A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus

Background

The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.

Results

We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel?) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.

Conclusions

Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.     

Studies on the interaction of C1q, a subcomponent of the first component of complement, with porins from Salmonella minnesota incorporated into artificial membranes.

Purified outer membrane proteins (OMP) of Salmonella minnesota, Re-form, were incorporated into liposomes. These induced in macrophages a chemiluminescence signal identical to that of the intact Re-form. This signal was abolished by preincubation of porin-containing liposomes with purified C1q. Incorporation of isolated OMP into black lipid membranes (BLM) resulted in channel-formation which could not be inhibited by isolated C1q. Additionally, incubation of OMP-containing liposomes with BLM resulted in pore-formation within the BLM. This was amplified when lipid A was present within the liposomes. Preincubation of OMP-containing liposomes with purified C1q abolished pore-formation within the BLM.     

The use of enhanced polymer one-step staining reagents for immunoenzyme double-labelling

Summary The newly developed peroxidase-labelled Enhanced Polymer One-Step (EPOS) reagents were applied, together with an unlabelled primary mouse antibody, in a multistep double-labelling protocol. Enzyme label reporter combinations consisted of either peroxidase and alkaline phosphatase in red and blue, respectively, or β-galactosidase and alkaline phosphatase in turquoise and red, respectively. The latter enzyme combination was introduced using a rabbit antiperoxidase antibody and an enzyme-labelled anti-rabbit immunoglobulin antibody. The multistep procedure was tested using five different antibody combinations on cryostat and Carnoy- or formalin-fixed, paraffin-embedded sections. In each instance, clear and distinct labelling was obtained, either with the two antigens at separate sites, or with an overlap in distribution. In the latter situation, the sites of co-localization were marked by mixed colours, which were distinct and readily discriminated from the two basic colours.     

The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.     

More fibrosis and thrombotic complications but similar expression patterns of markers for coagulation and inflammation in symptomatic plaques from DM2 patients.

OBJECTIVE: One of the possible pathological mechanisms behind the increased vascular injury in diabetes mellitus type 2 (DM2) is the formation of advanced glycation end products (AGEs). The aim of this study was to investigate whether the presence of AGEs and specific markers for coagulation and inflammation in symptomatic atherosclerotic plaques from DM2 patients differs from plaques from nondiabetics. METHODS AND RESULTS: Carotid atherectomies were obtained from DM2 patients (n=11) and controls without DM2 matched for age and other cardiovascular risk factors (n=12) who were treated for symptomatic carotid artery stenosis. Plaques were graded according to the American Heart Association classification of lesions. More fibrosis and more thrombotic complications (p=0.007) were observed in carotid atherectomies from DM2 patients. Percentages of immunostained smooth muscle cells and macrophages in the lesions, quantified planimetrically, did not differ between the two groups. No differences were found in the immunostaining for T cells, tissue factor (TF), endothelial protein C receptor (EPCR), nuclear factor kappaB, and the AGE carboxymethyllysine. CONCLUSIONS: These findings demonstrate that DM2 is associated with increased plaque complications; however, a local changed presence of AGEs, TF, and EPCR seems not to be involved in this end stage of atherosclerosis.     

Strategies to enable the adoption of animal biotechnology to sustainably improve global food safety and security

The ability to generate transgenic animals has existed for over 30 years, and from those early days many predicted that the technology would have beneficial applications in agriculture. Numerous transgenic agricultural animals now exist, however to date only one product from a transgenic animal has been approved for the food chain, due in part to cumbersome regulations. Recently, new techniques such as precision breeding have emerged, which enables the introduction of desired traits without the use of transgenes. The rapidly growing human population, environmental degradation, and concerns related to zoonotic and pandemic diseases have increased pressure on the animal agriculture sector to provide a safe, secure and sustainable food supply. There is a clear need to adopt transgenic technologies as well as new methods such as gene editing and precision breeding to meet these challenges and the rising demand for animal products. To achieve this goal, cooperation, education, and communication between multiple stakeholders—including scientists, industry, farmers, governments, trade organizations, NGOs and the public—is necessary. This report is the culmination of concepts first discussed at an OECD sponsored conference and aims to identify the main barriers to the adoption of animal biotechnology, tactics for navigating those barriers, strategies to improve public perception and trust, as well as industry engagement, and actions for governments and trade organizations including the OECD to harmonize regulations and trade agreements. Specifically, the report focuses on animal biotechnologies that are intended to improve breeding and genetics and currently are not routinely used in commercial animal agriculture. We put forward recommendations on how scientists, regulators, and trade organizations can work together to ensure that the potential benefits of animal biotechnology can be realized to meet the future needs of agriculture to feed the world.