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Cell compatibility of biomaterials is one of the most important issues in Tissue Engineering. It is affected by the catalyst, the chemical composition and the surface topography of the material. Ou...

The investigation of complex biological processes in vivo often requires defined multiple bioconjugation and positioning of functional entities on 3D structures. Prominent examples include spatially defined protein complexes in nature, facilitating efficient biocatalysis of multistep reactions. Mimicking natural strategies, synthetic scaffolds should comprise bioorthogonal conjugation reactions and allow for absolute stoichiometric quantification as well as facile scalability through scaffold reproduction. Existing in vivo scaffolding strategies often lack covalent conjugations on geometrically confined scaffolds or precise quantitative characterization. Addressing these shortcomings, we present a bioorthogonal dual conjugation platform based on genetically encoded artificial compartments in vivo, comprising two distinct genetically encoded covalent conjugation reactions and their precise stoichiometric quantification. The SpyTag/SpyCatcher (ST/SC) bioconjugation and the controllable strain-promoted azide-alkyne cycloaddition (SPAAC) were implemented on self-assembled protein membrane-based compartments (PMBCs). The SPAAC reaction yield was quantified to be 23% ± 3% and a ST/SC surface conjugation yield of 82% ± 9% was observed, while verifying the compatibility of both chemical reactions as well as enhanced proteolytic stability. Using tandem mass spectrometry, absolute concentrations of the proteinaceous reactants were calculated to be 0.11 ± 0.05 attomol/cell for PMBC surface-tethered mCherry-ST-His and 0.22 ± 0.09 attomol/cell for PMBC-constituting pAzF-SC-E20F20-His. The established in vivo conjugation platform enables quantifiable protein-protein interaction studies on geometrically defined scaffolds and paves the road to investigate effects of scaffold-tethering on enzyme activity.

Modern biotechnological laboratories are equipped with advanced parallel mini-bioreactor facilities that can perform sophisticated cultivation strategies (e.g. fed-batch or continuous) and generate significant amounts of measurement data. These systems require not only optimal experimental designs that find the best conditions in very large design spaces, but also algorithms that manage to operate a large number of different cultivations in parallel within a well-defined and tightly constrained operating regime. Existing advanced process control algorithms have to be tailored to tackle the specific issues of such facilities such as: a very complex biological system, constant changes in the metabolic activity and phenotypes, shifts of pH and/or temperature, and metabolic switches, e.g. by product induction, to name a few. In this work we implement a model-predictive control (MPC) approach based framework to demonstrate: 1) the challenges in terms of mathematical model structure, state and parameter estimation, and optimization under highly nonlinear and stiff constraints in biological systems, 2) the adaptations required to enable its application in High Throughput Bioprocess Development (HTBD), and 3) the added value of MPC implementations when operating parallel mini-bioreactors aiming to maximize the biomass concentration while coping with hard constrains on the Dissolved Oxygen Tension profile.Modern biotechnological laboratories are equipped with advanced parallel mini-bioreactor facilities that can perform sophisticated cultivation strategies (e.g. fed-batch or continuous) and generate significant amounts of measurement data. These systems require not only optimal experimental designs that find the best conditions in very large design spaces, but also algorithms that manage to operate a large number of different cultivations in parallel within a well-defined and tightly constrained operating regime. Existing advanced process control algorithms have to be tailored to tackle the specific issues of such facilities such as: a very complex biological system, constant changes in the metabolic activity and phenotypes, shifts of pH and/or temperature, and metabolic switches, e.g., by induction of product formation, to name a few. In this work we implement a model predictive control (MPC) framework to demonstrate: 1) the challenges in terms of mathematical model structure, state and parameter estimation, and optimization under highly nonlinear and stiff dynamics in biological systems, 2) the adaptations required to enable the application of MPC in High Throughput Bioprocess Development (HTBD), and 3) the added value of MPC implementations when operating parallel mini-bioreactors aiming to maximize the biomass concentration while coping with hard constrains on the Dissolved Oxygen Tension profile.

Life in its molecular context is characterized by the challenge of orchestrating structure, energy and information processes through compartmentalization and chemical transformations amenable to mimicry of protocell models. Here we present an alternative protocell model incorporating dynamic membranes based on amphiphilic elastin-like proteins (ELPs) rather than phospholipids. For the first time we demonstrate the feasibility of combining vesicular membrane formation and biocatalytic activity with molecular entities of a single class: proteins. The presented self-assembled protein-membrane-based compartments (PMBCs) accommodate either an anabolic reaction, based on free DNA ligase as an example of information transformation processes, or a catabolic process. We present a catabolic process based on a single molecular entity combining an amphiphilic protein with tobacco etch virus (TEV) protease as part of the enclosure of a reaction space and facilitating selective catalytic transformations. Combining compartmentalization and biocatalytic activity by utilizing an amphiphilic molecular building block with and without enzyme functionalization enables new strategies in bottom-up synthetic biology, regenerative medicine, pharmaceutical science and biotechnology.

The nature of the first prebiotic compartments and their possible minimal molecular composition is of great importance in the origin of life scenarios. Current protocell model membranes are proposed to be lipid-based. This paradigm has several shortcomings such as limited membrane stability of monoacyl lipid-based membranes (e.g., fatty acids), missing pathways to synthesize protocell membrane components (e.g., phospholipids) under early earth conditions, and the requirement for different classes of molecules for the formation of compartments and the catalysis of reactions. Amino acids on the other hand are known to arise and persist with remarkable abundance under early earth conditions since the fundamental Miller-Urey experiments. They were also postulated early to form protocellular structures, for example, proteinoid capsules. Here, we present a protocell model constituted by membranes assembled from amphiphilic proteins based on prebiotic amino acids. Self-assembled dynamic protein membrane-based compartments (PMBCs) are impressively stable and compatible with prevalent cellular membrane constituents forming protein-only or protein-lipid hybrid membranes. They can embed processes essential for extant living cells, such as enclosure of molecules, membrane fusion, phase separation, and complex biosynthetic elements from modern cells demonstrating "upward" compatibility. Our findings suggest that prebiotic PMBCs represent a new type of protocell as a possible ancestor of current lipid-based cells. The presented prebiotic PMBC model can be used to design artificial cells, important for the study of structural, catalytic, and evolutionary pathways related to the emergence of life.

ZusammenfassungBestehende Weichteildefekte nach Unfallverletzungen, onkologischen Tumorresektionen, kongenitalen Anomalien oder chronischen Wunden stellen eine zentrale Herausforderung in der rekonstruktiven Chirurgie dar. Der derzeitige Goldstandard in der Therapie von Gewebedefekten besteht in der Gewebetrans plantation in Form von freien oder lokalen Lappenplastiken. Limitierend ist jedoch die unumgängliche Morbidität an der Spenderstelle nach dem Gewebetransfer. Vor diesem Hintergrund wurden in den letzten Jahren aufgrund der günstigen Gewebetextur und hohen Plastizität große Anstrengungen im Bereich des Tissue Engineerings von vaskularisierten, langzeitstabilen autologen Fettgewebskonstrukten unternommen. Dabei stellt der enge Zusammenhang von Adipogenese und Angiogenese eine entscheidende Hürde in der de novo Erzeugung von Fettgewebe dar. Eine besondere Rolle kommt hierbei bioabbaubaren Biomaterialien (Scaffolds) als Trägereinheiten für Zellen und der damit verbundenen Zell-Matrix-Interaktion zu. Ein ideales Biomaterial sollte die Zellproliferation, -adhäsion und -differenzierung unterstützen, und gleichzeitig unbedenklich in seiner Biokompatibilität sein. Die vorliegende Übersichtsarbeit gewährt einen Überblick über derzeitige Ansätze des Tissue Engineerings von Fettgewebe vor dem Hintergrund der aktuell verfügbaren Evidenz.Die Problematiken bisheriger Modelle sind einerseits hohe Resorptionsraten der gezüchteten Gewebekonstrukte und andererseits der fehlende Nachweis von klinisch relevanten Gewebevolumina. Das Tissue Engineering von Fettgewebe in einem Wachtumskammermodell in Kombination mit Scaffolds bietet eine weitere Möglichkeit zur in vivo Gewebezüchtung. Hier zeigen aktuelle Ergebnisse, dass eine de novo Fettgewebezüchtung mit klinisch relevanten und langzeitstabilen Volumina in vivo möglich ist. Dieses Modell besitzt, unserer Auffassung nach, das Potential die Therapie großer Weichteildefekte signifikant zu verbessern.

Kollek et al. show that transient inhibition of apoptosis by short-term BCL-XL overexpression increases the viability of hematopoietic stem cells (HSCs) during engraftment and improves the outcome of HSC transplantation without signs of adverse pathologies. This strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability., During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.

The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self-assembly of supramolecular structures with desired shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self-assembly of tailored amphiphilic elastin-like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced. The described assembly protocols efficiently yield protein membrane-based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.

Atomic force microscopy is not only a high-resolution imaging device but also a mechanical machine, which can be used either to indent or stretch (soft) biomaterials. Due to the statistical nature of such materials (i.e., hydrogels or polymers) hundreds of force-distance curves are required to describe their mechanical properties. In this manuscript, we present an automated system for polymer unfolding detection based on continuous wavelet analysis. We have tested the automated program on elastin, which is an important protein that provides elasticity to tissues and organs. Our results show that elastin changes its mechanical behavior in the presence of electrolytes. In particular, we show that NaCl has a different effect on the contour length than CaCl2 for similar unfolding forces. In addition, we provide the program in the supporting information for the researches facing such kind of problem.

The potential of the copolymer polycaprolactone-co-poly-d,l-lactic acid (PCLLA) as a biomaterial for scaffold-based therapy for breast tissue engineering applications was assessed. First, the synthesized PCLLA was evaluated for its processability by means of additive manufacturing (AM). We found that the synthesized PCLLA could be fabricated into scaffolds with an overall gross morphology and porosity similar to that of polycaprolactone. The PCLLA scaffolds possessed a compressive Young's modulus (ca 46 kPa) similar to that of native breast (0.5 − 25 kPa), but lacked thermal stability and underwent thermal degradation during the fabrication process. The PCLLA scaffolds underwent rapid degradation in vitro which was characterized by loss of the scaffolds' mechanical integrity and a drastic decrease in mass-average molar mass (Mw) and number-average molar mass (Mn) after 4 weeks of immersion in phosphate buffer solution maintained at 37 °C. The tin-catalysed PCLLA scaffold was also found to have cytotoxic effects on cells. Although the initial mechanical properties of the PCLLA scaffolds generally showed potential for applications in breast tissue regeneration, the thermal stability of the copolymer for AM processes, biocompatibility towards cells and degradation rate is not satisfactory at this stage. Therefore, we conclude that research efforts should be geared towards fine-tuning the copolymer synthesizing methods. © 2016 Society of Chemical Industry

Hybrid nanoparticle (NP) structures containing organic building units such as polymers, peptides, DNA and proteins have great potential in biosensor and electronic applications. The nearly free modification of the polymer chain, the variation of the protein and DNA sequence and the implementation of functional moieties provide a great platform to create inorganic structures of different morphology, resulting in different optical and magnetic properties. Nevertheless, the design and modification of a protein structure with functional groups or sequences for the assembly of biohybrid materials is not trivial. This is mainly due to the sensitivity of its secondary, tertiary and quaternary structure to the changes in the interaction (e.g., hydrophobic, hydrophilic, electrostatic, chemical groups) between the protein subunits and the inorganic material. Here, we use hemolysin coregulated protein 1 (Hcp1) from Pseudomonas aeruginosa as a building and gluing unit for the formation of biohybrid structures by implementing cysteine anchoring points at defined positions on the protein rim (Hcp1_cys3). We successfully apply the Hcp1_cys3 gluing unit for the assembly of often linear, hybrid structures of plasmonic gold (Au NP), magnetite (Fe3O4 NP), and cobalt ferrite nanoparticles (CoFe2O4 NP). Furthermore, the assembly of Au NPs into linear structures using Hcp1_cys3 is investigated by UV–vis spectroscopy, TEM and cryo-TEM. One key parameter for the formation of Au NP assembly is the specific ionic strength in the mixture. The resulting network-like structure of Au NPs is characterized by Raman spectroscopy, showing surface-enhanced Raman scattering (SERS) by a factor of 8·104 and a stable secondary structure of the Hcp1_cys3 unit. In order to prove the catalytic performance of the gold hybrid structures, they are used as a catalyst in the reduction reaction of 4-nitrophenol showing similar catalytic activity as the pure Au NPs. To further extend the functionality of the Hcp1_cys3 gluing unit, Fe3O4 and CoFe2O4 NPs are aligned in a magnetic field and connected by utilization of cysteine-modified Hcp1. After lyophilization, a fiber-like material of micrometer scale length can be observed. The Fe3O4 Hcp1_cys3 fibers show superparamagnetic behavior with a decreasing blocking temperature and an increasing remanent magnetization leading to a higher squareness value of the hysteresis curve. Thus the Hcp1_cys3 unit is shown to be very versatile in the formation of new biohybrid materials with enhanced magnetic, catalytic and optical properties.

Phospholipid membranes are essential constituents of extant cells rendering them preferred candidates as membrane components in origin of life scenarios. These models greatly neglect stability requirements and their problematic synthetic complexity necessary to access such lipid membrane constituents under early life conditions. Here we present an alternative protocell model, based on amphiphilic protein membranes constituted of prebiotic amino acids. These self-assembled dynamic Protein Membrane Based Compartments (PMBC) are impressively stable and compatible with prevalent protocell membrane constituents. PMBCs can enclose functional proteins, undergo membrane fusion, phase separate, accommodate anabolic ligation reactions and DNA encoded synthesis of their own membrane constituents. Our findings suggest that prebiotic PMBC represent a new type of protocell as plausible ancestor of current lipid-based cells. They can be used to design simple artificial cells important for the study of structural and catalytic pathways related to the emergence of life.

Soft tissue defects resulting from injuries, tumor resection, congenital anomalies or chronic wounds pose a great challenge to reconstructive surgery. The current gold standard in therapy of such defects is the tissue transplantation in terms of free or local flaps. Unfortunately, donor site morbidity remains a considerable risk of flap surgery. Therefore, tissue engineering of autologous vascularized long term stable adipose tissue constructs could enrich the therapeutic possibilities of soft tissue defects. De novo adipose tissue growing requires fundamental knowledge about this kind of tissue and its synthesis, closely linked to angiogenesis. Bioresorbable biomaterials (scaffolds) are of crucial importance for adipose tissue engineering. Simulation or replacement of extracellular matrix for tissue growth by scaffold application requires a profound understanding of cell-matrix interactions. A proper biomaterial should be capable of supporting cell adherence, proliferation and differentiation. Important features are biocompatibility and resorption without toxic metabolites. In this review, various scaffold materials are discussed and novel achievements are presented. Persisting problems of de novo adipose tissue formation are high resorption rates and small tissue volumes of adipose constructs. Adipose tissue formation in a tissue engineering chamber is an additional possibility for in vivo tissue engineering. Recent studies proof that long term stable de novo adipose tissue formation of clinically relevant tissue volumes is possible. This method, in our opinion, has the potential to improve therapeutic strategies of soft tissue defects significantly.Bestehende Weichteildefekte nach Unfallverletzungen, onkologischen Tumorresektionen, kongenitalen Anomalien oder chronischen Wunden stellen eine zentrale Herausforderung in der rekonstruktiven Chirurgie dar. Der derzeitige Goldstandard in der Therapie von Gewebedefekten besteht in der Gewebetrans plantation in Form von freien oder lokalen Lappenplastiken. Limitierend ist jedoch die unumgängliche Morbidität an der Spenderstelle nach dem Gewebetransfer. Vor diesem Hintergrund wurden in den letzten Jahren aufgrund der günstigen Gewebetextur und hohen Plastizität große Anstrengungen im Bereich des Tissue Engineerings von vaskularisierten, langzeitstabilen autologen Fettgewebskonstrukten unternommen. Dabei stellt der enge Zusammenhang von Adipogenese und Angiogenese eine entscheidende Hürde in der de novo Erzeugung von Fettgewebe dar. Eine besondere Rolle kommt hierbei bioabbaubaren Biomaterialien (Scaffolds) als Trägereinheiten für Zellen und der damit verbundenen Zell-Matrix-Interaktion zu. Ein ideales Biomaterial sollte die Zellproliferation, -adhäsion und -differenzierung unterstützen, und gleichzeitig unbedenklich in seiner Biokompatibilität sein. Die vorliegende Übersichtsarbeit gewährt einen Überblick über derzeitige Ansätze des Tissue Engineerings von Fettgewebe vor dem Hintergrund der aktuell verfügbaren Evidenz.Die Problematiken bisheriger Modelle sind einerseits hohe Resorptionsraten der gezüchteten Gewebekonstrukte und andererseits der fehlende Nachweis von klinisch relevanten Gewebevolumina. Das Tissue Engineering von Fettgewebe in einem Wachtumskammermodell in Kombination mit Scaffolds bietet eine weitere Möglichkeit zur in vivo Gewebezüchtung. Hier zeigen aktuelle Ergebnisse, dass eine de novo Fettgewebezüchtung mit klinisch relevanten und langzeitstabilen Volumina in vivo möglich ist. Dieses Modell besitzt, unserer Auffassung nach, das Potential die Therapie großer Weichteildefekte signifikant zu verbessern.

Biogenic nucleation and crystallization occur in confined spaces with defined interfacial properties. However, the regulatory functions of organic players in the stabilization and transport of inorganic precursors such as ion clusters, liquid‐condensed phases, and amorphous particles are unclear. Given the prevalence of unstructured proteins in biogenic materials, the present study investigates the effects of biomineral‐associated, intrinsically disordered protein domains with simple and repetitive amino acid compositions on mineral nucleation and their capability to form distinct supramolecular assemblies. The quantitative assessment and structural evaluation of the nucleation process reveal that disordered regions confine hydrated mineral precursors within vesicles, transiently suppressing mineral precipitation. Stabilization of the amorphous mineral is attributed to protein self‐association and restructuration toward β‐configurations, triggered by specific bioinorganic interactions. In consequence, the conditioned macromolecules localize at phase boundaries formed upon liquid–liquid demixing of mineral precursors and stabilize the fluidic mineral precursors against crystallization. Thus, the conformational plasticity and self‐association of intrinsically disordered sequences in response to crystallization environments mediates the selection of functional macromolecular subensembles dedicated to biomaterial growth. published

Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials. Amphiphilic proteins act as building blocks for the de novo formation of membrane-based organelles within Escherichia coli. The organelles can be selectively functionalized in vivo with unnatural amino acids and hence may permit chemical reactions inside the cell that have not been possible so far.

The access to defined protein-based material systems is a major challenge in bionanotechnology and regenerative medicine. Exact control over sequence composition and modification is an important requirement for the intentional design of structure and function. Herein structural- and matrix proteins provide a great potential, but their large repetitive sequences pose a major challenge in their assembly. Here we introduce an integrative "one-vector-toolbox-platform" (OVTP) approach which is fast, efficient and reliable. The OVTP allows for the assembly, multimerization, intentional arrangement and direct translation of defined molecular DNA-tecton libraries, in combination with the selective functionalization of the yielded protein-tecton libraries. The diversity of the generated tectons ranges from elastine-, resilin, silk- to epitope sequence elements. OVTP comprises the expandability of modular biohybrid-materials via the assembly of defined multi-block domain genes and genetically encoded unnatural amino acids (UAA) for site-selective chemical modification. Thus, allowing for the modular combination of the protein-tecton library components and their functional expansion with chemical libraries via UAA functional groups with bioorthogonal reactivity. OVTP enables access to multitudes of defined protein-based biohybrid-materials for self-assembled superstructures such as nanoreactors and nanobiomaterials, e.g. for approaches in biotechnology and individualized regenerative medicine.

Biodegradable biopolymers are versatile materials with a wide range of applications including suture, scaffolds, growth chambers and matrix materials for tissue-engineering and regenerative medicine. Their synthesis is usually based on tin-salts at high temperatures. Strategies to replace tin are interesting due to the toxicity of tin for aquatic organisms and potentially problematic effects in higher organisms. In the framework of this study we investigate nontoxic iron catalysts (iron chloride, bromide and perchlorate) and their potential to facilitate ring-opening polymerizations (ROP) at room temperature. To find the best combination of a catalyst and an initiator for the ROP of e-caprolactone, three iron-catalysts were analysed with four different initiators (water, isopropyl alcohol, benzyl alcohol and 2-allyl phenol). Functional entities such as the allyl group are interesting for bioconjugation and may allow functional access to functional biohybrid materials. The polymerizations were carried out solvent free in bulk and yielded polymers in the range 10–20 kDa for large monomer to catalyst/initiator ratios analysed by GPC, NMR and MALDI-TOF. We find that commercial iron salts can replace tin salts in many cases and allow for the ROP of e-caprolactone at room temperature while tin(II) 2-ethylhexanoate Sn(Oct)2 does not facilitate ROP at room temperature. The use of iron based ROP catalysts opens interesting access to biopolymers utilizing a nontoxic metal ion at low temperatures in a solvent free reaction scheme saving energy and the environment.

Der heutige Standard fur die Deckung und Rekonstruktion groserer Haut-/Weichteildefekte mit freiliegenden Knochen, Nerven oder Gefasen, wie sie z. B. nach ausgedehnten Tumorresek­tionen oder komplexen Unfallverletzungen vorkommen, ist der lokale oder freie mikrochirurgische Gewebetransfer. Trotz Einfuhrung und Weiterentwicklung neuer chirurgischer Techniken sowie zahlreicher synthetischer Materialien, ist nach wie vor eine grose Anzahl von Limitationen und Komplikationen an der Hebe-/und Empfangerstelle vorhanden. Es besteht daher ein groser Bedarf an Entwicklung neuer Methoden und Materialien, welche einen dauerhaften korpereigenen Weichteilersatz ermoglichen. Ein vielversprechender Therapieansatz ist der Weichteilersatz mit korpereigenem Fettgewebe. Klinische und experimentelle Untersuchungen zur Wiederherstellung von Weichteilen mithilfe korpereigenen Fettgewebes zeigten allerdings bei den freien Fettgewebstransplantationen ohne einen direkten Gefasanschluss, v. a. im Hinblick auf das Langzeituberleben des Transplantates, bis dato eher enttauschende Ergebnisse. Haufig wurde ein Volumenverlust bzw. eine komplette Resorption des Transplantates aufgrund einer insuffizienten Gewebequalitat durch ausbleibende Zelldifferenzierung beobachtet. Diese Tatsache verweist auf die besondere Rolle der Unterhaltung und Entwicklung der transplantateigenen Blutversorgung (Vaskularisation und Angiogenese), entscheidend fur den Erhalt einer Volumenkonstanz des Gewebes und damit fur die Deckung groserer Weichteildefekte. Das schnell wachsende, interdisziplinare Gebiet des Tissue- Engineerings bietet alternative Losungsansatze zu den bisherigen Therapiemoglichkeiten. Die Zielsetzung ist hierbei Fettgewebe im Labor sowie zukunftig autolog im Patienten herzustellen, welches als permanenter Gewebeersatz an ­entsprechende Korperstellen volumenstabil transplantiert werden kann. Zahlreiche Untersuchungen haben die Vaskularisation als bedeutendsten und gleichzeitig kritischsten Faktor fur Qualitat, Volumen und Langzeituberleben der transplantierten und komplett neu generierten Fettgewebekonstrukte aufgezeigt. Und obwohl unser Verstandnis der regulatorischen Mechanismen der Adipogenese noch sehr eingeschrankt ist, bestehen klare Hinweise darauf, dass die komplexen Ablaufe der Zellinteraktionen bei der Proliferation und Differenzierung von Adipozyten in direkter Weise mit der Angiogenese korrelieren.

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein–ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-α-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. Proteins 2010. © 2010 Wiley-Liss, Inc.

Growing three-dimensional tissue within a chamber requires vigorous angiogenesis initiated by, for example, an arteriovenous fistula or a ligated vascular pedicle. Growth may also be enhanced by contact with the external environment. In this study tissue growth in a rat model, vascularized via an arteriovenous loop (AV Loop) or ligated pedicle, was compared in chambers that were either closed or perforated. Chambers were harvested at 4 weeks and tissue volume and histology compared. In perforated chambers, more tissue were generated using the ligated pedicle (0.75 ml+/-0.04) than the AV Loop (0.59 ml+/-0.01). Perforated chambers generated larger volumes of tissue than closed chambers because they encouraged tissue ingrowth through the perforations. Both vessel configurations supported tissue growth but, interestingly, the ligated pedicle resulted in significantly more tissue in the perforated chambers.

The covalent functionalization of "inert" polymers such as polypropylene with biomolecules for biocompatible or biosensor surfaces is challenging. Here we present a powerful approach to covalently modify "inert" macromolecular surfaces with biomacromolecules reusing old plastic material. A special emphasis was placed on easily accessible materials and a process which is easy, fast, efficient, cheap, and reliable. "Plastic trash" (lids from Eppendorf ® pipet tip containers) was used as a polymer substrate to demonstrate the use/reuse of commercial packing material to covalently modify this material with a thin reactive plasma polymerized maleic anhydride nano-layer network, which can be subsequently modified with biomolecules for various applications, e.g., in tissue engineering and as biochips.

For a monolayer of 2,3-di-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-a-lipoic acid ester lipid (DPTL) self-assembled (SAM) at a gold electrode surface we propose a new method to determine the charge number per adsorbed molecule and the packing density (area per molecule) in the monolayer. The method relies on chronocoulometry to measure the charge density at the SAM covered gold electrode surface. Two series of measurements have to be performed. In the first series, charge densities are measured for a monolayer transferred from the air-solution to the metal-solution interface using the Langmuir-Blodgett (LB) technique. This series of measurements allows one to determine charge numbers per adsorbed DPTL molecule. The second series is performed using a gold electrode covered with a self-assembled monolayer. The charge densities obtained in this series are then used to calculate the packing density with the help of charge numbers per adsorbed DPTL determined in the first series. The area per adsorbed molecule determined by the new method was compared to the area per molecule determined by the popular reductive desorption method. The molecular area determined with the new method is about 20% larger than the area calculated from the van der Waals model, which is a physically reasonable result. In contrast, the popular reductive desorption method gives an area per molecule 20% lower than the minimum estimated based on a van der Waals model. This is a physically unreasonable result. It is also shown that the charge numbers per adsorbed molecule depend on the electrode potential and may assume values smaller than the number of electrons participating in the reductive desorption step. An explanation of the origin of the "partial charge numbers" is provided. We recommend the new method be used in future studies of thiol adsorption at metal surfaces.

In order to incorporate integral proteins in a functionally active state, metal-supported lipid bilayers must have a hydrophilic region interposed between the bilayer and the metal. This region is realized with a hydrophilic molecule terminating at one end with a sulfhydryl or disulfide group that anchors this "hydrophilic spacer" to the surface of a metal, such as gold or mercury. The other end of the hydrophilic spacer may be covalently linked to the polar head of a phospholipid molecule, giving rise to a supramolecule called "thiolipid" (TL). With respect to gold, mercury has the advantage of providing a defect-free and fluid surface to the self-assembling spacer. Hydrophilic spacers consisting of a polyethyleneoxy or a hexapeptide chain, as well as thiolipids derived from these spacers, were employed to fabricate mercury-supported lipid bilayers. The formation of a lipid bilayer on top of a self-assembled monolayer of a hydrophilic spacer, or of a single-lipid monolayer on top of a self-assembled monolayer of a thiolipid, was realized by simply immersing the coated mercury electrode into an aqueous solution across a lipid film previously spread on its surface at its spreading pressure. Particularly stable mercury-supported lipid bilayers were obtained by using thiolipids. The biomimetic properties of these lipid bilayers were tested by incorporating channel-forming polypeptides (gramicidin and melittin) and proteins (OmpF porin). The effect of the transmembrane potential on the function of these channels was estimated by using a simple electrostatic model of the mercury-solution interphase.

We studied the interaction of the alpha-helical peptide acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)) with tethered bilayer lipid membranes (tBLM) and lipid monolayers formed at an air-water interface. The interaction of L(24) with tBLM resulted in adsorption of the peptide to the surface of the bilayer, characterized by a binding constant K(c)=2.4+/-0.6 microM(-1). The peptide L(24) an induced decrease of the elasticity modulus of the tBLM in a direction perpendicular to the membrane surface, E(radial). The decrease of E(radial) with increasing peptide concentration can be connected with a disordering effect of the peptide to the tBLM structure. The pure peptide formed a stable monolayer at the air/water interface. The pressure-area isotherms were characterized by a transition of the peptide monolayer, which probably corresponds of the partial intercalation of the alpha-helixes at higher surface pressure. Interaction of the peptide molecules with lipid monolayers resulted in an increase of the mean molecular area of phospholipids both in the gel and liquid crystalline states. With increasing peptide concentration, the temperature of the phase transition of the monolayer shifted toward lower temperatures. The analysis showed that the peptide-lipid monolayer is not an ideally miscible system and that the peptide molecules form aggregates in the monolayer.

Bilayer lipid membranes tethered to planar gold electrodes were prepared, based on a self assembled monolayer (SAM) of 2,3-di- O -phytanyl-sn-glycerol-1-tetraethylene glycol-DL-α-lipoic acid ester lipid (DPTL). When the SAM's were exposed to a suspension of liposomes made from diphytanoylphosphodatidyl choline (DPhyPC), tethered lipid bilayers (tBLMs) were formed with good sealing properties. The preformed tBLMs were doped with valinomycin, and the K + ion concentration in the bathing solution was increased stepwise by adding KCl. Electrical impedance spectra were recorded following every addition. These data were modelled by means of the network simulation program spice , using parameter values of the undoped membrane and a kinetic scheme for the K + /valinomycin system, whose rate constants were determined previously in independent measurements. Experimental and simulated data are in reasonable agreement despite some simplifying assumptions used in the spice simulations. Experimental data were also fitted to a conventional equivalent circuit, which reveals that the representation of the K + /valinomycin system by an ohmic resistor can be considered as no more than an approximation.

The nitroxide-mediated living free-radical polymerization of 1,2,5,6-di(iso-propylidene)-D-glucose-2-propenoate was achieved in dimethylformamide at 105 °C with an α-hydrido alkoxyamine initiator functionalized with a lipophilic N,N-di(octadecyl)amine group. The kinetics of the polymerization were investigated, and the mechanism was shown to be a living process allowing, after hydrolysis, controlled molecular weight, low-polydispersity lipo-glycopolymers to be prepared. The amphiphilic character of the macromolecule could be altered by either the exchange of the alkoxyamine at the chain end with hydrogen or the preparation of copolymers with lipophilic monomers such as N,N-di(octadecyl)acrylamide. The surface and membrane-forming properties of these novel lipopolymers demonstrate their amphiphilic character.

Inspired by nature, the synthesis of biohybrid nanocomposites containing inorganic nanoparticles (NPs) and biopolymers such as DNA and peptides as templates offers great potential for a wide range of applications. Using selective recognition schemes of 3D protein spaces for the assembly of magnetic nanocrystals is a challenge with great promise in the field of biomedicine and magnetic data storage. Here we apply the toroidal protein Hcp1 as an interparticle connector for the directed molecular assembly and ferrimagnetic coupling of biohybrid cobalt ferrite NP wires. The resulting biohybrid NP composites show bundles of nanofibers ranging from nano- to the microscale in length verified by TEM, EDX analysis and focused ion beam cut. Their magnetic characterization reveals an increase of the coercive field (+12%) reaching values of high-end Nd2Fe14B bulk magnets, enhanced saturation (+28%) and remanence magnetization (+38%) at 2 K compared to NPs lacking the protein connector. Thus, the combination of the nanoscale alignment of magnetic NPs with the molecular precision of the protein connectors leads to constructive addition of the magnetization reversal energy. This approach can be used to control magnetic properties for the design of materials with enhanced coercivity applicable for magnetic data storage, hyperthermia and theranostics. published

The expansion of cellular functions via novel modular building blocks, namely unnatural amino acids, their site-selective genetically encoded cotranslational incorporation into proteins, requires the redesign and expansion of the translational network with additional components, the orthogonal tRNA and tRNA synthetase. At the next level protein tectons (tecton = architectural building block) constitute complex genetically encoded “material libraries” inside the cell. These protein tectons are architectural building blocks allowing for complex supramolecular self-assembly inside the cell, forming cellular compartments or constituting 3D matrix mimicry of the extracellular matrix outside the cell. In addition they form the basis for biohybrid materials in protein/enzyme engineering, nanotechnology and regenerative medicine. The defined modification of protein tectons utilizing chemical biology allows for the selective bioconjugation e.g. of unnatural amino acids, via bioorthogonal chemical reactions introducing novel chemical entities expanding the repertoire of “posttranslational” protein modifications in vitro and in vivo for various applications.

Pulsed plasma polymerization has been used to deposit thin polymer films from maleic anhydride. By variations of the duty cycle (ton/(ton + toff)) employed during the plasma treatment, it has been possible to achieve a high retention of the anhydride groups within the film. Hydration of the anhydride groups in water created a carboxylic acid functionalized film, which was used to support a negatively charged DMPG lipid bilayer using Ca2+ as a chelating agent. The capacitance and resistance of adsorbed DMPG on three films prepared at different plasma duty cycles could be correlated with the relative amount of anhydride groups present in the plasma polymer films.

Conjugation of quantum dots (QDs) with proteins is applicable for biosensing and imaging in biomedical applications but also for the confined assembly of various nanostructures. Here, the conjugation of CdSe QDs ranging from 2.5 up to 9.5 nm with Hcp1, a yoctowell forming hexameric core protein of Pseudomonas aeruginosa, is described. Defined structures ranging from single QDs trapped within the donut-shaped hexameric protein cavity over “globular” multi-toroid architectures up to photonic wires of 850 nm in length and 9 nm in width are self-assembled while preserving the native hexameric structure of the protein conserving the photoluminescence intensity of the QDs in aqueous solution for more than 3 month.

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-alpha-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.

Growing three-dimensional tissue within a chamber requires vigorous angiogenesis initiated by, for example, an arteriovenous fistula or a ligated vascular pedicle. Growth may also be enhanced by contact with the external environment. In this study tissue growth in a rat model, vascularized via an arteriovenous loop (AV Loop) or ligated pedicle, was compared in chambers that were either closed or perforated. Chambers were harvested at 4 weeks and tissue volume and histology compared. In perforated chambers, more tissue were generated using the ligated pedicle (0.75 ml+/-0.04) than the AV Loop (0.59 ml+/-0.01). Perforated chambers generated larger volumes of tissue than closed chambers because they encouraged tissue ingrowth through the perforations. Both vessel configurations supported tissue growth but, interestingly, the ligated pedicle resulted in significantly more tissue in the perforated chambers.

This contribution describes the assembly and structural and functional characterization of various types of polymer-supported lipid bilayer membranes.We start with the description of the polymer-cushioned membrane that can be prepared by first attaching (covalently) polymer coils (as tethers or cushions) from solution to a reactive solid support, followed by the covalent coupling of a lipid monolayer containing reactive anchor lipids. Alternatively, a lipopolymer monolayer (if needed mixed with “normal” lipids) is pre-organized at the water-air interface in a Langmuir trough and then transferred to a solid substrate which is again pre-functionalized by a reactive coating. A special case discussed is the use of glycolipopolymers for the assembly of the proximal tethered monolayer. From all these interfacial architectures the final structure, the supported bilayer, is obtained by the fusion of vesicles forming the distal monolayer of the membrane.