Characterization of a microbial transglutaminase cross-linked type II collagen scaffold

This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of ε-(γ-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 ± 0.03 kPa) compared to controls (0.21 ± 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase cross-linked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications. © Mary Ann Liebert, Inc.

The microbial diagnosis of infective endocarditis

This review suggests an evidence-based algorithm for sequential testing in infective endocarditis. It discusses blood culture and the merits and drawbacks of serology in making the diagnosis. Newer techniques are briefly reviewed. The proposed algorithm will complement the Duke criteria in clinical practice. © 2003 The British Infection Society. Published by Elsevier Science Ltd. All rights reserved.

Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield:the use of a respiratory strain as a microbial cell factory

BACKGROUND: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. RESULTS: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. CONCLUSIONS: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.

Inhibition of microbial colonization of shipboard fuel systems

The aim of the investigation was to study the problem of colonization of shipboard fuel systems and to examine the effect of a number of environmental factors on microbial growth and survival in order to find potential preservative treatments. A variety of microbial species were isolated from samples taken from fuel storage tanks. Bacteria were more numerous than yeasts or fungi and most microorganisms were found at the fuel/water interface. 1he salinity, pH and phosphate concentration of some water bottoms were characteristic of sea water. Others were brackish, acidic and varied in phosphate content. Microorganisms were cultured under a number of environmental conditions. After prolonged incubation, the inoculum size had no effect on the final biomass of Cladosporium resinae but the time required to achieve the final mass decreased with increasing spore number. Undecane supported better growth of the fungus than diesel fuel and of four types of diesel fuel, two allowed more profuse growth. With sea water as the aqueous phase, a number of isolates were inhibited but the addition of nutrients allowed the development of many of the organisms. Agitation increased the growth of C. resinae on glucose but inhibited it on hydrocarbons. The optimum temperature fgr growth of C. resinae on surface culture lay between 25º C and 30º C and growth was evident at 5º C but not at 45º C. In aqueous suspension, 90% of spores were inactivated in around 60 hours at 45ºC and the same proportion of spores of C. resinae and Penicillium corylophilum were destroyed after about 30 seconds at 65ºC. The majority of bacteria and all yeasts in a water bottom sample were killed within 10 seconds at this temperature. An increase in the concentration of an organo-boron compound caused more rapid inactivation of C. resinae spores and raising the temperature from 25ºC to 45°C significantly enhanced the potency of the biocide.