Sulfate limitation increases specific plasmid DNA yield and productivity in E. coli fed-batch processes.

Plasmid DNA (pDNA) is a key biotechnological product whose importance became apparent in the last years due to its role as a raw material in the messenger ribonucleic acid (mRNA) vaccine manufacturing process. In pharmaceutical production processes, cells need to grow in the defined medium in order to guarantee the highest standards of quality and repeatability. However, often these requirements result in low product titer, productivity, and yield. In this study, we used constraint-based metabolic modeling to optimize the average volumetric productivity of pDNA production in a fed-batch process. We identified a set of 13 nutrients in the growth medium that are essential for cell growth but not for pDNA replication. When these nutrients are depleted in the medium, cell growth is stalled and pDNA production is increased, raising the specific and volumetric yield and productivity. To exploit this effect we designed a three-stage process (1. batch, 2. fed-batch with cell growth, 3. fed-batch without cell growth). The transition between stage 2 and 3 is induced by sulfate starvation. Its onset can be easily controlled via the initial concentration of sulfate in the medium. We validated the decoupling behavior of sulfate and assessed pDNA quality attributes (supercoiled pDNA content) in E. coli with lab-scale bioreactor cultivations. The results showed an increase in supercoiled pDNA to biomass yield by 33% and an increase of supercoiled pDNA volumetric productivity by 13 % upon limitation of sulfate. In conclusion, even for routinely manufactured biotechnological products such as pDNA, simple changes in the growth medium can significantly improve the yield and quality.

Bioinspired, biobased and living material designs: a review of recent research in architecture and construction.

This article provides an overview of recent advances in the development of nature-based material designs in architecture and construction fields. Firstly, it aims to classify existing projects and ongoing researches into three types: bioinspired, biobased and living building materials. Secondly, selected case studies absolving different functions in building, are analysed to identify new opportunities and contemporary challenges of different nature-based approaches. The main gaps are identified between the progression at a theoretical level in laboratories and real-world application. Particulary, the challenge is to implement existing and future bioinspired, biobased and living building materials in large scale designs and architectural contexts. The authors also discuss different aspects of the inspiration and the use of nature to improve better the design of materials properties, robustness, durability, including sustainable awareness. Finally, an outlook of promising avenues for future interdisciplinary research and specific questions associated with methods and techniques of implementation of the different types of bioinspired, biobased and living material designs and fabrications in architecture are highlighted.

Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load.

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution.

Growth and Mechanical Characterization of Mycelium-Based Composites towards Future Bioremediation and Food Production in the Material Manufacturing Cycle.

Today's architectural and agricultural practices negatively impact the planet. Mycelium-based composites are widely researched with the aim of producing sustainable building materials by upcycling organic byproducts. To go further, this study analyzed the growth process and tested the mechanical behavior of composite materials grown from fungal species used in bioremediation. Agricultural waste containing high levels of fertilizers serves as the substrate for mycelium growth to reduce chemical dispersal in the environment. Compression and three-point bending tests were conducted to evaluate the effects of the following variables on the mechanical behavior of mycelium-based materials: substrate particle size (with or without micro-particles), fungal species (Pleurotus ostreatus and Coprinus comatus), and post-growth treatment (dried, baked, compacted then dried, and compacted then baked). Overall, the density of the material positively correlated with its Young's and elastic moduli, showing higher moduli for composites made from substrate with micro-particles and for compacted composites. Compacted then baked composites grown on the substrate with micro-particles provided the highest elastic moduli in compression and flexural testing. In conclusion, this study provides valuable insight into the selection of substrate particle size, fungal species, and post-growth treatment for various applications with a focus on material manufacturing, food production, and bioremediation.

Root Systems Research for Bioinspired Resilient Design: A Concept Framework for Foundation and Coastal Engineering.

The continuous increase in population and human migration to urban and coastal areas leads to the expansion of built environments over natural habitats. Current infrastructure suffers from environmental changes and their impact on ecosystem services. Foundations are static anchoring structures dependent on soil compaction, which reduces water infiltration and increases flooding. Coastal infrastructure reduces wave action and landward erosion but alters natural habitat and sediment transport. On the other hand, root systems are multifunctional, resilient, biological structures that offer promising strategies for the design of civil and coastal infrastructure, such as adaptivity, multifunctionality, self-healing, mechanical and chemical soil attachment. Therefore, the biomimetic methodology is employed to abstract root strategies of interest for the design of building foundations and coastal infrastructures that prevent soil erosion, anchor structures, penetrate soils, and provide natural habitat. The strategies are described in a literature review on root biology, then these principles are abstracted from their biological context to show their potential for engineering transfer. After a review of current and developing technologies in both application fields, the abstracted strategies are translated into conceptual designs for foundation and coastal engineering. In addition to presenting the potential of root-inspired designs for both fields, this paper also showcases the main steps of the biomimetic methodology from the study of a biological system to the development of conceptual technical designs. In this way the paper also contributes to the development of a more strategic intersection between biology and engineering and provides a framework for further research and development projects.

Bio-inspired evaporation from shaped interfaces: an experimental study.

Evaporative interfaces help process heat and substances in a variety of technical realms, from electronic to architectural applications. Because geometry affects the hydraulics, thermal properties and aerodynamics of evaporative devices, their performance can be tuned through design. While non-smooth interfaces are widely exploited to enhance transfer passively, surface area extension in packed volumes is a predominant line of research. This leaves aerodynamic structure-transfer relations and the impact of geometry itself unclear. Meanwhile, protrusions in leaves such as lobes and toothed margins have been associated with enhanced vapor dissipation. This experimental study explores the design space of leaf-inspired structures with evaporating protrusions. Three sets of water-absorbing models with fixed evaporating surface area and unlimited hydraulic supply were tested: (1) paper strips with dimension-equivalent protrusions of varied shape and degree of elongation; (2) cellulose sponges with the same designs as their cross-sectional profile, extruded three-dimensionally; (3) ceramic tiles with grooves of varied cross-section, conceived as building elements for evaporative cooling. Overall, results demonstrate that protrusions affect mass transfer rate and surface temperatures and can be integrated in the design of evaporative exchangers with non-smooth geometries. For the paper models, evaporation rate correlated with protrusion aspect ratio, supporting a functional interpretation of leaf design and its utilization in low-wind plate-fin exchangers. However, the same transfer enhancement was not regained from simply extruding an effective design into three-dimensions. For the ceramic tiles, geometry-driven differences in evaporation depended on the aerodynamic roughness and size of the grooved pattern, and on ventilation. Their outdoor thermal behavior was complex due to a multifaceted interaction with the environment and geometry-related factors such as self-shading and thermal mass. Ultimately, this design effort illustrates the potential of structured interfaces for evaporative exchange and thermoregulating the built environment.

Biomimetic Groundwork for Thermal Exchange Structures Inspired by Plant Leaf Design.

Geometry is a determining factor for thermal performance in both biological and technical systems. While biology has inspired thermal design before, biomimetic translation of leaf morphology into structural aspects of heat exchangers remains largely unaddressed. One determinant of plant thermal endurance against environmental exposure is leaf shape, which modulates the leaf boundary layer, transpiration, evaporative cooling, and convective exchange. Here, we lay the research groundwork for the extraction of design principles from leaf shape relations to heat and mass transfer. Leaf role models were identified from an extensive literature review on environmentally sensitive morphology patterns and shape-dependent exchange. Addressing canopy sun-shade dimorphism, sun leaves collected from multiple oak species exceeded significantly in margin extension and shape dissection. Abstracted geometries (i.e., elongated; with finely toothed edges; with few large-scale teeth) were explored with paper models of the same surface area in a controlled environment of minimal airflow, which is more likely to induce leaf thermal stress. For two model characteristic dimensions, evaporation rates were significantly faster for the dissected geometries. Shape-driven transfer enhancements were higher for the smaller models, and finely toothed edges reached local cooling up to 10 °C below air temperature. This investigation breaks new ground for solution-based biomimetics to inform the design of evaporation-assisted and passively enhanced thermal systems.

Unfolding Crease Patterns Inspired by Insect Wings and Variations of the Miura-ori with a Single Vein.

In many disciplines, professionals are interested in folding patterns for their packing and shape changing capabilities. Many insects have folded wings fitting to their body morphology that can unfold to fly, support their weight and withstand external forces. This paper focuses on the main characteristics emerging from folding patterns inspired and adapted from both insect wings and Miura-ori patterns, along with the actuation mechanism. Pneumatic actuators, similar to the venations on insect wings, are used to unfold these patterns. Depending on one vein's placement, its inflation can unfold models with many creases. While a single vein cannot fold the model back, a snapping behavior, observed in some folding patterns, could be used to trigger the folding mechanism of a model. By presenting the characteristics of each folding pattern studied in this work, one could come forth with an application and choose the most efficient folding patterns based on the most suitable characteristics for this application. These folding patterns can then be optimized to address specific requirements by adapting their different parameters.

Active immunization against complement factor C5a: a new therapeutic approach for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by neuronal loss due to amyloid beta aggregations, neurofibrillary tangles, and prominent neuroinflammation. Recently, interference with neuroinflammation as a new therapeutic approach for AD treatment gained great interest. Microglia cells, one of the major contributors in neuroinflammation, are activated in response to misfolded proteins such as amyloid ß and cell debris leading to a sustained release of pro-inflammatory mediators. Especially, complement factor C5a and its receptor have been found to be up-regulated in microglia in the immediate surroundings of cerebral amyloid plaques and blocking of C5aR resulted in a reduction of pathological markers in a model of AD. Here, we investigate the effect of active vaccination against the complement factor C5a to interfere with neuroinflammation and neuropathologic alterations in a mouse model of AD. METHODS: Short antigenic peptides AFF1 and AFF2, which mimic a C-terminal epitope of C5a, were selected and formulated to vaccines. These vaccines are able to induce a highly specific antibody response to the target protein C5a. Tg2576 mice, a common model of AD, were immunized with these two C5a-peptide vaccines and the induced immune response toward C5a was analyzed by ELISA and Western blot analysis. The influence on memory retention was assessed by a contextual fear conditioning test. Microglia activation and amyloid plaque deposition in the brain was visualized by immunohistochemistry. RESULTS: Both C5a-targeting vaccines were highly immunogenic and induced sustained antibody titers against C5a. Tg2576 mice vaccinated at early stages of the disease showed significantly improved contextual memory accompanied by the reduction of microglia activation in the hippocampus and cerebral amyloid plaque load compared to control mice. Late-stage immunization also showed a decrease in the number of activated microglia, and improved memory function, however, had no influence on the amyloid ß load. CONCLUSION: C5a-peptide vaccines represent a safe and well-tolerated immunotherapy, which is able to induce a strong and specific immune response against the pro-inflammatory molecule C5a. In a mouse model of AD, C5a-peptide vaccines reduce microglia activation and thus neuroinflammation, which is supposed to lead to reduced neuronal dysfunction and AD symptomatic decline.

Tailoring the antibody response to aggregated Aß using novel Alzheimer-vaccines.

Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aß1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study.

Dewaxed ECM: A simple method for analyzing cell behaviour on decellularized extracellular matrices.

Decellularization techniques have been used on a wide variety of tissues to create cell-seedable scaffolds for tissue engineering. Finding a suitable decellularization protocol for a certain type of tissue can be laborious, especially when organ perfusion devices are needed. In this study, we report a quick and simple method for comparing decellularization protocols combining the use of paraffin slices and two-dimensional cell cultures. We developed three decellularization protocols for adult murine kidney that yielded decellularized extracellular matrices (ECMs) with varying histological properties. The resulting paraffin-embedded ECM slices were deparaffinized and reseeded with murine embryonic stem cells (mESCs). We analyzed cell attachment four days post seeding via determination of cell numbers, and used quantitative Real-Time PCR 13 days post seeding to measure gene expression levels of two genes associated with renal development, Pax2 and Pou3f3. The three decellularization protocols produced kidney-matrices that showed clearly distinguishable results. We demonstrated that formerly paraffin-embedded decellularized ECMs can effectively influence differentiation of stem cells. This method can be used to identify optimal decellularization protocols for recellularization of three-dimensional tissue-scaffolds with embryonic stem cells and other tissue-specific cell types.

ECM modulated early kidney development in embryonic organ culture.

The use of exogenous signals is gaining importance in renal regenerative therapies. We wanted to explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. We used a recently established organ culture setup to expose embryonic kidney rudiments directly to a large set of surface-immobilized or dissolved ECM molecules and growth factors. Organ culture was also performed on immobilized adult kidney ECM extracts and on reactive polymer films without any biomolecular components. The applied conditions resulted in distinct differences of organ phenotypes, underlining the multifaceted role of exogenous signals during kidney development. Specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. ECM biopolymers, e.g. hyaluronic acid, were found to determine the fate of developing explants in a concentration- and molecular weight-dependent manner. The organ culture system used was an effective and robust means to identify exogenous signals that direct kidney development. This system can provide valuable insight for future regenerative therapies of kidney diseases.

Role of the polypeptide backbone and post-translational modifications in cross-reactivity of Art v 1, the major mugwort pollen allergen.

Artemisia vulgaris (mugwort) is one of the main causes of late summer pollinosis in Europe, with >95% of patients sensitized to the glycoallergen Art v 1. Despite the importance of this allergen, little is known about its cross-reactive behavior. Here we investigated the occurrence of conserved Art v 1 antigenic determinants in sources known to display clinically relevant cross-reactivity with mugwort pollen. For this purpose, monoclonal antibodies specific for a cysteine-stabilized epitope of the Art v 1 defensin domain and for carbohydrates attached to the proline domain were produced by hybridoma and phage display technologies. Using polyclonal Art v 1-specific rabbit sera and antibodies against both the Art v 1 carbohydrate and polypeptide moieties, we could identify cross-reactive structures in pollen from botanically related Asteraceae weeds (Artemisia absinthium, Helianthus annuus and Ambrosia sp.). Homologous allergens were also recognized by IgE from mugwort-sensitized patients and the reactivity could be decreased by serum pre-incubation with natural and recombinant Art v 1. As no cross-reactive structures could be found in foods associated with mugwort pollinosis, we conclude that Art v 1 is poorly involved in mugwort cross-reactivity to food allergens.

The signs of life in architecture.

Engineers, designers and architects often look to nature for inspiration. The research on 'natural constructions' is aiming at innovation and the improvement of architectural quality. The introduction of life sciences terminology in the context of architecture delivers new perspectives towards innovation in architecture and design. The investigation is focused on the analogies between nature and architecture. Apart from other principles that are found in living nature, an interpretation of the so-called 'signs of life', which characterize living systems, in architecture is presented. Selected architectural projects that have applied specific characteristics of life, whether on purpose or not, will show the state of development in this field and open up future challenges. The survey will include famous built architecture as well as students' design programs, which were carried out under supervision of the author at the Department of Design and Building Construction at the Vienna University of Technology.

FAS (CD95) mutations are rare in gastric MALT lymphoma but occur more frequently in primary gastric diffuse large B-cell lymphoma.

A loss of FAS (CD95) function has been proposed to constitute an important step in early mucosa-associated lymphoid tissue (MALT) lymphoma development and FAS mutations have been recognized in malignant lymphomas, in particular at extranodal sites. Since primary gastric lymphomas frequently exhibit resistance to FAS-mediated apoptosis, we investigated whether FAS is mutated in 18 gastric MALT lymphomas and 28 diffuse large B-cell lymphomas (DLBCL). We detected seven mutations in five lymphomas, one MALT lymphoma and four DLBCL; two DLBCL had two mutations. The MALT lymphoma exhibited a point mutation in the splice donor region of intron 3. Three DLBCL had missense mutations in exon 2, which encodes a signal peptide and a portion of the extracellular FAS ligand-binding domain. One DLBCL carried a point mutation in the splice donor region of intron 8, which would result in exon skipping. Two DLBCL harbored a missense mutation in exon 9, which encodes the intracellular death domain. The two death domain mutations inhibited FAS ligand-induced apoptosis in a dominant-negative mode, when transiently expressed in human T47D breast carcinoma and Jurkat T cells. A signal peptide and an extracellular domain mutation, however, failed to inhibit apoptosis in these transfection assays. They are likely to reduce apoptosis in lymphoma cells solely by a loss of function. In summary, our data show that FAS mutations are rare in primary gastric MALT lymphomas (5.6%) but occur in a subset of primary gastric DLBCL (14.3%) and suggest that these mutations contribute to the pathogenesis of gastric lymphomas by rendering lymphocytes resistant to apoptosis.

Lab scale and medium scale production of recombinant allergens in Escherichia coli.

Recombinant products have become invaluable tools for diagnostic as well as therapeutic purposes in modern medicine. Especially in cases where raw naturally derived products are difficult to standardize, well-defined recombinant single components represent the matter of choice. In the recent past, much effort has been undertaken to define individual proteins derived from various sources like pollen, spores of moulds, pet dander, and food causing Type 1 allergic reactions in humans. Therefore, methods for cloning, sequencing, and expressing cDNAs coding for allergens in Escherichia coli became of great interest to allergologists. For the recombinant production of allergens, suitable expression systems, growing conditions, and purification steps have to be established for each individual product. Finally, the purified recombinant allergen has to be carefully investigated for the biochemical, biophysical, and immunological properties. In the following paper, several prokaryotic expression systems, purification strategies, and analytical methods will be presented and pitfalls discussed.