Study on the correlation of bisphenol A exposure, pro-inflammatory gene expression, and C-reactive protein with potential cardiovascular disease symptoms in young adults.

Bisphenol A (BPA) is a plasticizer used in the manufacture of polycarbonate and epoxy resins. It was found that higher urinary BPA levels are more likely to be associated with coronary artery disease (CVD). In recent years, the increasing incidence of CVD among young people is observed, which may be related with inflammation rather than the traditional triple-H risk factors. BPA is an endocrine-disrupting chemical, and can induce oxidative stress and chronic inflammation since its estrogenic effect. Inflammatory responses could come from the stimulation of IκB kinases (IKKs) by estrogen receptors (ERs). Therefore, this study investigated the association of BPA exposure with the gene expression of pro-inflammatory response (ERs and IKKs), an inflammation biomarker of CVD (C-reactive protein, CRP), and physiologic index potency of CVD development symptoms in young adults. This study divided BPA exposure levels into high and low groups based on the median plasma BPA level (4.34 ng/mL), and found that the high BPA group obviously had higher BMI, blood pressure, plasma CRP levels, and gene expression of ERß and IKKß. BMI and gene expression of IKKß were also positively correlated with plasma CRP secretion. Furthermore, the study subjects with potential CVD development symptoms had the increased levels of BPA (OR 2.10, 95% CI 0.83-5.39), CRP (OR 2.61, 95% CI 1.03-10.6) and IKKß (OR 4.29, 95% CI 1.51-15.6). These results indicated that exposure to BPA is potentially associated with expression of pro-inflammatory genes related to CRP secretion, which may promote the risk of CVD development symptoms in young adults. This study highlighted the possible connection between BPA exposure and CVD development but the mechanism between them needs to be further explored.

Variation of baculovirus-harbored transgene transcription among mesenchymal stem cell-derived progenitors leads to varied expression.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs) and MSCs-derived adipogenic, chondrogenic, and osteogenic progenitors without compromising the differentiation capacity. Remarkably, the transgene expression level and duration varied widely with the differentiation states at which the progenitors were transduced. However, whether the variation was a general phenomenon and what caused the variation were unclear. Here we demonstrated that transduction of the MSCs and MSC-derived progenitors using baculoviruses carrying egfp driven by CMV, EF-1alpha or CAG promoter resulted in a general trend of varied expression, that is, the chondrogenic progenitors allowed for the poorest expression while the adipogenic progenitors conferred the best expression. Quantification of the nuclear and cytoplasmic egfp gene copy numbers by quantitative real-time PCR revealed that the varied expression did not arise from the discrepancies in gene delivery efficiency nor was it due to the disparities in nuclear transport efficiency. In contrast, the transcription levels paralleled the overall expression levels. These data suggested that although the egfp genes could be efficiently delivered into the nuclei of chondrogenic progenitors, they were not transcribed as well as they were in the adipogenic progenitors. In conclusion, the rapidly altering cellular transcription machinery in the course of differentiation progression predominantly led to the varied expression levels.

Combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering.

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- or gene-based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported. METHODS: A recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) was constructed to transduce human MSCs derived from umbilical cord blood (uMSCs) or bone marrow (bMSCs). RESULTS: In this study, we demonstrated for the first time that human uMSCs or bMSCs could be transduced by baculovirus with high efficiencies (up to approximately 72.8% and 41.1%, respectively) and significantly elevated transgene (enhanced green fluorescent protein, EGFP) expression upon incubation with unconcentrated virus and phosphate-buffered saline for 4 h at 25 degrees C. The transduction efficiency into bMSCs could be further increased to approximately 72.2% by lowering the cell density. The improved transgene expression was partly attributed to the enhanced virus uptake upon transduction, as determined by quantitative real-time polymerase chain reaction (Q-PCR). MSC growth was not obstructed by baculovirus transduction itself, but was somewhat hampered by EGFP expression. Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic lineage. The adipogenic progenitors appeared more permissive to baculovirus transduction than the undifferentiated bMSCs, thus allowing for the maintenance and enhancement of transgene expression by repeated transduction after subculture. CONCLUSIONS: These findings implicate the potential applications of baculovirus as an alternative vector to genetically modify MSCs for ex vivo gene therapy.

Baculovirus as a highly efficient gene delivery vector for the expression of hepatitis delta virus antigens in mammalian cells.

Baculovirus has been employed for a wide variety of applications. In this study, we further expanded the application to the high-level expression of hepatitis delta virus (HDV) antigens and the formation of virus-like particles (VLP) in transduced mammalian cells. To this end, two recombinant baculoviruses were constructed to express large hepatitis delta antigen (L-HDAg) and hepatitis B surface antigen (HBsAg) under mammalian promoters. With a simplified transduction protocol using unconcentrated virus, high transduction efficiencies were achieved in hepatoma cells, in which L-HDAg and HBsAg were expressed abundantly, allowing for easy colorimetric detection in Western blots. L-HDAg alone was nucleus-bound and HBsAg alone was secreted; formation and secretion of HDV-like particles were readily detected upon coexpression, indicating that the baculovirus-expressed proteins were processed correctly as the authentic proteins. Quantitative real-time PCR (Q-PCR) analyses quantitatively revealed that baculovirus transduction was more efficient than plasmid transfection with respect to DNA uptake and DNA transport to the nucleus. Furthermore, superinfection introduced more baculovirus DNA into cells in the long-term culture as revealed by Q-PCR, thereby enhancing and prolonging the expression. In summary, baculovirus transduction can be an attractive method as an alternative to the plasmid transfection commonly employed for HDV research thanks to the significantly higher gene delivery efficiencies as well as the abundant expression and proper processing. Baculovirus can also be envisaged as a useful tool for investigating protein-cell interactions and virus assembly.

Highly efficient baculovirus-mediated gene transfer into rat chondrocytes.

To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.

Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells.

Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings that: 1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; 2) the half-life of baculovirus dramatically decreased at 37 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eukaryotic protein production on a larger scale.

Formation of enterovirus-like particle aggregates by recombinant baculoviruses co-expressing P1 and 3CD in insect cells.

The assembly of enterovirus requires the cleavage of P1 polyprotein by protease 3CD into individual structural proteins. Two recombinant baculoviruses were constructed to encode P1 and 3CD of enterovirus 71 (EV71), respectively. The expressed 3CD successfully cleaved P1 in vitro and in vivo. Also, the co-infection in insect cells resulted in crystalline virus-like particle structures morphologically resembling the authentic EV71 aggregates, which are reported for the first time.

Hospital quality information for patients in Taiwan: can they understand it?

OBJECTIVES: To investigate Taiwanese patients' ability to judge hospital quality and to examine their knowledge of commonly used quality indicators. DESIGN: Survey of patients during their stay in hospital. SETTING: Internal medicine, surgery, and gynecology wards in seven hospitals in northern Taiwan. PARTICIPANTS: Sample of 661 patients who voluntarily completed a questionnaire. MAIN OUTCOME MEASURES: (1) Patients' ability to judge hospital quality in relation to medical equipment, technical competence, and medication; (2) patients' knowledge of seven quality indicators: patient satisfaction, hospital-acquired infection, accreditation level, percent specialists, malpractice claims, unscheduled readmission, and mortality rate 48 hours after surgery. RESULTS: A total of 31-50% of the participants claimed that they could judge a hospital's quality on the basis of medical equipment, technical competence, or medication. The most frequently mentioned reasons on which their judgments were based were related to their own experiences and to the hospital's reputation. The percentage of participants reporting that they understood the quality indicators was 6.7-42.1%. CONCLUSION: In general, patients lack the ability to judge hospital quality and are unfamiliar with the commonly used quality indicators. Public education should be enhanced, or more understandable indicators should be developed in the future.