Prismatic Silver Nanoparticles Decorated on Graphene Oxide Sheets for Superior Antibacterial Activity.

Spherical silver nanoparticles (Ag NPs) and silver nanoprisms (Ag NPrsms) were synthesized and decorated on graphene oxide (GO) nanosheets. The Ag contents were 29% and 23% in the GO−Ag NPs and GO−Ag NPrsms, respectively. The Ag NPrsms exhibited stronger (111) crystal signal than Ag NPs. The GO−Ag NPrsms exhibited higher Ag (I) content (75.6%) than GO-Ag NPs (69.9%). Increasing the nanomaterial concentration from 25 to 100 µg mL−1 improved the bactericidal efficiency, and the antibacterial potency was in the order: GO−Ag NPrsms > GO−Ag NPs > Ag NPrsms > Ag NPs > GO. Gram-positive Staphylococcus aureus (S. aureus) was more vulnerable than Gram-negative Escherichia coli (E. coli) upon exposure to these nanomaterials. The GO−Ag NPrsms demonstrated a complete (100%) bactericidal effect against S. aureus at a concentration of 100 µg mL−1. The GO−Ag composites outperformed those of Ag or GO due to the synergistic effect of bacteriostatic Ag particles and GO affinity toward bacteria. The levels of reactive oxygen species produced in the bacteria−nanomaterial mixtures were highly correlated to the antibacterial efficacy values. The GO−Ag NPrsms are promising as bactericidal agents to suppress biofilm formation and inhibit bacterial infection.

Size-Dependent Antibacterial Activity of Silver Nanoparticle-Loaded Graphene Oxide Nanosheets.

A series of graphene oxide (GO) suspensions with different particle sizes (<100 nm, ~100 nm, ~1 µm and >1 µm) were successfully fabricated after 0, 30, 60 and 120 min of sonication, respectively. The antibacterial properties of GO suspensions showed that >1 µm GO size resulted in a loss of nearly 50% of bacterial viability, which was higher than treatment by ~100 nm GO size (25%) towards Escherichia coli (E. coli). Complete entrapment of bacteria by the larger GO was observed in transmission electron microscopy (TEM). Silver nanoparticles (Ag NPs) were doped onto GO samples with different lateral sizes to form GO-Ag NP composites. Resulting larger GO-Ag NPs showed higher antibacterial activity than smaller GO-Ag NPs. As observed by Fourier transform infrared spectroscopy (FTIR), the interaction between E. coli and GO occurred mainly at the outer membrane, where membrane amino acids interact with hydroxyl and epoxy groups. The reactive oxygen species (ROS) and the considerable penetration of released Ag+ into the inner bacterial cell membrane result in loss of membrane integrity and damaged morphology. The present work improves the combined action of GO size effect with constant Ag loadings for potential antibacterial activity.

Synergistic Antibacterial Activity of Silver-Loaded Graphene Oxide towards Staphylococcus Aureus and Escherichia Coli.

In this study, the physicochemical and surface properties of the GO-Ag composite promote a synergistic antibacterial effect towards both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. Aureus) bacteria. GO-Ag NPs have a better bactericidal effect on E. coli (73%) and S. Aureus (98.5%) than pristine samples (pure Ag or GO). Transmission electron microscopy (TEM) confirms that the GO layers folded entire bacteria by attaching to the membrane through functional groups, while the Ag NPs penetrated the inner cell, thus damaging the cell membrane and leading to cell death. Cyclic voltammetry (CV) tests showed significant redox activity in GO-Ag NPs, enabling good catalytic performance towards H2O2 reduction. Strong reactive oxygen species (ROS) in GO-Ag NPs suggests that ROS might be associated with bactericidal activity. Therefore, the synergy between the physicochemical effect and ROS production of this material is proposed as the mechanism of its antibacterial activity.

Genetic engineering of transitory starch accumulation by knockdown of OsSEX4 in rice plants for enhanced bioethanol production.

Rice straw, a common agricultural waste, is used as a potential feedstock for bioethanol production. Currently, bioethanol is made mostly from the microbial fermentation of starch-containing raw materials. Therefore, genetically engineered starch-excess rice straw through interference of starch degradation as a potential strategy to enhance bioethanol production was evaluated in this study. Arabidopsis Starch Excess 4 (SEX4) encodes a chloroplast-localized glucan phosphatase and plays a role in transitory starch degradation. Despite the identification of a SEX4 homolog in rice, OsSEX4, its biological function remains uncertain. Ectopic expression of OsSEX4 complementary DNA complemented the leaf starch-excess phenotype of the Arabidopsis sex4-4 mutant. OsSEX4-knockdown transgenic rice plants were generated using the RNA interference approach. Starch accumulation was higher in OsSEX4-knockdown suspension-cultured cells, leaves, and rice straw compared with the wild type, suggesting that OsSEX4 plays an important role in degradation of transitory starch. The OsSEX4-knockdown rice plants showed normal plant growth and no yield penalty. Starch-excess OsSEX4-knockdown rice straw used as feedstock for fermentation resulted in improved bioethanol yield, with a 50% increase in ethanol production in a vertical mass-flow type bioreactor, compared with that of the wild-type straw.

Fabrication of Cellulose Nanocrystal/Silver/Alginate Bionanocomposite Films with Enhanced Mechanical and Barrier Properties for Food Packaging Application.

Eco-friendly cellulose nanocrystal/silver/alginate (CNC/Ag/Alg) bionanocomposite films were successfully prepared by blending of CNC with Ag/Alg solution. The CNC was fabricated from cellulose microcrystal (CMC) by acid hydrolysis method. The Ag nanoparticles (AgNPs) were generated by using Alg as a reducing agent through hydrothermal process. AgNPs-included composite films showed characteristic plasmonic effect of the AgNPs with the maximum absorption at 491 nm and they also showed high ultraviolet (UV) barrier properties. The CNC/Ag/Alg composite films were analyzed by using scanning electron microscopy, transmission electron microscopy, optical microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction technique. Depending on the type of nanofillers, tensile strength of the composite films increased by 39-57% and water vapor permeation decreased by 17-36% compared with those of the neat Alg films. The Ag/Alg and CNC/Ag/Alg films showed brown color as detected from the increase of both 'b' and 'a' parameters by colorimeter. The UV and water barrier properties of Alg based composite films were found higher than the Alg films. The obtained results suggested that the prepared composite films can be used in food packaging applications.

Antrodia cinnamomea mycelial fermentation broth inhibits the epithelial-mesenchymal transition of human esophageal adenocarcinoma cancer cells.

Esophageal cancer is associated with a high mortality rate and easy metastasis. The aim of this study is to investigate the effect of the bio-product Antrodia cinnamomea mycelial fermentation broth (AC-MFB) on the epithelial mesenchymal transition (EMT) of human esophageal cancer cells and the molecular mechanisms underlying these effects. Transforming growth factor ß (TGF-ß) was used to induce EMT in human esophageal BE3 cancer cells. Changes in cell morphology and migration potential were examined. The expression of E-cadherin, N-cadherin, vimentin, and other transcriptional factors was studied by western blot assay. The results showed that AC-MFB was not only able to upregulate the expression of Ecadherin and attenuate the TGF-ß-induced overexpression of vimentin and N-cadherin, but it also reversed the TGF-ß-induced changes in cell morphology from polygonal to spindle-shaped and delayed the migration potential of BE3 cells. Furthermore, AC-MFB treatment was able to inhibit the expression levels of both Twist and Twist1. Overall, AC-MFB was able to inhibit the EMT of esophageal cancer BE3 cells, which was accompanied by Twist and Twist1 downregulation.

Production of bioethanol from Napier grass via simultaneous saccharification and co-fermentation in a modified bioreactor.

The aim of this study was to use a modified bioreactor system for simultaneous saccharification of cellulose and bioethanol production. We tested Aspergillus niger and Trichoderma reesei for cellulose saccharification and Zymomonas mobilis for bioethanol production simultaneously in this modified bioreactor. The results showed that various carboxymethylcellulose (CMC) concentrations (10, 15, or 20 g/L) as a substrate for A. niger and T. reesei yielded bioethanol production of 0.51, 0.78, and 0.89 g/L and a CMC conversion rate of 10.2%, 10.7%, and 8.89%, respectively. These data suggested that at 10 g/L CMC as a substrate, the CMC conversion rate was higher than that at the other concentrations. When CMC concentration exceeded 15 g/L, bioethanol production was prolonged to 40 h. These results were attributed to the viscosity of CMC. This study also tested Napier grass (an agricultural byproduct) for bioethanol production. The results revealed bioethanol production and the theoretical Napier grass conversion rate were 0.38 g/L and 12.6%, respectively, for 13-h cultivation when the feeding concentration of Napier grass was 10 g/L. When Napier grass concentration was increased to 15 g/L, bioethanol production and the Napier grass conversion rate reached 0.51 g/L and 23%, respectively, after 14-h cultivation. Eventually, the experimental results indicated using agricultural waste for bioethanol production has been become a potential strategy.

The medicinal fungus Antrodia cinnamomea regulates DNA repair and enhances the radiosensitivity of human esophageal cancer cells.

This study investigated the adjunctive effects of Antrodia cinnamomea mycelial fermentation broth (AC-MFB), a Taiwanese medicinal fungus, in enhancing the radiosensitivity of esophageal cancer cells. Human CE81T/VGH squamous and BE3 adenocarcinoma esophageal cancer cells were used in this study. A colony formation assay showed that pretreatment with AC-MFB decreased the survival of irradiated esophageal cancer cells, with a maximum sensitizer enhancement ratio of 1.91% and 37% survival. A DNA histogram study showed that AC-MFB pretreatment enhanced cell cycle arrest at the G2/M phase, the most radiosensitive phase. An immunofluorescence assay and a Western blotting assay showed that AC-MFB delayed the abrogation of γ-H2AX, upregulated p21 expression, and attenuated the radiation-induced phosphorylation of ataxia telangiectasia-mutated kinase and checkpoint kinase 2. An in vivo validation study showed that AC-MFB treatment tended to have a synergistic effect with radiation on the tumor growth delay of CE81T/VGH cells in BALB/c mice. These data suggest that this edible fungus product could enhance the effect of radiotherapy against esophageal cancer.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

Enhancement of recombinant human serum albumin in transgenic rice cell culture system by cultivation strategy.

Fusion of the sugar-starvation-induced αAmy3 promoter with its signal peptide has enabled secretion of recombinant human serum albumin (rHSA) into the culture medium. To simplify the production process and increase the rHSA yield in rice suspension cells, a one-step strategem without medium change was adopted. The yield of rHSA was increased sixfold by this one-step approach compared with the two-step recombinant protein process, in which a change of the culture medium to sugar-free medium is required. The one-step strategem was applied to check repeated cycle of rHSA production, and the production of rHSA was also higher in each cycle in the one-step, as opposed to the two-step, production process. The use of the one-step process resulted in fewer damaged cells during the cell sugar starvation phase for recombinant protein production. Furthermore, we scaled up the rHSA production in a 2-L airlift and a 2-L stirred tank bioreactor by the one-step approach, and concluded that rHSA can be enriched to 45 mg L(-1) in plant culture commonly used MS medium by the airlift-type bioreactor. Our results suggest that rHSA production can be enriched by this optimized cultivation strategem.

Ethanol extracts of Cinnamomum kanehirai Hayata leaves induce apoptosis in human hepatoma cell through caspase-3 cascade.

Inducing apoptosis to susceptible cells is the major mechanism of most cytotoxic anticancer drugs in current use. Cinnamomum kanehirai Hayata (Lauraceae), a unique and native tree of Taiwan, is the major host for the medicinal fungus Antrodia cinnamomea which exhibits anti-cancer activity. Because of the scarcity of A. cinnamomea, C. kanehirai Hayata instead, is used as fork medicine in liver cancer. Here we observed the C. kanehirai Hayata ethanol extract could inhibit the cellular viability of both HepG2 and HA22T/VGH human hepatoma cell lines in a dose- and time-dependent manner. We found the mode of cell death was apoptosis according to cell morphological changes by Liu's stain, oligonucleosomal DNA fragmentation by gel electrophoresis, externalization of phosphotidyl serine by detecting Annexin V and hypoploid population by cell cycle analysis. Our results showed that the extracts caused cleavage of caspase-3 and increased enzyme activity of caspase-8 and caspase-9. Caspase 3 inhibitor partially reversed the viability inhibition by the extract. Furthermore, the up-regulation of Bax and down-regulation of Bcl-2 were also noted by the extract treatment. In conclusion, C. kanehirai Hayata ethanol extract induced intrinsic pathway of apoptosis through caspase-3 cascade in human hepatoma HA22T/VGH and HepG2 cells, which might shed new light on hepatoma therapy.

Medicinal Fungus Antrodia cinnamomea Inhibits Growth and Cancer Stem Cell Characteristics of Hepatocellular Carcinoma.

Background. Antrodia cinnamomea is an edible fungus commonly used in Asia as a well-known medicinal herb capable of treating drug intoxication and liver cancer. Methods. This study evaluated the anticancer activity of its biotechnological product, mycelial fermentation broth (AC-MFB) on hepatocellular carcinoma (HCC) by tetrazolium-based colorimetric assay in vitro and syngeneic Balb/c 1MEA.7R.1 tumor implantation model in vivo. Given that cancer stem cell characteristics, such as angiogenesis, invasiveness, and migration, are known to cause recurrence, we further evaluated the effect of AC-MFB on cellular viability inhibition of HCC cells, angiogenic activity and migration of endothelial cells, and the release of proangiogenic factors from HCC cells. Results. We found that AC-MFB markedly inhibited the growth of HCC without hepatic enzyme abnormality. This anti-HCC activity was validated by growth-inhibitory effects on both cultured murine 1MEA.7R.1 and human HA22T/VGH HCC cells. For cancer stem cell characteristics, AC-MFB inhibited the cellular viability, migration, and tube formation activity of EA. hy926 and SVEC4-10 endothelial cells. Production of extracellular vascular endothelial growth factor and intracellular hypoxia-inducible factor-1 alpha from HCC cells was suppressed by AC-MFB. Conclusion. Antrodia cinnamomea could inhibit the growth and cancer stem cell characteristics of HCC cells.

Producing bioethanol from cellulosic hydrolyzate via co-immobilized cultivation strategy.

Lignocellulose was converted into reducing sugars by using saccharification enzymes from cocultivated Trichoderma reesei and Aspergillus niger and reducing sugars as nutrients for Zymomonas mobilis to produce bioethanol in an immobilization system. After 96 h of cultivation, cocultivated T. reesei and A. niger had enzymatical synergistic effects that enabled a reducing sugar production of 1.29 g/L and a cellulose conversion rate of 23.27%. An 18% total inoculum concentration and a 1/1 inoculation ratio of T. reesei to A. niger obtained a reducing sugar production rate and a cellulose conversion rate of 2.57 g/L and 46.27%, respectively. The co-immobilization cultivation results showed that using polyurethane as a carrier optimized total saccharification enzyme activity at an inoculum ratio of 1/1 and a total inoculum concentration of 6.5×10(6)spores/mL. Based on the experimental results, the bioreactor design was further modified to enhance bioethanol production. The three strains (A. niger, T. reesei and Z. mobilis) were cocultivated with a co-immobilization cultivation system. The experimental results showed that, after 24 h cultivation, bioethanol production reached 0.56 g/L, and reducing sugar conversion rate reached 11.2% when using carboxymethylcellulose (CMC) substrates. The experimental results confirmed that the modified bioreactor enhances bioethanol production. However, further experiments are needed to determine how to prevent multi-stage failure of reducing medium volume.

Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture.

To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.

Using a fed-batch culture strategy to enhance rAAV production in the baculovirus/insect cell system.

Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI=5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be 1x10(6) cells/ml, and the highest rAAV yield (1.22x10(8) IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13x10(8) IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40x10(8) IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production.

Inhibition of acidic mammalian chitinase by RNA interference suppresses ovalbumin-sensitized allergic asthma.

Asthma, a chronic helper T cell type 2-mediated inflammatory disease, is characterized by airway hyperresponsiveness and inflammation. Growing evidence suggests that increased expression of acidic mammalian chitinase (AMCase) may play a role in the pathogenesis of asthma. In the present study, we sought to develop an RNA interference approach to suppress allergic asthma in mice through silencing of AMCase expression. Mice sensitized with ovalbumin (OVA) were intratracheally administered a recombinant adeno-associated virus expressing short hairpin RNA (rAAV-shRNA) against AMCase. In OVA-sensitized mice, the development of allergic symptoms was significantly associated with elevated AMCase expression. After administration of rAAV-shRNA, there was a significant reduction of AMCase expression in the lung and in bronchoalveolar lavage fluid (BALF) cells of sensitized mice. Sensitized mice receiving rAAV-shRNA showed a significant improvement in allergic symptoms, including airway hyperresponsiveness (AHR), eosinophil infiltration, eotaxin, interleukin-13 secretion in BALF, and serum OVA-specific IgE level. Our data suggest the hyperexpression of AMCase in asthma can be suppressed by rAAV-mediated shRNA. Silencing AMCase expression by shRNA may be a promising therapeutic strategy in asthma.

Production of human serum albumin by sugar starvation induced promoter and rice cell culture.

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice alpha Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the alpha Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2-4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the alpha Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.