Epigallocatechin-3-gallate-mediated cardioprotection by Akt/GSK-3β/caveolin signalling in H9c2 rat cardiomyoblasts

Background: Epigallocatechin-3-gallate (EGCg) with its potent anti-oxidative capabilities is known for its beneficialeffects ameliorating oxidative injury to cardiac cells. Although studies have provided convincing evidence tosupport the cardioprotective effects of EGCg, it remains unclear whether EGCg affect trans-membrane signalling incardiac cells. Here, we have demonstrated the potential mechanism for cardioprotection of EGCg againstH2O2-induced oxidative stress in H9c2 cardiomyoblasts.Results: Exposing H9c2 cells to H2O2 suppressed cell viability and altered the expression of adherens and gapjunction proteins with increased levels of intracellular reactive oxygen species and cytosolic Ca2+. These detrimentaleffects were attenuated by pre-treating cells with EGCg for 30 min. EGCg also attenuated H2O2-mediated cell cyclearrest at the G1-S phase through the glycogen synthase kinase-3β (GSK-3β)/β-catenin/cyclin D1 signalling pathway.To determine how EGCg targets H9c2 cells, enhanced green fluorescence protein (EGFP) was ectopically expressedin these cells. EGFP-emission fluorescence spectroscopy revealed that EGCg induced dose-dependent fluorescencechanges in EGFP expressing cells, suggesting that EGCg signalling events might trigger proximity changes of EGFPexpressed in these cells.Proteomics studies showed that EGFP formed complexes with the 67 kD laminin receptor, caveolin-1 and -3,β-actin, myosin 9, vimentin in EGFP expressing cells. Using in vitro oxidative stress and in vivo myocardial ischemiamodels, we also demonstrated the involvement of caveolin in EGCg-mediated cardioprotection. In addition,EGCg-mediated caveolin-1 activation was found to be modulated by Akt/GSK-3β signalling in H2O2-induced H9c2cell injury.Conclusions: Our data suggest that caveolin serves as a membrane raft that may help mediate cardioprotectiveEGCg transmembrane signalling

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials

Women with endometriosis have higher comorbidities: Analysis of domestic data in Taiwan

AbstractEndometriosis, defined by the presence of viable extrauterine endometrial glands and stroma, can grow or bleed cyclically, and possesses characteristics including a destructive, invasive, and metastatic nature. Since endometriosis may result in pelvic inflammation, adhesion, chronic pain, and infertility, and can progress to biologically malignant tumors, it is a long-term major health issue in women of reproductive age. In this review, we analyze the Taiwan domestic research addressing associations between endometriosis and other diseases. Concerning malignant tumors, we identified four studies on the links between endometriosis and ovarian cancer, one on breast cancer, two on endometrial cancer, one on colorectal cancer, and one on other malignancies, as well as one on associations between endometriosis and irritable bowel syndrome, one on links with migraine headache, three on links with pelvic inflammatory diseases, four on links with infertility, four on links with obesity, four on links with chronic liver disease, four on links with rheumatoid arthritis, four on links with chronic renal disease, five on links with diabetes mellitus, and five on links with cardiovascular diseases (hypertension, hyperlipidemia, etc.). The data available to date support that women with endometriosis might be at risk of some chronic illnesses and certain malignancies, although we consider the evidence for some comorbidities to be of low quality, for example, the association between colon cancer and adenomyosis/endometriosis. We still believe that the risk of comorbidity might be higher in women with endometriosis than that we supposed before. More research is needed to determine whether women with endometriosis are really at risk of these comorbidities

Field Observations of Changes in SST, Chlorophyll and POC Flux in the Southern East China Sea Before and After the Passage of Typhoon Jangmi

Severe tropical storms play an important role in triggering phytoplankton blooms, yet direct field observation of evidence of the effects of a typhoon is very rare. Sea surface temperature (SST), nitrate concentration, chlorophyll a (chl a) concentration, and particulate organic carbon (POC) flux were measured before and shortly after Typhoon Jangmi which affected the southern East China Sea (SECS) on September 28 ~ 29, 2008. In situ SST (27.5 ~ 28.0°C) on September 19 ~ 21, decreased to ~24.0°C (October 3 ~ 6) in the SECS 4 ~ 7 days after the passage of Typhoon Jangmi. In situ nitrate and chl a concentrations 7-days (on October 6) after the passage of Jangmi were 1.9 μM and 1.61 mg m-3, respectively, much higher than those (nitrate: 0.3 μM and chl a: 0.73 mg m-3) concentrations before the typhoon (September 21). The enhanced chl a concentration is thus caused by a nutrient supply via vertical mixing or upwelling in the euphotic zone. The POC flux 7-days after Jangmi¡¦s passage was 552 ± 28 mg-C m-2 d-1, a ~2.5-fold increases before the typhoon (224 ± 33 mg-C m-2 d-1, on September 21). Our results suggest that typhoons indeed can stimulate efficient POC export out of the euphotic zone, while it is still poorly understood with regard to the total effects of a typhoon on nutrient dynamics and detailed carbon sequestration due to sampling difficulty. Therefore, successional sea-going observations ought to be conducted in the affected area after the passage of typhoons

The optimization of the up-stream process for EV71 virus production.

<p>(A) The T-flask was seeded with either 1.0∼1.5×10⁶ or 2.0∼2.5×10⁶ Vero cells, then after 2–3 days the cells were infected by the different ratio of E59/EV71 virus. The effects of different M.O.I were detected in the kinetic profile of virus produced from Vero cell grown in the VP-SFM medium. (B) The consistency of 3 Lots of EV71 virus production in the roller bottles. Virus titer was detected every day by TCID50 for 5 days.</p

Stability profiles of EV71 vaccine products (70 µg of formalin-inactivated EV71 virion formulated with 9 mg of aluminum phosphate in 3 mL of PBS.

<p>Eight mice per group were immunized twice with 0.2 mL of EV71 vaccine products Lot #1 and 3 that were stored in stability study chambers with different pre-set temperatures. The immunization protocol, IgG tier and virus neutralization assay are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034834#s2" target="_blank">Materials and Methods</a> section.</p

The growth profiles of Vero cell grown in serum-contain medium (SC) and different serum-free media (SF).

<p>Vero cells (2×10⁵ cell/ml) was cultured in 75T-flask and counted for viable cell number every 24 hours (0, 24, 48, 72, 96, 120 and 144 hours). The conditions for cell culture were described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034834#s2" target="_blank">Materials and Methods</a> section.</p

Immunogenicity studies of EV71 vaccine bulks.

<p>Eight mice per group and 5 rats per group were immunized twice with formalin-inactivated EV71 virion formulated with alum. Two rabbits per group and individual macaques were immunized three times with formalin-inactivated EV71 virion formulated with alum. The immunization protocol, IgG tier and virus neutralization assay are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034834#s2" target="_blank">Materials and Methods</a> section.</p>*<p>not done.</p

The temperature effect in the kinetic of formalin-inactivation of EV71 virus.

<p>Purified E59/EV71 was inactivated by 0.025% (v/v) formalin at different temperature and the residual of virus infectivity was detected by plaque assay performed triplicate at different time points.</p