Sq-2, a biotinylated annonaceous acetogenin, induces apoptosis, autophagy and S-phase arrest by activating the MAPK pathway in breast cancer cells.

Squamocin, an annonaceous acetogenin isolated from plants in the Annonaceae family, has antitumour activity. In this study, we report that Sq-2, a biotinylated squamocin monomer, has a favorable antitumour effect on MDA-MB-231 and SKBR3 breast cancer cells in vitro. MTT assays show that Sq-2 has a better antitumour effect on MDA-MB-231 cells than Sq-5 and Sq-6. Furthermore, RNA-Seq and KEGG enrichment analyses reveal that Sq-2 activates the MAPK signaling pathway, and results of western blot analysis demonstrate that Sq-2 activates the JNK and p38 pathways in MDA-MB-231 and SKBR3 cells. Flow cytometry and western blot analysis reveal that Sq-2 induces cell apoptosis by increasing the expressions of cleaved Caspase-3 and cleaved PARP as well as the ratio of Bax/Bcl-2. Inhibition of the Caspase family by Z-VAD-FMK attenuates the viability of MDA-MB-231 cells, indicating that Sq-2 induces apoptosis in a Caspase-dependent manner. Additionally, pretreatment with the p38 inhibitor SB203580 or JNK inhibitor SP600125 partially reverses the increase in the apoptosis rate and decrease in cell viability prompted by Sq-2. Furthermore, Sq-2 treatment decreases the expression level of CyclinD1 and increases the expression levels of p21, p27, CyclinA1, and CDK2, causing S-phase arrest in MDA-MB-231 and SKBR3 cells. Further study indicates that Sq-2 stimulates autophagy in MDA-MB-231 and SKBR3 cells, and inhibition of autophagy by bafilomycin A1 increases cell viability and promotes cell survival. Sq-2, a novel biotin-squamocin compound, shows a significant inhibitory effect on the propagation of SKBR3 and MDA-MB-231 breast cancer cells. Furthermore, Sq-2 treatment not only induces S-phase arrest and activates the JNK and p38 pathways to trigger apoptosis but also causes autophagy to promote apoptosis in MDA-MB-231 and SKBR3 cells.

A recombinant influenza virus with a CTLA4-specific scFv inhibits tumor growth in a mouse model.

Oncolytic viruses (OV) have shown excellent safety and efficacy in preclinical and clinical studies. Influenza A virus (IAV) is considered a promising oncolytic virus. In this report, we generated a recombinant influenza virus expressing an immune checkpoint blockade agent targeting CTLA4. Using reverse genetics, a recombinant influenza virus, termed rFlu-CTLA4, encoding the heavy chain of a CTLA4 antibody on the PB1 segment and the light chain of the CTLA4 antibody on the PA segment was produced. RFlu-CTLA4 could replicate to high titers, and antibodies were produced in the allantoic fluid of infected eggs. Furthermore, the selective cytotoxicity of the virus was higher in various hepatocellular carcinoma cancer cell lines than in the normal cell line L02 in vitro, as indicated by MTS assays. More importantly, in a subcutaneous H22 mouse hepatocarcinoma model, intratumoral injections of rFlu-CTLA4 inhibited the growth of treated tumors and increased the overall survival of mice compared with injections of the PR8 virus. Taken together, these results warrant further exploration of this novel recombinant influenza virus for its potential use as a single or combination agent for cancer immunotherapy.

[Application and evaluation of loop-mediated isothermal amplification method for detecting of Listeria monocytogenes in food].

OBJECTIVE: The loop-mediated isothermal amplification (LAMP) detection method was applied to detect Listeria monocytogenes in food. The specificity and sensitivity of this method were evaluated through comparing it with Real-time PCR and conventional detection method. METHODS: The LAMP primers were designed based on hly gene of Listeria monocytogenes. The LAMP method was applied to detect 88 Listeria monocytogenes, 1 reference strain ATCC 15313 of Listeria monocytogenes and 33 non-targets bacteria strains; base-material addition test and practical food samples detection were also conducted. Both of Real-time PCR and ISO 11290-1 methods were used as parallel detection method in addition to LAMP. The three kinds of methods were compared by specificity, sensitivity, detection limit and the detection result of practical food samples. RESULTS: Both detection results of LAMP and Real-time PCR for 89 Listeria monocytogenes were positive (100%, 89/89), 33 non-targets bacteria strains were negative (100%, 33/33). The sensitivity of LAMP was 2 × 10² CFU/ml, which was consistent with Real-time PCR method (2 × 10² CFU/ml) and better than ISO 11290-1 method (2 × 10² CFU/ml). Base-material addition test result showed that the detection limit of the three kinds of methods were 3 CFU/25 g samples. And the result of practical food samples displayed the same detection rate of 4% in the three methods (2/45). CONCLUSION: The LAMP method of Listeria monocytogenes established in this study has good specificity and sensitivity, which can be applied to the rapid detection of Listeria monocytogenes.

[The establishment of a novel method of nano-immunomagnetic separation and Real-time PCR for detecting Vibrio cholerae from seafood].

OBJECTIVE: A novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae. METHODS: The Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156. RESULTS: The capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment. CONCLUSIONS: The Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.

An Engineered Influenza a Virus Expressing the Co-Stimulator OX40L as an Oncolytic Agent Against Hepatocellular Carcinoma.

Background: Oncolytic virus (OV) therapy has emerged as a promising novel form of immunotherapy. Moreover, an increasing number of studies have shown that the therapeutic efficacy of OV can be further improved by arming OVs with immune-stimulating molecules. Methods: In this study, we used reverse genetics to produce a novel influenza A virus, termed IAV-OX40L, which contained the immune-stimulating molecule OX40L gene in the influenza virus nonstructural (NS1) protein gene. The oncolytic effect of IAV-OX40L was explored on hepatocellular carcinoma (HCC)HCC cells in vitro and in vivo. Results: Hemagglutination titers of the IAV-OX40L virus were stably 27-28 in specific-pathogen-free chicken embryos. The morphology and size distribution of IAV-OX40L are similar to those of the wild-type influenza. Expression of OX40L protein was confirmed by Western blot and immunofluorescence. MTS assays showed that the cytotoxicity of IAV-OX40L was higher in HCC cells (HepG2 and Huh7) than in normal liver cells (MIHA) in a time- and dose-dependent manner in vitro. We found that intratumoral injection of IAV-OX40L reduced tumor growth and increased the survival rate of mice compared with PR8-treated controls in vivo. In addition, the pathological results showed that IAV-OX40L selectively destroyed tumor tissues without harming liver and lung tissues. CD4+ and CD8+ T cells of the IAV-OX40L group were significantly increased in the splenic lymphocytes of mice. Further validation confirmed that IAV-OX40L enhanced the immune response mainly by activating Th1-dominant immune cells, releasing interferon-γ and interleukin-2. Conclusion: Taken together, our findings demonstrate the novel chimeric influenza OV could provide a potential therapeutic strategy for combating HCC and improve the effectiveness of virotherapy for cancer therapy.

A Recombinant Oncolytic Influenza Virus Carrying GV1001 Triggers an Antitumor Immune Response.

Oncolytic viruses are able to lyse tumor cells selectively in the liver without killing normal hepatocytes, in addition to activating the immune response. Oncolytic virus therapy is expected to revolutionize the treatment of liver cancer, including hepatocellular carcinoma (HCC), one of the most frequent and fatal malignancies. In this study, reverse genetics techniques were exploited to load NA fragments of the A/PuertoRico/8/34 virus (PR8) with GV1001 peptides derived from human telomerase reverse transcriptase. An in vitro assessment of the therapeutic effect of the recombinant oncolytic virus was followed by an in vivo study in mice with HCC. The recombinant virus was verified by sequencing of the recombinant viral gene sequence, and viral virulence was detected by hemagglutination assays and based on the 50% tissue culture infectious dose (TCID50). The morphological structure of the virus was observed by electron microscopy, and GV1001 peptide was localized by cellular immunofluorescence. The selective cytotoxicity of the recombinant oncolytic virus in vitro was demonstrated in cultured HCC cells and normal hepatocytes, as only the tumor cells were killed; the normal cells were not significantly altered. Consistent with the in vitro results, the recombinant oncolytic influenza virus significantly inhibited liver tumor growth in mice in vivo, in addition to inducing an antitumor immune response, including an increase in the number of CD4+ and CD8+ T lymphocytes and, in turn, improving survival. Our results suggest that oncolytic influenza virus carrying GV1001 is a promising immunotherapy in patients with HCC.

Oncolytic Activity of a Chimeric Influenza A Virus Carrying a Human CTLA4 Antibody in Hepatocellular Carcinoma.

Oncolytic virotherapy belongs to a kind of active immunotherapy, which could trigger a potent antitumor immune response, showing great potential in clinical application. OVs could induce immune responses through the dual mechanisms of selective tumor killing without destroying normal tissues and induction of systemic antitumor immunity. In this study, we successfully rescued a chimeric oncolytic influenza virus carrying a human CTLA4 antibody in the background of the A/PR/8/34 (PR8) virus. The chimeric virus, called rFlu-huCTLA4, contained the heavy and light chains of the human CTLA4 antibody in the PB1 and PA segments of the PR8 virus, respectively. The first-generation hemagglutination (HA) titers of the rFlu-huCTLA4 virus ranged from 27 to 28, which could be passaged stably in specific pathogen-free (SPF) chicken embryos from P1 to P5. The morphology and size distribution of the chimeric virus were consistent with those of the wt influenza virus. The rFlu-huCTLA4 virus could effectively replicate in various cells in time- and dose-dependent manners. ELISA assay revealed that the secreted huCTLA4 antibody levels in chicken embryos increased gradually over time. Furthermore, MTS and crystal violet analysis showed that the selective cytotoxicity of the virus was higher in hepatocellular carcinoma cells (HepG2 and Huh7) than in normal liver cells (MIHA). In vivo experiments displayed that intratumoral injection with rFlu-huCTLA4 reduced tumor growth and increased the survival of mice compared with the PR8 group. More importantly, in the rFlu-huCTLA4 group, we found that CD4+ and CD8 +T cells were significantly increased in tumor-bearing BALB/c mice. Taken together, these findings demonstrated that the chimeric oncolytic virus rFlu-huCTLA4 could selectively destroy hepatocellular carcinoma cells in vitro and in vivo and may provide a promising clinical strategy for targeted immunotherapy of HCC with the oncolytic flu virus.

[The study on the value of using the chronic obstructive pulmonary disease and asthma physiology score to assess the severity of acute exacerbation in patients with chronic obstructive pulmonary disease complicated by type II respiratory failure].

OBJECTIVE: To explore the value of the chronic obstructive pulmonary disease (COPD) and asthma physiology score (CAPS) in evaluating the severity and prognosis of patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated by type II respiratory failure. METHODS: Eighty-two cases with AECOPD complicated by type II respiratory failure between January 2005 and March 2009 were retrospectively analyzed. The severity in survivors and non-survivors was evaluated by CAPS and acute physiology and chronic health evaluation system (APACHE II score, APACHE III score), and retrospective and statistical analyses of all data were performed. RESULTS: CAPS, APACHE II score, APACHE III score, duration of invasive positive pressure ventilation (IPPV) and days in intensive care unit of 19 cases in the death group were 34.21+/-9.89, 22.53+/-7.49, 75.11+/-18.07, (25.06+/-24.64) days, (32.42+/-25.49) days , respectively, while 63 cases of the survival group were 27.41+/-8.15, 18.65+/-5.34, 64.11+/-15.92, (5.23+/-5.50) days, (12.51+/-20.70) days, respectively, and there were significant differences between two groups (P<0.05 or P<0.01). The areas under receiver operating characteristic (ROC) curves of CAPS, APACHE II score and APACHE III score were 0.712 (P=0.005), 0.654 (P=0.043) and 0.655 (P=0.042), respectively. When CAPS score was 30.5, Youden index was the highest (0.435). The mortality rate had a positive correlation with CAPS. When the CAPS score was over 30, there was a tendency of increase in mortality rate. CONCLUSION: CAPS is very useful to evaluate the severity and prognosis of patients with AECOPD complicated by type II respiratory failure. It is easy to perform, and better than APACHE II and APACHE III.

Active Monomer RTR-1 Derived from the Root of Rhodomyrtus t omentosa Induces Apoptosis in Gastric Carcinoma Cells by Inducing ER Stress and Inhibiting the STAT3 Signaling Pathway.

PURPOSE: Rhodomyrtus tomentosa, a flowering plant from the Myrtaceae family, is considered  an antitumour substance with versatile biological and pharmacological activities. RTR-1 is an active monomer purified from the root of Rhodomyrtus tomentosa. However, the detail of mechanism involving in RTR-1 anti-cancer activity remains to be elucidated, and the effect on gastric cancer cells is unknown. METHODS: Cell proliferation was determined by MTT and clone formation assay. The effect of RTR-1 on cell cycle distribution and apoptosis was analysed utilizing flow cytometry, respectively. Moreover, Western blotting was used to detect the expression of cell cycle- and apoptosis-related protein. RESULTS: Based on MTT and clone formation assay, we noticed that RTR-1 inhibited the proliferation of gastric carcinoma (BGC823 and SGC7901) cells in a dose- and time-dependent manner. Furthermore, the results of flow cytometry and Western blotting showed that RTR-1 induced cell cycle arrest in the G2/M phase through the ATM-Chk2-p53-p21 signaling pathway and induced cell apoptosis by inhibiting the signal transducers and activators of transcription 3 (STAT3) pathway and activating the endoplasmic reticulum stress (ER stress) pathway. CONCLUSION: Taken together, these results demonstrate that RTR-1 induces cell cycle arrest and promotes apoptosis in gastric carcinoma, indicating its potential application for gastric cancer therapy.

Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loop-mediated isothermal amplification.

The objective of this study was to develop a method that combined nanoparticle-based immunomagnetic separation (IMS) with real-time loop-mediated isothermal amplification (LAMP) for the rapid detection of Vibrio parahaemolyticus. Magnetic nanoparticles were functionalized with monoclonal antibodies that were produced against flagella from V. parahaemolyticus to capture and separate the target cells from raw oysters. After optimization, the immunomagnetic nanoparticles (IMNPs) presented a capture efficiency of 87.3% for 10(5) colony-forming unit (CFU)/mL of V. parahaemolyticus using 2.5µg of IMNPs within 30min. Although a very low level of non-specific binding was seen among 8 non-V. parahaemolyticus Vibrio spp. and 5 non-Vibrio strains, the IMS-LAMP method identified 133 V. parahaemolyticus strains correctly without the amplification from 54 other strains. The detection limit was about 1.4×10(2)CFU/mL in pure culture and was unaffected by the presence of 10(8)CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9×10(3)CFU/g without enrichment. After enrichment for 6-8h, the limit of detectability could be improved to 1.9 to 0.19CFU/g. Hence, the IMS-LAMP assay provided a rapid, simple, and cost-effective method for total V. parahaemolyticus detection. This method will have important implications in the rapid detection of contaminated food in the early stage before distribution.

[Preparation of monoclonal antibodies against flagellin core protein of Vibrio cholerae and its application in establishing double-antibody sandwich ELISA for testing Vibrio cholerae from food products].

OBJECTIVE: To prepare the monoclonal antibodies (mAbs) against flagellin core protein of Vibrio cholerae and establish the double-antibody sandwich ELISA method for testing Vibrio cholerae from food products. METHODS: BALB/c mice were immunized with flagellin extracted from Vibrio cholerae Vc75 by differential centrifugation. The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells when the antibody titer in serum reached 1:32 000. The hybridoma cell lines were obtained by regular subcloning and used to generate ascites. And mAbs reacting to Vibrio cholerae flagellin were achieved by purified from the ascites. RESULTS: Six hybridoma cell lines stably secreting mAbs against Vibrio cholerae flagellin were taken and named VcNo.1-VcNo.6. The mAb titer in serum by indirect ELISA was 1:2 × 10(6). SDS-PAGE showed that the flagellin protein molecular weight (Mr) was 44 000 and the purity was high. Double-antibody sandwich ELISA method was set up using VcNo.6 antibody for detecting Vibrio cholerae. The sensitivity reached 10(3) CFU/mL. The ELISA method showed high specificity to Vibrio cholerae through testing 100 Vibrio cholerae (100% positive) and 101 non-Vibrio cholerae strains (100% negative). The detection limit was 1 CFU/g sample in artificial contaminated samples. CONCLUSION: The mAbs against flagellin core protein of Vibrio cholerae was successfully prepared and used to set up the double-antibody sandwich ELISA. The mAb of VcNo.6 was highly specific to Vibrio cholerae. The sensitivity of the established ELISA was as high as 10(3) CFU/mL. Moreover, it did not react to non-Vibrio cholerae strains. Therefore, the mAbs of VcNo.6 could be widely used in Vibrio cholerae detection from food samples as well as clinical samples.

[Preparation of monoclonal antibodies against flagellin core protein of Vibrio vulnificus and its application in testing Vibrio vulnificus from food products].

OBJECTIVE: To prepare the monoclonal antibodies (mAbs) against flagellin core protein of Vibrio (V.) vulnificus and establish the double-antibody sandwich ELISA for testing V. vulnificus from food products. METHODS: BALB/c mice were immunized by flagellin which was extracted by differential centrifugation method from V. vulnificus ATCC 1.1758. The splenocytes taken from the immunized mice were fused with Sp2/0 myeloma cells when the antibody titer reached 1:32 000 in serum. The hybridoma cell lines were prepared and screened by hybridoma technique and ELISA. The cells secreting mAbs were cloned through the limited dilution. The hybridoma cell lines were used to generate ascites. The mAbs were obtained by purification from the ascites. RESULTS: Five hybridoma cell lines which stably secreted mAbs against flagellin were isolated and named VVNo.1-VVNo.5. The mAb titer in serum reached 1:(2×10(6);). SDS-PAGE showed that the relative molecular mass (Mr;) of flagellin protein was 44 000, and that the purity was high. Double-antibody sandwich ELISA was set up using VVNo.5 antibody, and the sensitivity reached 10(3); CFU/mL culture broth. The ELISA showed that VVNo.5 antibody was highly specific to V. vulnificus. The detection limit was 2 CFU/25 g culture broth in artificial contaminated samples. CONCLUSION: The mAbs were obtained against flagellin core protein of V. vulnificus. The double-antibody sandwich ELISA was established using VVNo.5 mAb. The monoclonal antibody of VVNo.5 was highly specific to V. vulnificus, without cross reaction with non-target bacteria. Therefore the monoclonal antibodies of VVNo.5 could be widely used in detecting V. vulnificus from food samples as well as the clinical samples.

Abnormal glucose regulation in pyrethroid pesticide factory workers.

The purpose of this study was to investigate associations between pyrethroids occupational exposures, and risk of abnormal glucose regulation. Data from total of 3080 subjects in two pesticide factories were used. This was a population-based case-controlled study in China. In total, 18.3% of subjects with impaired glucose regulation (IGR) and 6.5% of subjects with diabetes, and the prevalence of abnormal glucose regulation was 24.8%, 86 subjects had known type 2 diabetes and 114 had newly diagnosed diabetes. The prevalence of subjects with abnormal glucose regulation increased from 21.3% in the controls to 29.3% in the exposures (χ² = 33.182, P < 0.001). Multivariate logistic regression was used to control potential confounders and calculate odd ratios as the estimate of effect. An indication of increased risk for abnormal glucose regulation was noted for exposure to pyrethroids (OR = 1.482, 95%CI = 1.238-1.774). Abnormal glucose regulation is common in subjects exposed to pyrethroids. The present investigation indicates the adverse health effects of pyrethroids are underestimated.