Comparative observation of freeze–thaw-induced damage in pig, rabbit, and human corneal stroma

Objective  Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze–thaw-induced damage in keratocytes and collagen in rabbit, pig, and human corneas.
Animals studied  Rabbit, pig, and human.
Procedures  We prepared 250-μm-thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250-μm-thick corneal stroma without epithelium was placed in a 50-mL tube, frozen with liquid N₂ for 15 min and taken out to thaw rapidly at 37 °C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin–eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze–thaw cycle into rabbit eyes and evaluated graft survival.
Results  On gross examination, rabbit corneas became opaque after the triple freeze–thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month.
Conclusions  Our study showed that rabbit corneal stroma was more susceptible to freeze–thaw injury than pig and human corneas.     

Epidemiology of H5N1 avian influenza

High pathogenic (HP) H5N1 avian influenza (AI) infection has been reported in domestic poultry, wildlife, and human populations since 1996. Risk of infection is associated with direct contact with infected birds. The mode of H5N1 spread from Asia to Europe, Africa and the Far East is unclear; risk factors such as legal and illegal domestic poultry and exotic bird trade, and migratory bird movements have been documented. Measures used to control disease such as culling, stamping out, cleaning and disinfection, and vaccination have not been successful in eradicating H5N1 in Asia, but have been effective in Europe.     

Schisandrin B co-treatment ameliorates the impairment on mitochondrial antioxidant status in various tissues of long-term ethanol treated rats

The effect of schisandrin B (Sch B) on long-term ethanol-induced oxidative stress in various rat tissues was investigated. Long-term ethanol treatment increased reactive oxygen metabolites (ROM) level in plasma. The ethanol-induced oxidative stress was assessed by mitochondrial glutathione and α-tocopherol levels, antioxidant enzyme activities, malondialdehyde (mtMDA) production and heat shock protein (Hsp) 25/70 levels. Liver was most susceptible to oxidative stress with a significant increase in mtMDA production. Long-term Sch B treatment enhanced mitochondrial antioxidant status in a tissue non-specific manner. Sch B co-treatment ameliorated the alterations in plasma ROM levels, mtMDA production and Hsp 25/70 expression in rat tissues.     

Mass production of rubusoside using a novel stevioside-specific β-glucosidase from Aspergillus aculeatus

Rubusoside (R) is a natural sweetener and a solubilizing agent with antiangiogenic and antiallergic properties. However, currently, its production is quite expensive, and therefore, we have investigated nine commercially available glycosidases to optimize an economically viable R-production method. A stevioside (ST)-specific β-glucosidase (SSGase) was selected and purified 7-fold from Aspergillus aculeatus Viscozyme L by a two-step column chromatography procedure. The 79 kDa protein was stable from pH 3.0 to pH 7.0 at 50-60 °C. Hydrolysis of ST by SSGase produced R and steviol monoglucosyl ester as determined by (1)H and (13)C nuclear magnetic resonance (NMR). Importantly, SSGase showed higher activity toward ST than other β-linked glucobioses. The optimal conditions for R production were 280 mM ST and 16.6 μL of SSGase at pH 5.1 and 63 °C. This is the first discussion detailing the production of R by enzymatic hydrolysis of ST and is useful for the food additive and pharmaceutical industries.     

High-efficiency regnerationin vitro from potato petioles with intact leaflets

The shoot/plantlet regenerationin vitro of seven potato (Solanum tuberosum L.) cultivars from petioles with intact leaflets was assessed using six treatment combinations-a basal medium with or without silver thiosul-phate (STS) or thidiazuron (TDZ) at two concentrations (2 or 0.5 mg/l) of the indoleacetic acid (IAA). The basal medium consisted of Murashige and Skoog (MS) salts and vitamins supplemented with 3 mg/1 6-benzylaminopurine, and 1 mg/1 gibberellic acid, 30 g/l sucrose, and 7.0 g/l PHYTOAGAR. Two full sets repeats and one partial set repeat of independent experiments were conducted and all produced similar results. Silver thiosulphate decreased the regeneration frequency and number of shoots per callus. No significant changes were observed with thidiazuron. Regeneration rates of (100% ) with up to 20 shoots/plantlets per callus were achieved at 2 mg/1 IAA with cultivars Désirée, Kennebec, Niska, and Lenape. These cultivars still showed high regeneration rates (87%–98% ) on media with 0.5 mg/1 IAA, and good regeneration rates were also achieved by the other three cultivars (48%, 94%, and 50% for Chieftain, Russet Burbank, and Shepody, respectively). Even with the single medium protocol (0.5% IAA without thiosulphate or thidiazuron), Désirée, Lenape, and Niska exhibited a regeneration rate of 98%. The use of petiole-with-leaflet explants could be ideal for the regeneration step of potato genetic transformation protocol because of their high regeneration efficiency and their small cut surface area forAgrobacterium elimination after co-incubation.     

Processing of microcellular nanocomposite foams by using a supercritical fluid

Polystyrene/layered silicate nanocomposites were prepared by melt intercalation. To examine the distribution of the clay in polymer matrix, small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) were used. Intercalated nanocomposites were obtained and their rheological properties were investigated. Microcellular nanocomposite foams were produced by using a supercritical fluid. As clay contents increased, the cell size decreased and the cell density increased. It was found that layered silicates could operate as heterogeneous nucleation sites. As the saturation pressure increased and the saturation temperature decreased, the cell size decreased and the cell density increased. Microcellular foams have different morphology depending upon the dispersion state of nanoclays.     

A Novel Pretreatment Method of Lignocellulosic Material as Adsorbent and Kinetic Study of Dye Waste Adsorption

Sulphuric acid-modified bagasse has been used as low-cost adsorbent for the removal of methylene blue (MB) dye from aqueous solution. In order to remove organic compounds that contribute to chemical oxygen demand (COD), pretreatment with thorough washing of adsorbent using boiling distilled water was performed instead of conventional washing using distilled water at room temperature only. This has resulted in the highest efficiency of color removal of 99.45% and COD reduction of 99.36% for MB dye solution at pH 9. Effects of initial pH, dye concentration, adsorbent dosage, temperature, and contact time have been studied. The adsorption of MB dye was pH dependent. Langmuir and Freundlich isotherm models were tested on the adsorption data. The kinetic experimental data were analyzed using pseudo-first order, pseudo-second order, and the intraparticle diffusion model in order to examine the adsorption mechanisms. The adsorption process followed the Langmuir isotherm as well as the Freundlich isotherm and pseudo-second-order kinetic model. The process was found to be endothermic in nature.     

Effects of AMF inoculation on growth of Panax ginseng C.A. Meyer seedlings and on soil structures in mycorrhizosphere

We aimed to investigate the effects of inoculating Panax ginseng C.A. Meyer seedlings with arbuscular mycorrhizal fungi (AMF) by examining the root colonization, plant nutrition uptake, growth characteristics, and soil aggregation of P. ginseng seedlings inoculated at the time of transplantation. At 16 weeks, the AMF spore density per 30 g of fresh mycorrhizosphere in seedlings inoculated with AMF (AMF+ seedlings) was 256.8 and that in seedlings not inoculated with AMF (AMF− seedlings) was 186.3, respectively. The colonization rate of AMF in the lateral roots of AMF+ seedlings was approximately 19% higher than that in the lateral roots of AMF− seedlings. The patterns of AMF colonization in ginseng roots were similar to those of the Paris-type mycorrhizal association. Plant growth characteristics, such as plant height, root length, leaf area, number of lateral roots, fresh weight of shoots and roots, and chlorophyll content, were significantly enhanced in AMF+ seedlings compared to AMF− seedlings. The macronutrient content (P, K, and Ca) and micronutrient content (Cu, Fe, and Zn) of both shoots and roots were also significantly higher in AMF+ seedlings compared to AMF− seedlings. Furthermore, glomalin content and soil aggregation were significantly enhanced in AMF inoculated areas. Our results indicate that AMF inoculation may enhance the growth of ginseng seedlings by improving the uptake of mineral nutrients and the soil structure in mycorrhizosphere.