control larvae that fed on non-specific dsRNA (dsEGFP). When larvae that previously ingested phosphate buffered saline (PBS)-, dsEGFP-, or five dsSeALPs-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities after six days were 70.0%, 71.8%, 49.1%, 54.9%, 65.3%, 52.5%, and 77.4%, respectively. ANOVA analysis revealed that the larvae that previously fed on the dsSeALP1-, dsSeALP2-, and dsSeALP4-overlaid diets had significantly lower mortalities than those that previously ingested the PBS-, dsEGFP-, dsSeALP3- and dsSeALP5-overlaid diets. The results suggest that SeALP1, SeALP2 and SeALP4 are involved in the intoxication of Cry1Ca to S. exigua larvae.

Ex vivo-expanded bone marrow stem cells home to the liver and ameliorate functional recovery in a mouse model of acute hepatic injury.

BACKGROUND: Stem cell transplantation provides a theoretical approach for liver regeneration medicine; it may promote liver regeneration and self-repair. However, the transplantation of bone marrow-mesenchymal stem cells expanded ex vivo as a therapy for liver disease has rarely been investigated. This study aimed to explore whether bone marrow stem cells expanded ex vivo home to the liver and foster hepatic recovery after CCl4 injury. METHODS: Bone marrow cells from BALB/c mice were expanded ex vivo by multiple-passage cultivation, characterized by cytoflow immunofluorescence, and pre-labeled with PKH26 before intravenous infusion into animals treated with CCl4. The integration of bone marrow cells into the liver was examined microscopically, and plasma hepatic enzymes were determined biochemically. RESULTS: Cultured bone marrow cells exhibited antigenic profiles comparable to those of primary medullary stem cells. Double immunofluorescence showed colocalization of these cells with proliferative activity and albumin expression in the liver of CCl4-treated mice. Densitometry showed increased in situ cell proliferation (50+/-14 vs 20+/-3 cells/high-power field, P<0.05) and albumin expression (149+/-25 vs 20+/-5 cells/high-power field, P<0.05) in the liver, as well as reduced serum aminotransferase levels (P<0.05) and better survival rates (P<0.05) in animals receiving cultured bone marrow cells relative to controls. CONCLUSIONS: Ex vivo-expanded bone marrow cells are capable of relocating to and proliferating in the chemically-injured liver. Transplantation of these pluripotent stem cells appears to improve serum indices of liver function and survival rate in mice after CCl4-induced hepatic damage.

Granulocyte colony-stimulating factor increases the therapeutic efficacy of bone marrow mononuclear cell transplantation in cerebral ischemia in mice.

BACKGROUND: Bone marrow mononuclear cell (BMMC) transplantation is a promising therapy for cerebral ischemia; however, little is known if its therapeutic efficacy may be improved by co-administration of potential modulatory factors in vivo. To explore this possibility, the present study examined the effect of BMMCs and G-CSF on cell proliferation, early neuronal development and neurological function recovery in experimental cerebral ischemia relative to controls that received neither treatment. RESULT: Ischemia/infarct area was significantly reduced in BMMCs+G-CSF group relative to animal groups treated with BMMCs only, G-CSF only or saline. Transplanted BMMCs were found to colocalize with the proliferative cell nuclear antigen (PCNA) and the immature neuronal marker doublecortin (DCX). The BMMCs+G-CSF group showed increased numerical density of cells expressing PCNA and DCX, improved performance in adhesive sticker removal test and reduced neurological function severity scores relative to other groups in a time-dependent manner. CONCLUSION: BMMCs and G-CSF co-administration exhibits synergistic beneficial effect over time. This effect could be at least partially related to increased proliferation and differentiation of bone marrow stem cells and enhanced host brain regeneration and functional recovery. The results suggest that G-CSF can increase the therapeutic efficacy of BMMCs transplantation in an experimental mouse model of cerebral ischemia.

[Promoter methylation of ASPP1 and ASPP2 genes in non-small cell lung cancers].

OBJECTIVE: To investigate the methylation status of CpG islands in the promoter region and the protein expression of ASPP1 and ASPP2 genes in non-small-cell lung cancer (NSCLC) and their relationship with cellular apoptosis and p53 gene expression. METHODS: The 5'CpG island methylation patterns of ASPP1 and ASPP2 were evaluated by methylation specific polymerase chain reaction (MSP) followed by confirmation of sequencing. Immunohistochemistry was used to detect the expression of ASPP1, ASPP2 and p53 in lung carcinoma tissue samples (n = 90) and adjacent non-neoplastic lung tissue samples (n = 25). TUNEL assay was used to detect the apoptotic activity. RESULTS: The presence of ASPP1 methylation was significantly higher in NSCLC than that in the adjacent non-neoplastic lung tissue [42.2% (38/90) vs. 16.0% (4/25), P = 0.019]. ASPP1 promoter methylation had a close relationship with TNM stage and lymph node metastasis (P = 0.031, P = 0.030), but was not related to the age, sex, histological types and the grades of tumor differentiation (P = 0.389, P = 0.278, P = 0.570, and P = 0.103). Tumors with ASPP1 promoter methylation demonstrated a lower expression of ASPP1 as compared with those without the methylation (P = 0.002). ASPP1 expression was associated with a higher apoptotic index (AI) (P = 0.022) and a decreased p53 expression (r = -0.259, P < 0.01). Methylation in the promoter region of ASPP2 gene was not detected in lung cancer (n = 90) or adjacent non-neoplastic lung tissue (n = 25). Expression of ASPP2 protein did not correlate with AI (P = 0.282) and p53 status in NSCLC. CONCLUSIONS: High methylation of ASPP1 gene promoter regions is one of the important mechanisms that down-regulate its protein expression in NSCLC. ASPP1 promoter methylation may be associated with the malignant progression of the tumor, and ASPP1 expression promotes cellular apoptosis.

The Yunnan national medicine Maytenus compound inhibits the proliferation of hepatocellular carcinoma (HCC) by suppressing the activation of the EGFR-PI3K-AKT signaling pathway.

Objective: To investigate the effects of Maytenus compound on the proliferation of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to explore the underlying mechanism. Methods: The half maximal inhibitory concentration (IC50) values of Maytenus compound in HepG2 and BEL-7402 cells were determined by the MTS assay. HepG2 and BEL-7402 cells were treated with different concentrations of Maytenus compound. MTS assays, colony formation assays and cell cycle analyses were performed to clarify the inhibitory effect of Maytenus compound on the proliferation of HepG2 and BEL-7402 cells in vitro. After subcutaneous injection of HepG2 cells, nude mice were randomly divided into a vehicle control group and a drug intervention group, which were intragastrically administered ddH2O or Maytenus compound, respectively. The inhibitory effect of Maytenus compound on the proliferation of HepG2 cells in vivo was analyzed using subcutaneous tumor growth curves, tumor weight, the tumor growth inhibition rate and the immunohistochemical detection of BrdU-labeled cells in S phase. The organ toxicity of Maytenus compound was initially evaluated by comparing the weight difference and organ index of the two groups of nude mice. The main proteins in the EGFR-PI3K-AKT signaling pathway were detected by Western blot after Maytenus compound intervention in vivo and in vitro. Results: Maytenus compound showed favorable antiproliferation activity against HepG2 and BEL-7402 cells with IC50 values of 79.42±11.71 µg/mL and 78.48±8.87 µg/mL, respectively. MTS assays, colony formation assays and cell cycle analyses showed that Maytenus compound at different concentration gradients within the IC50 concentration range significantly suppressed the proliferation of HepG2 and BEL-7402 cells in vitro and inhibited cell cycle progression from G1 to S phase. Additionally, Maytenus compound, at an oral dose of 2.45 g/kg, dramatically inhibited, without obvious organ toxicity, the proliferation of subcutaneous tumors formed by HepG2 cells in nude mice. In addition, the tumor growth inhibition rate for Maytenus compound was 66.94%. Furthermore, Maytenus compound inhibited the proliferation of liver orthotopic transplantation tumors in nude mice. Western blot analysis showed that Maytenus compound significantly downregulated the expression of p-EGFR, p-PI3K, and p-AKT and upregulated the expression of p-FOXO3a, p27, and p21 in vivo and in vitro. Conclusion: Maytenus compound significantly inhibited the proliferation of HCC cells in vitro and in vivo. The downregulation of the EGFR-PI3K-AKT signaling pathway and subsequent inhibition of cell cycle progression from G1 to S phase is one of the possible mechanisms. Maytenus compound has a high tumor growth inhibition rate and has no obvious organ toxicity, which may make it a potential anti-HCC drug, but the results from this study need to be confirmed by further clinical trials in HCC patients.

Switching from ticagrelor to clopidogrel in patients with ST-segment elevation myocardial infarction undergoing successful percutaneous coronary intervention in real-world China: Occurrences, reasons, and long-term clinical outcomes.

BACKGROUND: Although switching between ticagrelor and clopidogrel is common in clinical practice, the efficacy and safety of this de-escalation remain controversial. HYPOTHESIS: We assessed the occurrences, reasons, and outcomes of switching from ticagrelor to clopidogrel in patients with ST-segment elevation myocardial infarction (STEMI) undergoing successful primary percutaneous coronary intervention (PCI). METHODS: A total of 653 patients with STEMI were randomly assigned to receive loading dose of ticagrelor or clopidogrel before PCI and then received maintenance dose, respectively, for 12 months follow-up. The primary outcome was major adverse cardiac events (MACE), including cardiovascular death, nonfatal myocardial infarction, and stroke. The secondary outcome included unexpected rehospitalization for angina, coronary revascularization, and stent thrombosis. The safety outcome was bleeding described by the Bleeding Academic Research Consortium (BARC) criteria. RESULTS: A total of 602 participants completed the study. The rate of switching from ticagrelor to clopidogrel was 48.6% and the main reason was financial burden. The rate of secondary ischemic events in the de-escalation group was higher than that in the ticagrelor group (15.1% vs 5.6%, P = 0.008), but lower than that in the clopidogrel group (15.1% vs 24.6%, P = 0.03), while there were no significant differences in MACE among the three groups (P = 0.16). De-escalation, ticagrelor, and clopidogrel did not cause significant differences in the rates of major bleeding among the three groups (BARC ≥ 2, P = 0.34). CONCLUSION: Switching from ticagrelor to clopidogrel is very common in patients with STEMI in China. De-escalation might be safe but associated with high risk of ischemic events as compared to ticagrelor.

The Spodoptera exigua (Lepidoptera: Noctuidae) ABCC2 Mediates Cry1Ac Cytotoxicity and, in Conjunction with Cadherin, Contributes to Enhance Cry1Ca Toxicity in Sf9 Cells.

In insects, the mode of Cry1A toxins action has been studied in detail and many receptors that participate in the process are known. Recent evidence has revealed that an ABC transporter (ABCC2) is involved in conferring resistance to Cry1A toxins and that ABCC2 could be a receptor of Cry1A. However, it is not known whether Cry1Ca interacts with the same receptor proteins as Cry1A. In this study, we report the cloning of an ABC transporter gene, SeABCC2b, from the midgut of Spodoptera exigua (Hübner) larvae, and its expression in Sf9 cells for a functional analysis. The addition of Cry1Ca and Cry1Ac to Sf9 cell culture caused swelling in 28.5% and 93.9% of the SeABCC2-expressing cells, respectively. In contrast, only 7.4% and 1.3% of the controls cells swelled in the presence of Cry1Ca and Cry1Ac. Thus, SeABCC2b-expressing Sf9 cells had increased susceptibility to Cry1Ca and Cry1Ac. Similarly, S. exigua cadherin (SeCad1b) expressed in Sf9 cells caused 47.1% and 1.8% of the SeCad1b-expressing cells to swell to Cry1Ca and Cry1Ac exposure. Therefore, Sf9 cells expressing SeCad1b were more sensitive to Cry1Ca than Cry1Ac. Together, our data suggest that SeABCC2b from S. exigua mediates Cry1Ac cytotoxicity and, in conjunction with SeCad1b, contributes to enhance Cry1Ca toxicity in Sf9 cells.

Purification and identification of antioxidative peptides from dry-cured Xuanwei ham.

This study mainly focused on the purification and identification of antioxidative peptides generated in dry-cured Xuanwei ham. Based on scavenging effect on free radicals and ferrous ion, the antioxidant activity of crude peptides from Xuanwei ham was assessed. From the scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH) radicals and superoxide anion (O2(-)), it was suggested that XHP generated during the ripening period had a strong antioxidant activity. By using size exclusion chromatography, anion exchange column and reversed-phase HPLC, fractions with a strong antioxidative activity were separated based on their molecular weight and polarity differences. The fraction with strong antioxidant effect was further characterized by LC-MS/MS. The results suggest that antioxidative peptides are produced during the long processing of Xuanwei ham among which the tetrapeptide Asp-Leu-Glu-Glu could be one of the main peptides that play key role in the antioxidant activity.

A novel Wolbachia strain from the rice moth Corcyra cephalonica induces reproductive incompatibility in the whitefly Bemisia tabaci: sequence typing combined with phenotypic evidence.

Wolbachia are a group of maternally inherited bacteria frequently found in arthropods and filarial nematodes. They have recently attracted attention for their ecological roles in manipulating host reproduction, their potential use in biological control of pest insects and medical significance. Classification of Wolbachia strains is currently solely based on molecular methods. However, the strains even with identical sequence types may induce different host phenotypes. Here we isolated a Wolbachia strain from the rice moth Corcyra cephalonica (designated as wCcep_B_BJ), which was shown to share multilocus sequence typing and Wolbachia surface protein hypervariable region profiles with a cytoplasmic incompatibility (CI)-inducing strain in supergroup B, but the phenotype wCcep_B_BJ may induce needs to be determined. We thus transinfected it into the whitefly Bemisia tabaci harbouring an A-Wolbachia through nymphal microinjection. Fluorescent in situ hybridization demonstrated that wCcep_B_BJ was successfully transinfected into B. tabaci and transmitted to offspring through host eggs. Reciprocal cross showed that wCcep_B_BJ induced a strong bidirectional CI in the transinfected host without imposing a significant cost on female fecundity. Our results suggest that wCcep_B_BJ may be a promising strain for biocontrol of B. tabaci, an important agricultural pest insect.