Development of SRAP, SRAP-RGA, RAPD and SCAR markers linked with a Fusarium wilt resistance gene in eggplant

Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melongena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F₂ and BC₁ populations of eggplant segregating for fusarium wilt resistance was screened with 2,316 primer combinations to detect polymorphism. Three markers were linked within 2.6 cM of the gene. The codominant SRAP marker Me8/Em5 and dominant SRAP-RGA marker Em12/GLPL2 were tightly linked to each other and mapped 1.2 cM from the resistance gene, whereas RAPD marker H12 mapped 2.6 cM from the gene and on the same side as the other two markers. The SRAP marker was converted into two dominant SCAR markers that were confirmed to be linked to the resistance gene in the F_2, BC₁ and F₂ of BC₃ generations of the same cross. These markers provide a starting point for mapping the eggplant FOM resistance gene in eggplant and for exploring the synteny between solanaceous crops for fusarium wilt resistance genes. The SCAR markers will be useful for identifying fusarium wilt-resistant genotypes in marker-assisted selection breeding programs using segregating progenies of the resistant eggplant progenitor used in this study.     

Pilot-scale adenovirus seed production through concurrent virus release and concentration by hollow fiber filtration

Recovery of recombinant adenoviruses from infected mammalian cell cultures often requires multiple unit operations such as cell lysis for virus release, microfiltration for clarification, and ultrafiltration for concentration. While development of these multiple unit operations is relatively straightforward, implementation under aseptic conditions in a closed system can be challenging for the production of virus seed at industrial scales. In this study, we have developed a simple, single-step, scaleable process to effectively recover adenoviruses from infected PER.C6 cell cultures for the production of concentrated adenovirus seeds under aseptic conditions. Specifically, hollow fiber tangential flow filtration technology was applied to maximize cell lysis of infected cultures for virus release while simultaneously concentrating the virus to an appropriate level of volume reduction. Hollow fiber filters with small lumen diameter of 0.5 mm were chosen to maximize the wall shear for a highly effective cell lysis and virus release. Cell lysis and virus release were shown to correlate with the exposure time in the hollow fiber cartridge: the shear zone. In most cases, a virus recovery yield of more than 80% and a 15- to 20-fold concentration (or up to 95% volume reduction) was achieved in less than 2 h of processing time. The virus seeds prepared using this process at lab scale and at 300-L scale without clarification have been successfully tested for sterility and potency and used for subsequent infection with consistent virus productivity. This process should enable rapid production of adenovirus seeds with minimal unit operations and high efficiency recovery for adenovirus production at 1000-L scale.     

Immobilization of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> β-galactosidase on low-pressure plasma-modified cellulose acetate membrane using polyethyleneimine for production of galactooligosaccharide

The aim of this study was to produce galactooligosaccharides (GOS) from lactose using β-galactosidase from Aspergillus oryzae immobilized on a low-pressure plasma-modified cellulose acetate (CA) membrane. Specifically, a novel method was developed for multilayer enzyme immobilization involving polyethyleneimine (PEI)-enzyme aggregate formation and growth on a CA membrane. A large amount of enzyme (997 μg/cm² membrane) was immobilized with 66% efficiency. The K _m value for the immobilized enzyme was estimated to be 48 mM, which indicates decreased affinity for the substrate, whereas the Vmax value was smaller. The immobilized enzyme showed good storage and operational stability. The half-life of the immobilized enzyme on the membrane was about 1 month at 30°C and ∼ 60 h at 60°C. Maximum GOS production of 27% (w/w) was achieved with 70% lactose conversion from 320 g/L of lactose at pH 4.5 and 60°C. Trisaccharides were the major types of GOS formed and accounted for about 75% of the total GOS produced. Based on these results, immobilized enzyme technology could be applied to GOS production from lactose.     

Determination of oxidant stress in plasma of rheumatoid arthritis and primary osteoarthritis patients

Determination of oxidant stress in plasma of rheumatoid arthritis (RA) and primary osteoarthritis (POA) patients is important in understanding the pathogenesis of these diseases. In this study, we examined the relationship between oxidant stress and inflammation by measuring protein carbonyl content, thiol levels and plasma protein fractions as the oxidation markers and erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) tests as inflammation markers. Protein carbonyls content was higher in RA and POA patients, as compared to controls (p<0.0001), while the plasma thiol levels in both groups of patients were significantly lower than controls (p<0.0001). Increased levels of proteins under 40 kDa molecular mass were detected in the RA and POA patients compared to that of controls (p<0.0001) both in HPLC and SDS-PAGE analysis. Total protein concentration in plasma of RA patients was higher than the controls (p<0.001), while in POA patients was lower than that of controls (p<0.001). ESR and CRP levels were higher in both the patient groups than the normal group (p<0.001). These results suggested that alterations in the oxidant stress markers could be the cause of inflammation in these diseases. Thus, while working for RA/POA treatment strategies, consideration of the relationship between oxidant stress and inflammation would be worth evaluating.