Bioengineering bacterial protein nanocompartments as modular platforms for vaccines and drug delivery

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Dynamic changes in connexin expression correlate with key events in the wound healing process.

Wound healing is a complex process requiring communication for the precise co-ordination of different cell types. The role of extracellular communication through growth factors in the wound healing process has been extensively documented, but the role of direct intercellular communication via gap junctions has scarcely been investigated. We have examined the dynamics of gap junction protein (Connexins 26, 30, 31.1 and 43) expression in the murine epidermis and dermis during wound healing, and we show that connexin expression is extremely plastic between 6 hours and 12 days post-wounding. The immediate response (6 h) to wounding is to downregulate all connexins in the epidermis, but thereafter the expression profile of each connexin changes dramatically. Here, we correlate the changing patterns of connexin expression with key events in the wound healing process

Developing bacterial microcompartments for the recombinant production of proteins

In prokaryotes, supramolecular self-assembling protein structures, known as bacterial microcompartments (BMCs), have evolved to encapsulate proteins associated with a number of different metabolic processes, providing a physical diffusion barrier whilst increasing local enzyme concentrations. The modular nature of these structures makes them promising biological platforms for the engineering of synthetic compartmentation within the bacterial cell that have potential to be used as novel nano-bioreactors [1-3]. We are interested in the development of robust synthetic BMC technologies that can be utilised for industrially relevant applications, specifically the spatial segregation of synthetic enzyme cascades for the enhanced production of valuable chemical compounds. BMCs may also be valuable in the production of recombinant proteins. Many therapeutic proteins and antibody fragments require disulphide-bonds for correct folding and activity. Here, we are exploring the potential of BMCs to serve as synthetic cellular organelles within the bacterial cytoplasm of E. coli that promote correct protein folding and disulphide bond formation of recombinant proteins, providing an alternative method to traditional approaches (folding in the eukaryotic endoplasmic reticulum and in the periplasm of prokaryotes). Previously, it has been shown that proteins of interest can be compartmentalised by fusing them to targeting peptides, which direct the proteins to the microcompartment shell [4, 5]. In this work, both, the shell (PduABJKNU) and the targeting peptides (P18/ D18) are derived from the 1,2- propanediol utilisation (Pdu) BMC from Citrobacter freundii [4]. We first determined the effect of fusing short targeting peptides onto the E. coli alkaline phosphatase PhoA, a protein widely used to examine disulfide bond formation in vivo, and the sulfhydryl oxidase Erv1p, a catalytic enzyme for the formation of disulphide bonds. The most active fusion proteins were selected for co-production with the BMC shell. For efficient recruitment of these proteins to the BMC, gene expression levels were controlled using tunable promoters and recombinantly produced BMC variants were analysed in vivo and in vitro using biochemical and biophysical methods. We demonstrated that both, PhoA and Erv1p, are targeted to recombinant BMCs and determined disulphide bond formation of PhoA in the presence and absence of Erv1p when targeted to the microcompartments. Using this approach, a range of other proteins of industrial interest will be tested and the potential for the production and purification of bio-therapeutic proteins and antibody fragments will be determined. References: [1] Lawrence, A. D., Frank, S., Newnham, S., Lee, M. J., Brown, I. R., Xue, W. F., Rowe, M.L. & Warren, M. J. (2014). Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor. ACS synthetic biology, 3(7), 454-465. [2] Wagner, H. J., Capitain, C. C., Richter, K., Nessling, M., & Mampel, J. (2017). Engineering bacterial microcompartments with heterologous enzyme cargos. Engineering in Life Sciences, 17(1), 36-46. [3] Plegaria, J. S., & Kerfeld, C. A. (2018). Engineering nanoreactors using bacterial microcompartment architectures. Current opinion in biotechnology, 51, 1-7. [4] Parsons, J. B., Frank, S., Bhella, D., Liang, M., Prentice, M. B., Mulvihill, D. P., & Warren, M. J. (2010). Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement. Molecular cell, 38(2), 305-315. [5] Aussignargues, C., Paasch, B. C., Gonzalez-Esquer, R., Erbilgin, O., & Kerfeld, C. A. (2015). Bacterial microcompartment assembly: the key role of encapsulation peptides. Communicative & integrative biology, 8(3

Simultaneous measurements of heat of hydration and chemical shrinkage on hardening cement pastes

Isothermal calorimetry and chemical shrinkage measurements are two independent techniques used to study the development of hydration in cementitious systems. In this study, calorimetry and chemical shrinkage measurements were combined and simultaneously performed on hydrating cement paste samples. Portland cement pastes with different water to cement ratios and a cement paste containing calcium sulfoaluminate clinker and anhydrite were studied. The combined calorimetry/chemical shrinkage test showed good reproducibility and revealed the different hydration behavior of sealed samples and open samples, i.e., samples exposed to external water during hydration. Large differences between sealed and open samples were observed in a Portland cement paste with low water to cement ratio and in the calcium sulfoaluminate paste; these effects are attributed to self-desiccation of the sealed pastes. Once the setup is fully automatized, it is expected that combined calorimetry/chemical shrinkage measurements can be routinely used for investigating cement hydratio

Selective in vitro loading of proteins into protein nanocompartments for applications in the bioindustry

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Population density and habitat use of the Green Woodpecker Picus viridis in Donau-Auen National Park (Lower Austria)

In einer 1170 ha großen Probefläche im Nationalpark Donau- Auen (Niederösterreich) wurden Siedlungsdichten und Habitatpräferenzen des Grünspechts Picus viridis untersucht. Im Rahmen einer rationalisierten Revierkartierung zwischen Februar und April 2008 wurden 14 Reviere ermittelt (Revierdichte: 0,12 Reviere/10 ha). Basierend auf dem Vorkommen der Art in 400 m x 400 m Rastern wurde der Einfluss der vorherrschenden Baumarten, des Bestandesalters, der Länge der Waldrandgrenze sowie der Länge der Seitenarme auf das Vorkommen des Grünspechts mittels verallgemeinerter linearer Modelle analysiert. Der beste Prädiktor für das Vorkommen der Art war der Grenzlinienanteil zwischen Wald und Nicht-Wald-Bereichen. Die meisten Reviere befanden sich in Bereichen des Untersuchungsgebiets, die durch einen Damm vor Hochwasser geschützt sind. Die Harte Au wurde im Vergleich zur Weichen Au signifikant bevorzugt. Dies ist höchstwahrscheinlich mit einer besseren Nahrungsverfügbarkeit (Ameisen) in den trockeneren Gebieten zu erklären. Es konnten keine signifikanten Präferenzen für bestimmte Baumarten festgestellt werden, Hybridpappeln und Weiden (Arten der Weichen Au) wurden jedoch scheinbar gemieden, was aber wohl eher auf die weniger günstigen Bedingungen in feuchteren Lebensräumen zurückzuführen ist. Alle Reviere lagen im Waldrandbereich und beinhalteten Wiesen, Teile des Damms, aber auch landwirtschaftlich genutzte Felder. Bei fünf Revieren dürften intensiv genutzte Getreidefelder den einzigen Offenlandanteil darstellen.Population densities and habitat use of the Green Woodpecker Picus viridis were studied in a 1,170 ha study area in the Donau- Auen National Park (Lower Austria). Territory mapping (three visits) between February and April 2008 yielded a minimum of 14 territories, which corresponds to 0.12 territories/10 ha. Based on the incidence of the species in 400 m x 400 m grids, generalized linear models were constructed and the presence of the species was related to dominant tree species, tree age, length of the forest boundary and length of river sidearms. The best predictor for the presence of P. viridis was the length of the forest boundary. Most territories were located in areas protected from flooding by a dyke. Hardwood forest was significantly preferred to softwood forest. This can be explained by a better food supply (ants) in drier areas. No significant preferences for particular tree species were found; however, hybrid poplars and willows were apparently avoided, which can be attributed to less favourable conditions in wetter habitats. All territories were located at the forest’s edges and contained meadows or agricultural fields. In five territories, agricultural fields seemed to constitute the only open land

Expression of K1 Toxin Derivatives in Saccharomyces cerevisiae Mimics Treatment with Exogenous Toxin and Provides a Useful Tool for Elucidating K1 Mechanisms of Action and Immunity

Killer toxin K1 is a heterodimeric protein toxin secreted by Saccharomyces cerevisiae strains infected with the M1 double-stranded RNA ‘killer’ virus. After binding to a primary receptor at the level of the cell wall, K1 interacts with its secondary plasma membrane receptor Kre1p, eventually leading to an ionophoric disruption of membrane function. Although it has been under investigation for decades, neither the particular mechanisms leading to toxicity nor those leading to immunity have been elucidated. In this study, we constructed derivatives of the K1α subunit and expressed them in sensitive yeast cells. We show that these derivatives are able to mimic the action of externally applied K1 toxin in terms of growth inhibition and pore formation within the membrane, leading to a suicidal phenotype that could be abolished by co-expression of the toxin precursor, confirming a mechanistic similarity of external and internal toxin action. The derivatives were successfully used to investigate a null mutant completely resistant to externally applied toxin. They provide a valuable tool for the identification of so far unknown gene products involved in K1 toxin action and/or immunity