High-level production of animal-free recombinant transferrin from saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin.</p> <p>Results</p> <p>The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from <it>Saccharomyces cerevisiae </it>strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin^®USP-NF) and albumin fusion proteins (Novozymes' albufuse^®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin.</p> <p>Conclusions</p> <p>A <it>Saccharomyces cerevisiae </it>expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.</p

The effect of riparian restoration on channel complexity and soil nutrients

International audienceRestoration of riparian vegetation may reduce nutrient and sediment contamination of waterways while potentially enhancing stream channel complexity. Accordingly, the present study used a paired-site approach to investigate the effects of mature regrowth riparian vegetation on river channel morphology and soil nutrients (i.e. nitrogen and phosphorus), comparing four sites of degraded (pasture) and reforested reaches. A revised rapid assessment of riparian condition (RARC) was used to validate the site pairings. Riparian soil nutrient and elemental geochemistry were compared between paired sites, along with two parameters of channel width complexity and two for channel slope complexity. The RARC analysis confirmed the validity of the paired site design. The elemental geochemistry results indicated that underlying geology may affect the paired site analyses. Reaches with mature regrowth vegetation had greater channel width complexity but no difference in their riverbed slope complexity. In addition, degraded reaches had higher soil nutrient (i.e. nitrogen and phosphorus) concentrations, potentially indicative of the greater nutrient retention of pasture grass sites compared with mature regrowth forested reaches with less ground cover. Overall, the present study indicates that restoring mature regrowth riparian vegetation may increase river channel width complexity, although it may require canopy management to optimise the nutrient retention potential necessary to maximise the effect of riparian restoration strategies on freshwater environments

Prioritizing management actions for the conservation of freshwater biodiversity under changing climate and land-cover

Freshwater ecosystems are declining under climate change and land-use change. To maximize the return on investment in freshwater conservation with limited financial resources, managers must prioritize management actions that are most cost-effective. However, little is known about what these priorities may be under the combined effects of climate and land-cover change. We present a novel decision-making framework for prioritizing conservation resources to different management actions for the conservation of freshwater biodiversity. The approach is novel in that it has the ability to model interactions, rank management options for dealing with conservation threats from climate and land-cover change, and integrate empirical data with expert knowledge. We illustrate the approach using a case study in South East Queensland (SEQ), Australia under climate change, land-cover change and their combined effects. Our results show that the explicit inclusion of multiple threats and costs results in quite different priorities than when costs and interactions are ignored. When costs are not considered, stream and riparian restoration, as a single management strategy, provides the greatest overall protection of macroinvertebrate and fish richness in rural and urban areas of SEQ in response to climate change and/or urban growth. Whereas, when costs are considered, farm/land management with stream and riparian restoration are the most cost-effective strategies for macroinvertebrate and fish conservation. Our findings support riparian restoration as the most effective adaptation strategy to climate change and urban development, but because it is expensive it may often not be the most cost-efficient strategy. Our approach allows for these decisions to be evaluated explicitly. (C) 2016 Elsevier Ltd. All rights reserved

An evaluation of owner expectation on apparent treatment effect in a blinded comparison of 2 homeopathic remedies for firework noise sensitivity in dogs.

A blinded and randomized study was carried out to investigate whether dog owners would report different treatment effects depending on whether they knew they might be administering a placebo, versus if they knew they were definitely administering a homeopathic remedy. A secondary aim was to determine the consistency of owner reports of treatment effect across multiple trial periods. A total of 73 dogs with a stable, predictable, and easily assessable response to firework noises were enrolled and randomly allocated to receive 1 of the 2 homeopathic preparations along with a basic behavior modification program. Treatment A was a homeopathic treatment formulated for firework noise sensitivity that had previously been tested in a placebo-controlled study by the authors, and Treatment B was a different formulation for the same condition. The same allocated treatment was trialed on 2 occasions by all participants to allow assessment of owner-report reliability. It was found that knowledge of participating in a placebo-controlled trial had no effect on the owners’ perception of treatment effect, and that their reports of effect were consistent across both trial periods. No specific effect of homeopathic treatment was found in this study; however, it was observed that the reported behavioral effects that followed each treatment were similar across the 2 firework periods, but that there was a consistently different pattern of behavioral effects reported between Treatment groups A and B. These results might be ascribed to either a treatment or population effect. We suggest that examination of the consistency of owner-reported effects within and between treatments may be used as part of the suite of methodologies available to investigate whether any specific effect can be ascribed to homeopathic intervention

Anomalous Diffusion Reports on the Interaction of Misfolded Proteins with the Quality Control Machinery in the Endoplasmic Reticulum

A multitude of transmembrane proteins enters the endoplasmic reticulum (ER) as unfolded polypeptide chains. During their folding process, they interact repetitively with the ER's quality control machinery. Here, we have used fluorescence correlation spectroscopy to probe these interactions for a prototypical transmembrane protein, VSVG ts045, in vivo. While both folded and unfolded VSVG ts045 showed anomalous diffusion, the unfolded protein had a significantly stronger anomaly. This difference subsided when unfolded VSVG ts045 was in a complex with its chaperone calnexin, or when a mutant form of VSVG ts045 with only one glycan was used. Our experimental data and accompanying simulations suggest that the folding sensor of the quality control (UGT1) oligomerizes unfolded VSVG ts045, leading to a more anomalous/obstructed diffusion. In contrast, calnexin dissolves the oligomers, rendering unfolded VSVG ts045 more mobile, and hence prevents poisoning of the ER