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Bony injuries lead to compromised skeletal functional ability which further increase in aging population due to decreased bone mineral density. Therefore, we aimed to investigate the therapeutic potential of platelet-derived biomaterials (PDB) against bone injury. Specifically, we assessed the impact of PDB on osteo-inductive characteristics and migration of mouse embryonic fibroblasts (MEFs). Osteogenic lineage, matrix mineralization and cell migration were determined by gene markers (RUNX2, OPN and OCN), alizarin Red S staining, and migration markers (FAK, pFAK and Src) and EMT markers, respectively. The therapeutic impact of TGF-β1, a key component of PDB, was confirmed by employing inhibitor of TGF-β receptor I (Ti). Molecular imaging-based in vivo cellular migration in mice was determined by establishing bone injury at right femurs. Results showed that PDB markedly increased expression of osteogenic markers, matrix mineralization, migration and EMT markers, revealing higher osteogenic and migratory potential of PDB-treated MEFs. In vivo cell migration was manifested by expression of migratory factors, SDF-1 and CXCR4. Compared to control, PDB-treated mice exhibited higher bone density and volume. Ti treatment inhibited both migration and osteogenic potential of MEFs, affirming impact of TGF-β1. Collectively, our study clearly indicated PDB-rescued bone injury through enhancing migratory potential of MEFs and osteogenesis.

Porcine circovirus associated diseases (PCVADs) are among the most important diseases affecting the worldwide swine industry. Vaccination against porcine circovirus type 2 (PCV2) infection has been utilized for disease control and effectively reduces clinical signs of PCVADs. To evaluate the efficacy of the PCV2 vaccine in field farms, we conducted a trial using conventional pigs immunized with the subunit PCV2 vaccine followed by PCV2 challenge. Immunized pigs demonstrated lower serum viral loads, less viral antigen staining in lymph nodes, and higher average daily weight gain, confirming the protective efficacy of the vaccine. However, low levels of PCV2 infection were still detected in vaccinated pigs after challenge, suggesting that the PCV2 vaccine was unable to eradicate the virus, which could lead to asymptomatic PCV2 subclinical infection (PCV2-SI) in pig farms. Additionally, PCV2 infection is a risk factor for impaired pig immune response development during the weaning to growth stages, which is a crucial period to receive vaccines against classical swine fever (CSF). Therefore, the impact of PCV2-SI or PCV2-systemic disease (PCV2-SD) on live attenuated CSF vaccine was investigated. After PCV2 challenge, there was no difference in levels of classical swine fever virus (CSFV) neutralizing antibodies (NA) between pigs with PCV2-SD and PCV2-SI, suggesting that the efficacy of CSF vaccine was compromised. Moreover, results of long-term monitoring of CSFV NA titers in PCV2-SI pigs with minimized interference by maternally-derived antibodies suggested that serum PCV2 viral loads greater than 102 copies/mL may compromise the efficacy of CSF vaccine. Overall, a conventional pig model was established to demonstrate the impaired efficacy of the subunit PCV2 vaccine and its impact on the CSF vaccine in vaccination-challenge trials. Additionally, the impaired efficacy of the PCV2 vaccine resulted in increased PCV2-SI, eventually leading to compromised the live attenuated CSF vaccine induced NA response in field farm applications.

In the present study, low-temperature plasma combined with allylamine polymerization approach was used to coat amino-groups on oxide layer of plasma-oxidized biomedical titanium (Ti) for protein immobilization. Scanning electron microscopy, X-ray diffractometer, transmission electron microscopy, X-ray photoemission spectroscopy, secondary ion mass spectrometer and 2, 4, 6-trinitrobenzenesulfonic acid assay were utilized to investigate the surface and microstructural properties of the plasma-oxidized, plasma-polymerized and protein-immobilized samples. Analytical results indicated that the presence of a nanostructured rutile-TiO2 thin layer could be found on the plasma-oxidized samples. The thickness of nanostructured rutile-TiO2 layer increased with increasing plasma treatment power and period. As the plasma-oxidized samples underwent plasma polymerization with allylamine, amino-groups (NH2) were uniformly coated on nanostructured rutile-TiO2 layer. It was also found that Ti surface with thick oxide layer exhibited higher amounts of amino-groups deposition. After protein immobilization, the plasma-polymerized samples presented a formation of uniform streak-like immobilized protein clusters. Therefore, biomedical Ti with nanostructured rutile-TiO2 layer is a promising biomaterial that can be applied to cross-link with other biomolecules for promoting the bone healing and regeneration.

The microstructure and surface properties of the optimal nanostructured-gold (ONG) film were characterized using scanning electron microscopy, transmission electron microscopy, X-ray diffraction, nanoindentation and static contact angle evaluation in the present study. A three-dimensional model of human liver was reconstructed using magnetic resonance imaging to simulate the thermomechanical performance of the ONG film by means of finite element model analysis. Moreover, a monopolar electrosurgical unit equipped with needle type uncoated stainless steel (U-SS) and ONG film coated stainless steel (ONG-SS) electrodes was used to investigate the thermal distribution in the liver of rats. Experimental results showed that the temperature decreased significantly when using the ONG-SS electrode. The ONG-SS electrode also created a relatively small thermal injury area and lateral thermal effect. Furthermore, thermography indicated that the ONG-SS electrode exhibited a significantly lower surgical temperature as compared with the U-SS electrode during electrosurgery process in rats. Therefore, it could be demonstrated that the ONG-SS electrode is a promising biomedical device for reducing excessive thermal injury and decreasing adherence tissue in electrosurgery.

In previous (k,n) secret image sharing scheme, the threshold k is decided by dealer according to the security requirement, and this threshold value is fixed without considering the dynamic secure environment in future. In this work, we propose a novel threshold changeable secret image sharing scheme where the threshold value can be changed according to the changeable security requirement. In our scheme, each participant only needs to keep one initial shadow. When reconstructing image, the dealer decides the threshold according to security level. If the threshold is unchanged, any k or more initial shadows can recover the image; else if the threshold is increased or decreased, dealer publishes additional information, each participant update their shadows accordingly such that the threshold of updated shadows is changed correspondingly. The contribution of our work is that the threshold of shadows can be changed flexibly to satisfy the dynamic secure environment, and each participant only need to keep one initial shadows. The feature of threshold changeable makes our scheme more practical than previous secret image sharing in some complicated applications.

Background: Classical swine fever (CSF) is one of the most devastating pig diseases that affect the swine industry worldwide. Besides stamping out policy for eradication, immunization with vaccines of live attenuated CSF or the CSF-E2 subunit is an efficacious measure of disease control. However, after decades of efforts, it is still hard to eliminate CSF from endemically affected regions and reemerging areas. Most of previous studies demonstrated the efficacy of different CSF vaccines in laboratories under high containment conditions, which may not represent the practical performance in field farms. The inadequate vaccine efficacy induced by unrestrained factors may lead to chronic or persistent CSF infection in animals that develop a major source for virus shedding among pig populations. In this study, a vaccination-challenge-cohabitation trial on specific-pathogen-free (SPF) pigs and long-term monitoring of sows and piglets were used to evaluate the efficacy and the impact of maternally derived antibody (MDA) interference on CSF vaccines in farm applications.Results: The trials demonstrated higher neutralizing antibody (NA) titers with no clinical symptoms and significant pathological changes in the CSF-E2 subunit vaccine immunized group after CSFV challenge. Additionally, none of the sentinel pigs were infected during cohabitation indicating that the CSF-E2 subunit vaccine could provoke adequate acquired immunity to prevent horizontal transmission. In field farm applications, the CSF-E2 subunit vaccine significantly reduced CSF viral RNA detection via saliva monitoring in a sow population that was routinely immunized with live attenuated CSF vaccine long term. Moreover, no adverse effects and an average of higher, long-lasting and consistent NA level could be detected in sows immunized with CSF-E2 subunit vaccine before parturition. Furthermore, early application of the CSF-E2 subunit vaccine in 3-week-old piglets illustrated no MDA interference on primary immunization whereas interference of MDA was noted in piglets vaccinated with live attenuated CSF.Conclusions: The CSF-E2 subunit vaccine demonstrated more flexibility and efficacy for immunization in both pregnant sows and piglets. These advantages could provide a novel approach to avoid possible virus shedding in sow population and MDA interference in piglets for control of CSF in field farm applications.

In this study, the microstructural, mechanical and biological characterizations of the Ti-25Ta (wt.%) alloy were investigated by means of optical microscope, X-ray diffraction, electron microscope, microhardness test, contact angle goniometer and in vitro cytotoxicity assay. As the alloy underwent heat-treatment at temperatures between 700 °C and 1000 °C for 30 min, its microstructure was a mixture of β phase and needle-like α′′ martensite phase. The analytical results indicated that the volume fraction of the needle-like α′′ martensite phase decreased with increasing heat-treatment temperature. The β transus temperature of the investigated Ti-25Ta alloy was below 700 °C. Moreover, the alloy heat-treated at 900 °C exhibited the maximum hardness Hv 743 ± 12.93. For wettability evaluation, all investigated samples possessed surface hydrophilicity. The cytotoxicity assay results also demonstrated that the heat-treated Ti-25Ta samples have well cell proliferation and adhesion behaviors. Therefore, these results and features could be used to further understand the relationship between the high temperature microstructure, mechanical behavior and in vivo biocompatibility of the Ti-25Ta alloy, and develop as a potential biomedical alloy.

Porcine circovirus type 2 (PCV2) is the primary causative agent of an economically important swine disease, now known as porcine-associated disease (PCVAD). The only structural protein of viral capsid, Cap has become the major target for development of PCV2 subunit vaccines. The purpose of this study is to express Cap of PCV2 using a recombinant pseudorabies virus (PRV) that is gE gene deficient, which is a widely used PRV marker vaccine. The recombinant PRV, gE(-)/PCV2cap(+)PRV, was constructed using homologous recombination techniques, in order to replace the upstream of the gE gene with the PCV2 cap gene. The expression of Cap during virus replication was confirmed using immunofluorescence and Western blotting analysis. The expressed Cap protein self-assembled into virus-like particles (VLPs), which was demonstrated using electromicrography. The immunization of mice or guinea pigs with purified VLPs could induce significant, specific antibody responses to PCV2 Cap. These results demonstrate an alternative to PCV2 for the development of a VLP-based subunit vaccine.

This study aimed to investigate whether α-tocopherol is able to protect keratinocytes against ultraviolet A (UVA) radiation by increasing glutathione (γ-glutamylcysteinylglycine; GSH) levels or decreasing lipid peroxidation and reactive oxygen species (ROS) generation. The cell survival fraction was 43.6% when keratinocytes were irradiated with UVA at a dose of 8 J/cm2. α-Tocopherol was added prior to UVA irradiation and the cell viability was assayed. The cell survival fractions were 60.2, 77.1, 89.0, 92.9 and 96.2% when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. These results suggested that α-tocopherol is capable of protecting keratinocytes against UVA irradiation. Furthermore, the levels of GSH, lipid peroxidation and ROS were measured. The levels of GSH were 0.354 and 0.600 mmol/g protein in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 0.364, 0.420, 0.525, 0.540 and 0.545 mmol/g protein when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. The levels of lipid peroxidation were 20.401 or 5.328 μmol/g [malondialdehyde (MDA)/protein] in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 11.685, 6.544, 5.847, 4.390 and 2.164 μmol/g (MDA/protein) when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. The levels of ROS were 3,952.17 or 111.87 1/mg protein in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 742.48, 579.36, 358.16, 285.63 and 199.82 1/mg protein when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. These findings suggested that α-tocopherol protects keratinocytes against UVA irradiation, possibly through increasing the levels of GSH or decreasing the levels of lipid peroxidation and ROS generation.

Retrospective radiographical review.To evaluate the outcome of selective thoracic fusion (STF) by using the Guan-Din method for the treatment of major thoracic compensatory lumbar (MTCL) curves.Performing STF for MTCL curves is to minimize the loss of lumbar motion and the risk of lumbar degeneration or pain. Surgical treatment of MTCL curves aims to maximize the rate of STF for MTCL curves while optimizing instrumental thoracic and compensatory lumbar correction. The Guan-Din method has been demonstrated to be able to enhance the lumbar curve's capacity for spontaneous correction and broaden the current curve criteria of MTCL curves for STF.Between 2004 and 2010, 510 consecutive surgically treated MTCL curves were reviewed. Of these MTCL curves, who met the criteria of lumbar side bending Cobb 35° or less and without global thoracic hyperkyphosis and/or thoracolumbar kyphosis (T10-L2 ≤20°), were treated with STF using the Guan-Din method. Radiographs were analyzed before surgery, immediately after surgery, and at the most recent follow-up (range, 2-8 yr).Curve types of 510 MTCL curves according to Lenke system were as follows: 1A (n = 91), 2A (n = 74), 3A (n = 6), 4A (n = 2), 1B (n = 93), 2B (n = 34), 3B (n = 8), 4B (n = 5), 1C (n = 84), 2C (n = 26), 3C (n = 72), and 4C (n = 15). Of the 510 MTCL curves, 458 (90%) curves were treated with STF. A mean 73% thoracic correction and 63% lumbar correction was obtained at the most recent follow-up. Of the 197 surgically treated MTCL curves with a lumbar C modifier, 148 (75%) curves that contained 57 Lenke 1C and 2C curves and 40 Lenke 3C and 4C curves that did not meet Lenke curve criteria for STF, were successfully treated with STF. A mean 67% thoracic correction and 57% lumbar correction was obtained at the most recent follow-up. The rate of STF and the magnitude of correction of MTCL curves in this study were significantly greater than those in all other reports. No significant change in global coronal and sagittal imbalance was observed.The rate of STF and the compensatory correction of MTCL curves could be maximized by using the Guan-Din method as the method for STF.4.

Knee osteoarthritis (OA) is one of the most prevalent disorders in elderly population. Among various therapeutic alternatives, we employed stromal vascular fraction (SVF), a heterogeneous cell population, to regenerate damaged knee cartilage. OA patients were classified on the basis of age, gender, body mass index (BMI), and x-ray-derived Kellgren&ndash, Lawrence (KL) grade. They were treated with SVF and followed-up for 24 months. Visual analogue scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index were used to determine treatment efficacy. Cartilage healing was assessed using the MRI-based Outerbridge score (OS) and evaluation of bone marrow edema (BME) lesions, while a placebo group was used as a control. Time- and KL-dependent changes were also monitored. We observed a decreasing trend in VAS score and WOMAC index in the SVF-treated group up to 24 months, as compared with the placebo group. Besides, a significant increase and decrease in Lysholm and OS, respectively, were observed in the treatment group. Compared with the values before treatment, the greatly reduced WOMAC scores of KL3 than KL2 groups at 24 months, indicate more improvement in the KL3 group. Highly decreased BME in the treated group was also noted. In conclusion, the SVF therapy is effective in the recovery of OA patients of KL3 grade in 24 months.

WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matrix cellular proteins. Osteosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effect of WISP-1 on migration activity in human osteosarcoma cells is mostly unknown. In this study, we first found that the expression of WISP-1 in osteosarcoma patients was significantly higher than that in normal bone and corrected with tumor stage. Exogenous treatment of osteosarcoma cells with WISP-1 promoted cell motility and matrix metalloproteinase (MMP)-2 and MMP-9 expression. In addition, the Ras and Raf-1 inhibitor or siRNA abolished WISP-1-induced cell migration and MMP expression. On the other hand, activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after WISP-1 treatment was demonstrated, and WISP-1-induced expression of MMPs and migration activity were inhibited by the specific inhibitor, and mutant of MEK, ERK, and NF-κB cascades. Taken together, our results indicated that WISP-1 enhances the migration of osteosarcoma cells by increasing MMP-2 and MMP-9 expression through the integrin receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway.

There are trace amounts of heavy metals in cosmetics. Heavy metals such as mercury (Hg), which is added to skin-whitening cosmetics, may cause acute or chronic damage to human cells. The aim of this study was to investigate the cytotoxicity of mercury chloride (HgCl2) to human keratinocytes. The keratinocytes were treated with various concentrations of HgCl2 and the cell survival fractions were found to be 38.08, 17.59, 12.76, 3.29 and 0.77% when the cells were treated with 0.25, 0.5, 0.75, 1 and 1.5 μM of HgCl2, respectively. Moreover, we observed that the greatest damage was to the cell membrane. The metallothionein (MT) protein expression was also investigated. MT expression levels increased with increasing concentrations of HgCl2. The results indicated that MT protects the keratinocytes against HgCl2-induced toxicity.

Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

The stable, water-soluble, and nonfluorescent FA-OMe can sense nitric oxide (NO) and form the intensely fluorescent product dA-FA-OMe via reductive deamination of the aromatic primary amine. The reaction is accompanied by a notable increase of the fluorescent quantum yield from 1.5 to 88.8%. The deamination mechanism of FA-OMe with NO was proposed in this study. The turn-on fluorescence signals were performed by suppression of photoinduced electron transfer (PeT), which was demonstrated by density functional theory (DFT) calculations of the components forming FA-OMe and dA-FA-OMe. Furthermore, FA-OMe showed water solubility and good stability at physiological pHs. Moreover, the selectivity study indicated that FA-OMe had high specificity for NO over other reactive oxygen/nitrogen species. In an endogenously generated NO detection study, increasing the incubation time of FA-OMe with lipopolysaccharide (LPS) pretreated Raw 264.7 murine macrophages could cause an enhanced fluorescence intensity image. In addition, a diffusion/localization cell imaging study showed that FA-OMe could be trapped in Raw 264.7 cells. These cell imaging results demonstrated that FA-OMe could be used as a turn-on fluorescent sensor for the detection of endogenously generated NO.

This study explores post-recovery satisfaction and post-purchase intentions with service recovery of online shopping customers from the perspectives on perceived justice and emotions based on twenty scenarios that combine five service failures and four recoveries. Analytical results indicating that in addition to enhancing post-purchase intentions and post-recovery satisfaction among customers, distributive justice increases positive emotions and decreases negative ones. Additionally, procedural justice enhances post-recovery satisfaction as well as increases positive emotions and decreases negative ones, while interactional justice only increases post-recovery satisfaction of customers. Moreover, while positive emotions increase post-recovery satisfaction, and vice versa. Post-recovery satisfaction positively affects post-purchase intentions. Results of this study provide a valuable reference for operators and managers of online shopping websites.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. Insulin-like growth factor-I (IGF)-I plays an important role in regulating cell growth, proliferation, survival, and metabolism. However, the effects of IGF-I in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IGF-I increased the migration and the expression of α5β1 integrin in human chondrosarcoma cells. Pretreatment of cells with IGF-I receptor antibody reduced IGF-I-induced cell migration and integrin expression. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor-κB (NF-κB) pathways after IGF-I treatment were demonstrated, and IGF-I-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and NF-κB cascades. Taken together, our results indicated that IGF-I enhances the migration of chondrosarcoma cells by increasing α5β1 integrin expression through the IGF-I receptor/PI3K/Akt/NF-κB signal transduction pathway. J. Cell. Physiol. 226: 3270–3277, 2011. © 2011 Wiley Periodicals, Inc.

Actinobacillus pleuropneumoniae exotoxins (Apx) are major virulence factors that play important roles in the pathogenesis of pleuropneumonia in swine. A previous study has demonstrated that native ApxI at low concentrations induces apoptosis in primary porcine alveolar macrophages (PAMs) via a caspase-3-dependent pathway. However, the molecular mechanisms underlying ApxI-induced apoptosis remain largely unknown. In this study, it was shown that ApxI treatment in PAMs rapidly induced phosphorylation of both p38 and JNK, members of the mitogen-activated protein kinase family. Application of a selective p38 or JNK inhibitor significantly reduced ApxI-induced apoptosis, indicating the involvement of p38 and JNK pathways in this event. Furthermore, activation of both caspase-8 and -9 were observed in ApxI-stimulated PAMs. Inhibition of caspase-8 and caspase-9 activity significantly protected PAMs from ApxI-induced apoptosis. In addition, Bid activation was also noted in ApxI-treated PAMs, and inhibition of caspase-8 suppressed the activation of Bid and caspase-9, suggesting that ApxI was able to activate the caspases-8–Bid–caspase-9 pathway. Notably, inhibition of p38 or JNK pathway greatly attenuated the activation of caspases-3, -8, and -9. This study is the first to demonstrate that ApxI-induced apoptosis of PAMs involves the activation of p38 and JNK, and engages the extrinsic and intrinsic apoptotic pathways.

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. This study is the first to investigate the anti-cancer effects of the new benzimidazole derivative (5-methyl-2(pyridine-3-yl)-1-(3,4,5-trimethoxybenzyl)benzimidazole; MPTB) in human chondrosarcoma cells. MPTB-induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but not in primary chondrocytes. MPTB-induced upregulation of Bax and Bak and dysfunction of mitochondria in chondrosarcoma. MPTB triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels, and increased glucose-regulated protein (GRP) expression. MPTB also increased calpain expression. Transfection of cells with GRP78 or calpain siRNA reduced MPTB-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 44% reduction in tumor volume after 21 d of treatment. This study demonstrates novel anti-cancer activity of MPTB against human chondrosarcoma cells and in murine tumor models. Mol. Carcinog. ©2011 Wiley-Liss, Inc.

The purposes of this study are to construct an instrument to evaluate service quality of mobile value-added services and have a further discussion of the relationships among service quality, perceived value, customer satisfaction, and post-purchase intention. Structural equation modeling and multiple regression analysis were used to analyze the data collected from college and graduate students of 15 major universities in Taiwan. The main findings are as follows: (1) service quality positively influences both perceived value and customer satisfaction; (2) perceived value positively influences on both customer satisfaction and post-purchase intention; (3) customer satisfaction positively influences post-purchase intention; (4) service quality has an indirect positive influence on post-purchase intention through customer satisfaction or perceived value; (5) among the dimensions of service quality, ''customer service and system reliability'' is most influential on perceived value and customer satisfaction, and the influence of ''content quality'' ranks second; (6) the proposed model is proven with the effectiveness in explaining the relationships among service quality, perceived value, customer satisfaction, and post-purchase intention in mobile added-value services.

Transforming growth factor-beta1 (TGF-beta1) plays a crucial role in adhesion and migration of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found that TGF-beta1 increased the migration and cell surface expression of beta1 integrin in human lung cancer cells (A549 cells). TGF-beta1 stimulation increased phosphorylation of p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) and Ser(473) of Akt was determined. Besides, we performed that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed the TGF-beta1-induced migration activities of A549 cells. Treatment of A549 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also repressed TGF-beta1-induced cells migration and beta1 integrins expression. In addition, treatment of A549 cells with TGF-beta1 induced IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the TGF-beta1-mediated increases in IKKalpha/beta, IkappaBalpha phosphorylation and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85alpha and Akt mutants also reduced the TGF-beta1-induced kappaB-luciferase activity. Taken together, our results suggest that TGF-beta1 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 integrins and contributing the migration of human lung cancer cells.

A green fluorescent protein (gfp) gene was ligated to the Lactobacillus reuteri-specific nisin-inducible expression-secretion vector pNIES, generating a pNIES-GFP vector capable of secreting the cloned gene as a GFP-fusion protein with fluorescent activity. To develop this system as a live vehicle carrying the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LT(B)) of the enterotoxigenic Escherichia coli (ETEC), a recombinant 5'-ST-LT(B)-3' DNA fragment was cloned into pNIES-GFP. The resulting L. reuteri/pNIES-GFP:STLT(B) system was found to possess the capability of adhering to the mice gut, secreting GFP:STLT(B) product at 0.14 and 0.026 pgcell(-1) under induced and noninduced conditions, respectively. Further analysis of the GFP:STLT(B) product confirmed its ganglioside-binding ability, LT(B) antigenicity and relative freedom from the ST-associated toxicity, making it suitable for use as an oral vaccine in mice. Oral inoculation of the L. reuteri/pNIES-GFP:STLT(B) culture in mice elicited significant (P

The purpose of this study is to develop a three-dimensional (3D) finite element model of the temporomandibular joint (TMJ) and to further investigate the stress distributions after occlusal splint therapy. Six patients with anterior disc displacement without reduction were reconstructed before and after six months of occlusal splint treatments from computed topography (CT) and magnetic resonance imaging (MRI). One must focus on the magnitude and location of the maximum stresses under physiological loading. In the 3D model studies, each mandibular condyle, disc, and articular eminence was divided into five regions: anterior, posterior, central, medial, and lateral. The results of this study show that the abnormal von Mises stresses occurred in the condyle during jaw closing, and overloading of the anterior condyle and posterior disc during jaw opening occurred before treatment. Stresses of all regions became well distributed after treatment, and an approximately 30% stress reduction in the condyle and a 35% stress reduction in the disc were found during jaw opening (p < 0.05), a remarkable difference. This result shows the potential clinical benefits in terms of proven superior biomechanical behavior according to occlusal splint treatment.

Mutations in codons 12 and 13 of the K-RAS oncogene are detected at a remarkably high frequency in colorectal adenocarcinoma and are believed to be a critical event in oncogenesis. In the present study, we evaluated colorectal tumor specimens from Taiwan for mutations in K-RAS codons 12 and 13 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct DNA sequencing. Mutations were found in 48 of 181 (26.5%) tumors, 30 mutations were G--A transitions (62.5% of all mutations), 14 were G--T transversions (29.2%), and only 4 were G--C transversions (8.3%). Similar relative mutation frequencies and spectra were found regardless of the sex of the patient, the tumor grade, or the tumor stage. The high frequency of transitions among K-RAS mutation suggests that G/T mismatches play an important role in the oncogenesis of colorectal adenocarcinoma, implying that alkylating carcinogens may be involved in the colorectal carcinogenesis. Although the frequency of mutation (26.5%) appears to be lower than those reported in the United States (40%), France (49%), and the Netherlands (34%), the spectrum of point mutations in codons 12 and 13 of the K-RAS gene in the Taiwan Chinese population appears to be similar. The reason for these results may be that diet and ethnicity are not rate limit factors in controlling the spectra of mutations but influence on the frequency of K-RAS mutations in human colorectal adenocarcinomas.

It is believed that human progesterone receptor (PR) contains a ligand binding subunit A (83 kDa) or subunit B (120 kDa) and 2 copies of heat shock proteins (hsp90) of molecular weight 90 kDa. To elucidate the mechanism of hormone binding, we employed radiation inactivation to determine its functional size. The functional masses determined in the presence of glycerol, molybdate and potassium chloride were 120 \pm 14, 124 \pm 13 and 130 \pm 20 kDa, respectively. From scatchard plot analysis, the radiation decreased the binding sites and increased the binding affinity of PR with ligand. The functional masses of PR dissolved in the three variant buffers were similar to the molecular weight of PR subunit B. The results implied that PR subunit B could bind with ligand despite hsp90 and hsp90 was not involved in the PR binding to progesterone.

One hundred and thirty-eight stage II and III colorectal cancer patients were included to evaluate the prognostic significance of genetic markers (including mutations of the p53, K- ras genes, and microsatellite instability) on the response to 5-fluorouracil (FU)-based post-operative adjuvant therapies (PAT). When stratified by PAT and adjusting for other prognostic variables, presence of p53 mutation was associated with a poor outcome (hazard ratio (HR)=3.1, 95% confidence interval (CI), 0.9–11.0) among patients without PAT. Our data confirmed that p53 mutation is an independent pre-treatment factor in stage II and III colorectal cancer after curative resection.

The electrodeposition of various copper deposits was carried out by means of cyclic voltammetric, potentiostatic, galvanostatic, pulse-rest and pulse-reverse plating modes in a simple acidic CuSO4 bath without any additives, although the 8 mM CuSO4 solution employed in this work was not practically applicable for electroplating. The morphologies of these Cu deposits were systematically compared by atomic force microscopy (AFM), and scanning electron microscopy (SEM). The average grain size and roughness of Cu deposits were found to strongly depend on the deposition modes. From the results of SEM and AFM, the Cu deposit plated by cyclic voltammetry showed the smoothest and most compact morphology with the smallest average grain size. When the deposition potentials were negative to −0.17 V, nucleation of Cu seeds and growth of Cu grains occurred simultaneously, resulting in the presence of relatively small Cu clusters on the deposit grains. The anodic process through means of either cyclic voltammetry or reverse plating rendered the significant dissolution of grain peaks and these small Cu clusters on the deposit, resulting in a relatively smooth surface.

Study design Retrospective radiographical review. Objective To demonstrate that the structural and noncompensatory Lenke 3 and 4C lumbar curves could be nonstructural and compensatory. Summary of background data Historically, Lenke 3 and 4C curves were not recommended for selective thoracic fusion (STF) because the lumbar curve was considered structural and noncompensatory. However, consecutive series of Lenke 3 and 4C curves suggest successful treatment with STF. Methods Between 2001 and 2004, 2005 and 2008, and 2010 and 2012, 3 consecutive series of 108, 134, and 78 surgically treated Lenke 1, 2, 3, and 4C curves were reviewed, respectively. The coronal curve criteria for the curves treated with STF during each period were lumbar side bending Cobb angle less than 25° and meeting the Lenke ratio criteria, lumbar side bending Cobb angle 35° or less, and lumbar side bending Cobb angle 45° or less, respectively. The sagittal curve criteria for STF during each period was absence of junctional thoracolumbar kyphosis 20° or more between T10 and L2. The technique used for STF was the Guan-Din method. Radiographs of all the curves treated with STF were analyzed before and after surgery. Results Optimal instrumented thoracic and compensatory lumbar correction was obtained for all Lenke 1, 2, 3, and 4C curves treated with STF in each period. As the coronal criteria for STF were broadened, the extent of feasibility of STF was expanded and the rate of STF increased. Although Cobb angle, apical vertebral translation, and apical vertebral rotation magnitudes of Lenke 3 and 4C curves were larger and more severe than those of Lenke 1 and 2C curves, optimal compensatory correction could still be obtained for Lenke 3 and 4C curves. Conclusion The structural and noncompensatory Lenke 3 and 4C lumbar curves were proven to be nonstructural and compensatory. Lenke 1, 2, 3, and 4C curves have similar natures and similar responses to the same technique (Guan-Din method) used for STF and could be considered collectively as a single indication for STF. The extent of feasibility of STF could be expanded from Lenke 1 and 2 curves to Lenke 1, 2, 3, and 4 curves. Level of evidence 2.

Positron emission tomography (PET) is an important tool for enabling quantification of human brain function. However, quantitative studies using tracer kinetic modeling require the measurement of the tracer time-activity curve in plasma (PTAC) as the model input function. It is widely believed that the insertion of arterial lines and the subsequent collection and processing of the biomedical signal sampled from the arterial blood are not compatible with the practice of clinical PET, as it is invasive and exposes personnel to the risks associated with the handling of patient blood and radiation dose. Therefore, it is of interest to develop practical noninvasive measurement techniques for tracer kinetic modeling with PET. In this paper, a technique is proposed to extract the input function together with the physiological parameters from the brain dynamic images alone. The identifiability of this method is tested rigorously by using Monte Carlo simulation. The results show that the proposed method is able to quantify all the required parameters by using the information obtained from two or more regions of interest (ROI's) with very different dynamics in the PET dynamic images. There is no significant improvement in parameter estimation for the local cerebral metabolic rate of glucose (LCMRGlc) if the number of ROI's are more than three. The proposed method can provide very reliable estimation of LCMRGlc, which is our primary interest in this study.

Background Si-Wu-Tang (SWT), a Traditional Chinese Medicine (TCM) formula, is widely used for the treatment of gynopathies diseases such as menstrual discomfort, climacteric syndrome, dysmenorrhea, and other estrogen-related diseases. Recent studies have shown that SWT can treat primary dysmenorrhea, have anti-pruritic anti-inflammatory effects, and protect against radiation-induced bone marrow damage in an animal model. It has been reported that anti-inflammatory and anti-oxidant agents have the potential to treat osteoporosis by increasing bone formation and/or suppressing bone resorption. However, the effect of SWT on bone cell function has not yet been reported. Methods Alkaline phosphatase (ALP), bone morphogenetic proteins (BMP)-2, and osteopontin (OPN) mRNA expression was analyzed by qPCR. The mechanism of action of SWT extract was investigated using western blotting. The in vivo anti-osteoporotic effect of SWT extract was assessed in ovariectomized mice. Results Here, we report that SWT increases ALP, BMP-2, and OPN expression as well as bone mineralization. In addition, we show that the PI3K, Akt, and NF-κB signaling pathways may be involved in the SWT-mediated increase in gene expression and bone mineralization. Notably, treatment of mice with SWT extract prevented bone loss induced by ovariectomy in vivo. Conclusion SWT may be used to stimulate bone formation for the treatment of osteoporosis.

This is the first report of 0 eV or negative Schottky barrier height for electrons down to 100 K, even on lightly doped Si. It is achieved by energy barrier engineering with Yb doping at NiSi/Si interface. Ideal unity rectification ratio and reasonable sheet resistance are also achieved in this study. This technology can potentially boost future MOSFET performance.

Interleukin-11 (IL-11) was originally identified as the cytokine that could induce the proliferation of human cells. Recent studies have shown that IL-11 plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of IL-11 on human chondrosarcoma cells are largely unknown. Here, we found that IL-11 increased the migration and expression of intercellular adhesion molecule-1 (ICAM)-1 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of the IL-11 which was higher than that in primary chondrocytes. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by IL-11 treatment, and the IL-11-induced expression of ICAM-1 and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. Taken together, our results indicate that IL-11 enhanced the migration of the chondrosarcoma cells by increasing ICAM-1 expression through the IL-11Rα receptor, PI3K, Akt, and NF-κB signal transduction pathway. J. Cell. Biochem. 113: 3353–3362, 2012. © 2012 Wiley Periodicals, Inc.

ObjectivesWe investigated the effect of Cistanche deserticola Ma. (CD) on bone formation by cultured osteoblasts.MethodsThe mineralized nodule formation assay was used to examine the in-vitro effects of CD on bone formation. Alkaline phosphatase (ALP), bone morphogenetic proteins (BMP)-2 and osteopontin (OPN) mRNA expression was analysed by quantitative real-time polymerase chain reaction. The mechanism of action of CD extract was investigated using Western blotting. The in-vivo anti-osteoporotic effect of CD extract was assessed in ovariectomized mice.Key findingsCD extract had no effect on the proliferation, migration or wound healing of cultured osteoblasts, but increased ALP, BMP-2 and OPN mRNA and bone mineralization. Mitogen-activated protein kinase (MAPK) or nuclear factor (NF)-κB inhibitors reduced CD extract-induced bone formation and ALP, BMP-2 and OPN expression. However, CD extract did not affect osteoclastogenesis. In addition, CD extract prevented the bone loss induced by ovariectomy in vivo.ConclusionsCD may be a novel bone formation agent for the treatment of osteoporosis.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5′-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.

The aim of this study was to investigate whether magnesium ascorbyl phosphate (MAP) and coenzyme Q10 (CoQ10) can protect keratinocytes against ultraviolet (UV)A irradiation by increasing the levels of glutathione (GSH). The cell survival fraction was 89.9% when the keratinocytes were irradiated with UVA at a dose of 4 J/cm2. The cell survival fractions were 48.4, 9.1 and 4.8%, at doses of 8, 16 and 32 J/cm2, respectively. MAP was added to the cells prior to UVA irradiation at a dose of 8 J/cm2 and then the cell viability was assayed. The cell survival fractions were 51.6, 55.5, 64.8 and 76.7%, when MAP was added at concentrations of 125, 250, 500 µM and 1 mM, respectively. The results showed that MAP is capable of protecting keratinocytes against UVA irradiation. The cell survival fractions were 77.2, 89.4 and 90.1%, when CoQ10 was added at concentrations of 2.5, 5 and 10 µM, respectively. The results revealed that CoQ10 is capable of protecting keratinocytes against UVA irradiation. At the same time, the levels of GSH within cells were detected. The level of GSH within cells was 0.3 mmol/g protein when the keratinocytes were irradiated with UVA at a dose of 8 J/cm2. We measured the levels of GSH within the cells after MAP or CoQ10 was added prior to UVA irradiation at a dose of 8 J/cm2. The levels of GSH within the cells were 0.344, 0.388, 0.456 and 0.5 mmol/g protein, when MAP was added at concentrations of 125, 250, 500 µM and 1 mM, respectively. The levels of GSH within the cells were 0.328, 0.35 and 0.394 mmol/g protein, when CoQ10 was added at concentrations of 2.5, 5 and 10 µM, respectively. These results imply that MAP and CoQ10 can protect the keratinocytes against UVA irradiation, possibly via increasing the levels of GSH.

A colorless and non-fluorescent rhodamine derivative, rhodamine B hydrazide (RH), is applied to detect nitric oxide and form fluorescent rhodamine B (RB). The reaction mechanism of RH with NO is proposed in this study. The probe shows good stability over a broad pH range (pH>4). Furthermore, fluorescence intensity of RH displays an excellent linearity to the NO concentration and the detection limit is as low as 20 nM. A 1000-fold fluorescence turn-on from a dark background was observed. Moreover, the selectivity study indicated that the fluorescence intensity increasing in the presence of NO was significantly higher than those of other reactive oxygen/nitrogen species. In exogenously generated NO detection study, clear intracellular red fluorescence was observed in the presence of S-nitroso-N-acetyl-D,L-penicillamine (SNAP, a kind of NO releasing agent). In endogenously generated NO detection study, increasing incubation time of RH with lipopolysaccharied (LPS) pre-treated cells could obtain a highly fluorescent cell image. These cell imaging results demonstrated that RH can efficiently penetrate into Raw 264.7 cells and be used for detection of exogenously and endogenously generated nitric oxide.

AIM: To investigate how a complex network of CC chemokine ligands (CCLs) and their receptors influence the progression of tumor and metastasis. METHODS: In the present study, we used immunohistochemistry to examine the expression of CCL7, CCL8 and CCL21 in 194 gastric cancer samples and adjacent normal tissues. We analyzed their correlation with tumor metastasis, clinicopathologic parameters and clinical outcome. RESULTS: We found that the higher expression of CCL7 and CCL21 in cancer tissues than in normal tissues was significantly correlated with advanced depth of wall invasion, lymph node metastasis and higher tumor node metastasis stage. Moreover, Kaplan-Meier survival analysis revealed that CCL7 and CCL21 overexpression in cancer tissues was correlated with poor prognosis. CONCLUSION: These results suggest that overexpression of these two CC chemokine ligands is associated with tumor metastasis and serves as a prognostic factor in patients with gastric cancer.

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. This study is the first to investigate the anti-cancer effects of the new benzimidazole derivative (5-methyl-2(pyridine-3-yl)-1-(3,4,5-trimethoxybenzyl)benzimidazole; MPTB) in human chondrosarcoma cells. MPTB-induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but not in primary chondrocytes. MPTB-induced upregulation of Bax and Bak and dysfunction of mitochondria in chondrosarcoma. MPTB triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels, and increased glucose-regulated protein (GRP) expression. MPTB also increased calpain expression. Transfection of cells with GRP78 or calpain siRNA reduced MPTB-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 44% reduction in tumor volume after 21 d of treatment. This study demonstrates novel anti-cancer activity of MPTB against human chondrosarcoma cells and in murine tumor models.

To develop an economical, easy technique for producing recombinant E2 glycoprotein (rE2) of classical swine fever virus (CSFV) as a candidate immunogen, a bi-cistronic baculovirus/larvae expression vector was constructed using p10 promoter, an internal ribosome entry site, and the gfp gene. Trichoplusia ni larvae were successfully infected with the occluded recombinant baculovirus via feed, and the characteristics of rE2 were confirmed by immunoblot and glycosylation stain. rE2 at a concentration of 0.6-0.8 mg/ml without degradation was obtained from hemolymphs of infected larvae that emitted high levels of green fluorescence. Immunization assays indicated that mice and piglets immunized with rE2-containing hemolymph elicited high titers of anti-CSFV E2 antibodies with virus-neutralizing activity. This is the first study to indicate that baculovirus/T. ni larvae-expressed rE2 can be served as a vaccine candidate. This system provides an economical alternative for the production of vaccine components in the veterinary industry.

Claudin-4 is a member of the claudin family, a large family of transmembrane proteins that are essential in the formation and maintenance of tight junctions. Matrix metal-loproteinase (MMP)-2 and -9 degrade type IV collagen of the extracellular matrix and basal membranes. Claudin-4 activates MMP-2, indicating that claudin-mediated increased cancer cell invasion may result from the activation of MMP proteins. In the present study, we used immunohistochemistry to examine the expression levels of claudin-4, MMP-2 and MMP-9 in 189 gastric cancer samples, and analyzed their correlation with tumor invasion, clinicopathologic parameters and clinical outcome. The relationship between claudin-4 expression and MMP-2 and -9 expression was also investigated. The expression of claudin-4 was found to be significantly higher in gastric cancer cases with advanced depth of wall invasion, lymph node metastasis, lymphatic invasion and higher TNM stage. Further analysis revealed claudin-4 expression to be significantly correlated with the expression of MMP-2 and -9. Kaplan-Meier survival analysis indicated that MMP-9 expression was correlated with poor prognosis. These results suggest that claudin-4 expression is associated with tumor invasion and with MMP-2 and -9 expression in gastric cancer. Additionally, MMP-9 expression was demonstrated to serve as a prognostic factor in patients with gastric cancer.

Bone morphogenetic protein-2 (BMP-2), a member of transforming growth factor-beta superfamily, plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that BMP-2 directed the migration and increased cell surface and mRNA expression of beta1 integrin in human chondrosarcoma cancer cells (JJ012). Pretreated of JJ012 cells with phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002) or Akt inhibitor inhibited the BMP-2-mediated migration and integrin expression. BMP-2 increased the phosphorylation of p85 subunit of PI3K and serine 473 of Akt. In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited BMP-2-mediated cells migration and integrin upregulation. Stimulation of JJ012 cells with BMP-2 induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the BMP-2-mediated increasing of IKKalpha/beta phosphorylation, IkappaB phosphorylation, and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85 and Akt mutants also reduced the BMP-2-induced kappaB-luciferase activity. Taken together, these results suggest that the BMP-2 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 integrin and contributing the migration of human chondrosarcoma cells.

Actinobacillus pleuropneumoniae (AP) is the causative agent of swine pleuropneumonia, a fibrinous, exudative, hemorrhagic, necrotizing pleuropneumonia affecting all ages of pigs. Actinobacillus pleuropneumoniae exotoxins (Apx) are one of the major virulence factors of AP. Due to the complex nature of Apx toxins produced by AP, little is known regarding the interactions of individual species of Apx toxin with target cells. The objective of this study was to examine whether AP serotype 10-derived exotoxin, ApxI, caused apoptosis in porcine alveolar macrophages (PAMs) and to delineate the underlying signaling pathways. Isolated PAMs were stimulated with different concentrations of native ApxI and monitored for apoptosis using Hoechst staining, TUNEL, and DNA laddering assays. The ApxI-stimulated PAMs exhibited typical morphological features of apoptosis, including condensation of chromatin, formation of apoptotic bodies and DNA laddering. ApxI-induced apoptosis in a concentration- and time-dependent manner. Furthermore, to delineate the signaling events involved in ApxI-induced apoptosis, it was observed that caspase 3 was activated in ApxI-stimulated PAMs. Ablation of caspase 3 activity via specific inhibitors protected PAMs from apoptosis by ApxI. This study is the first to demonstrate that native ApxI causes apoptosis in PAMs at low concentrations and that these apoptotic events are mediated via a caspase 3-dependent pathway. These findings suggest a role of ApxI in AP infection as it might impair the host defense system through the induction of apoptosis in PAMs.

Matrix metalloproteinases (MMPs) MMP-2 and MMP-9 can degrade type IV collagen of extracellular matrix and basal membranes. Claudin-4 is a member of a large family of transmembrane proteins, claudins, essential in the formation and maintenance of tight junctions. Claudin-4 has been shown to activate MMP-2, indicating that claudin-mediated increased cancer cell invasion might be mediated through the activation of MMP proteins. To explore the roles of MMP-2, MMP-9 and claudin-4 in gastric cancer, we selected 88 cases and then analyzed the expression of these proteins using immunohistochemistry. We found that all of MMP-2, MMP-9 and claudin-4 expressions were significantly higher in intestinal-type than in diffuse-type gastric cancer. On further analysis, testing the relationship between MMP-2 and MMP-9 expression with claudin-4 expression, claudin-4 expression was significantly associated with MMP-9 expression, but not with MMP-2 expression. The results showed that MMP-2, MMP-9 and claudin-4 expression may be phenotypic features, distinguishing intestinal-type and diffuse-type gastric cancer. Possibly, claudin-4 played a role in determining MMP-9 activity which favored intestinal-type gastric cancer to distal metastasis.

The mushrooms of diverse Lingzhi species have been traditionally consumed as luxurious functional food supplements in Chinese society. FIP-gts, a fungal immunomodulatory protein found in Song-Shan Lingzhi (Ganodera tsugae) has been proposed to possess therapeutic effects on cancer and autoimmune diseases. To produce active FIP-gts for evaluation of oral administration, a recombinant FIP-gts (rFIP-gts) fused with a 6His-tag at its C-terminus was expressed in Sf21 insect cells by the baculovirus expression system. High yield (about 70%) and purity (about 90%) of rFIP-gts was obtained by one-step nickel-affinity chromatography. The correctness of the harvested rFIP-gts was verified by Western blot and MALDI-MS analyses. Optimal expression of rFIP-gts was observed when the Sf21 cells were infected with multiplicity of infection of 10 for 72 h, and the yield was up to 47.2 microg/3 x 10(6) infected cells. The immunomodulatory activity of the purified rFIP-gts was detected as the induction of interleukin 2 released from murine splenocytes. Compared with the rFIP-gts produced in Escherichia coli cells, the rFIP-gts produced in Sf21 cells possessed evidently higher specific immunomodulatory activity.

Our previous microarray analysis of gastric cancer found that claudin-4 was differentially expressed between intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC). Claudin-4 is a member of a large family of transmembrane proteins, claudins, essential in the formation and maintenance of tight junctions. To explore the roles of claudin-4 in the two histologically distinct types of gastric cancer, we selected 45 IGC and 48 DGC cases and then analyzed the expression of the protein using immunohistochemistry. We found that the overexpression of claudin-4 was greater in IGC than in DGC. A trend was observed between the overexpression of claudin-4 and lymph node metastasis, however, this association was not statistically significant. The results showed that the expression of claudin-4 was lower in DGC. Possibly it played a role in determining the diffuse phenotype and loose cohesion of cells in DGC in a similar manner as E-cadherin.

The Nisin-controlled gene expression (NICE) system, which was discovered in Lactococcus lactis, was adapted to Lactobacillus reuteri by ligating nisA promoter (PnisA) and nisRK DNA fragments into the Escherichia coli-Lb. reuteri shuttle vector pSTE32. This chimerical plasmid (pNICE) was capable of expressing the heterologous amylase gene (amyL) under nisin induction. Optimization of induction factors for this Lb. reuteri/pNICE system, including nisin concentration (viz. 50 ng/ml), growth phase of culture at which nisin be added (viz. at the early exponential phase), and the best time for analyzing the gene product after inoculation (viz. at the 3rd h), allowed the amylase product to be expressed in high amounts, constituting up to about 18% of the total intracellular protein. Furthermore, the signal peptide (SP) of amyL gene (SPamyL) from Bacillus licheniformis was ligated to the downstream of PnisA in pNICE, upgrading this vector to a NICE-secretion (NIES) level, which was then designated pNIES (Sec+, secretion positive). Characterization of pNIES using an amyL-SPDelta gene (amyL gene lacking its SP) as a reporter revealed the 3rd h after induction as the secretion peak of this system, at which the secretion efficiency and the amount of alpha-amylase being secreted into the culture supernatant were estimated to reach 77.6% and 27.75 mg/l. Expression and secretion of AmyL products by pNIES in Lb. reuteri was also confirmed by SDS-PAGE and immunoblotting analysis.

To compare the gene expression profiling between intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), cDNA microarray containing 7334 gene elements was performed on 12 paired IGC specimens/its' normal epithelial tissue and 11 paired DGC specimens/its' normal epithelial tissue. Twenty-seven genes were co-over-expressed in IGC and DGC. These overexpressed genes were related to transcription and translation, DNA replications and mitosis, calcium binding, apoptosis and mitochondria protein. Twelve genes were co-underexpressed in IGC and DGC. These underexpressed genes were associated with cell adhesion and migration, organelle movement and intracellular transport, and matrix metalloproteinase. A clustering dendrogram of IGC and DGC with 27 genes significantly differed between IGC and DGC. Nineteen genes were more overexpressed in DGC than in IGC, including annexin Al (ANXA1), chemokine ligand 7 (CCL7), and chemokine ligand 8 (CCL8). Eight genes were more overexpressed in IGC than in DGC, including claudin 4 (CLDN4). The results of quantitative real-time PCR and immunohistochemical staining confirmed the microarray finding. The gene expression profiling between IGC and DGC suggested that they might have unique genetic pathways which share some of the same and some different genetic alterations.

A signal peptide (SP)-probe vector pNICE-gfpSP, which employed a green fluorescent protein (Gfp) as the SP-selection marker, was constructed for use in Lactobacillus reuteri. This chimerical plasmid allowed cloning and screening DNA fragments with the SP function by direct visualization of the expressed fluorescence activity around cells. Assay of fluorescent intensity in their culture supernatant with spectrofluorometry further enabled quantifying the secretion efficiency of the identified SP fragment.