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Reconstruction of Ultra-thin Alveolar-capillary Basement Membrane Mimics
Alveolar-capillary basement membrane (BM) is ultra-thin (<2 µm) extracellular matrix that maintains integral epithelial-endothelial cell layers. In vitro reconstructions of alveolar-capillary barrier supported on synthetic scaffolds closely resembling the fibrous and ultra-thin natural BM are essential in mimicking the lung pathophysiology. Although BM topology and dimensions are well known to significantly influence cellular behavior, conventionally used BM mimics fail to recreate this natural niche. To overcome this, electrospun ultra-thin 2 µm poly(caprolactone) (PCL) nanofibrous mesh is used to establish an alveolar-capillary barrier model of lung endothelial/epithelial cells. Transepithelial electrical resistance (TEER) and permeability studies reveal integral tight junctions and improved mass transport through the highly porous PCL meshes compared to conventional dense membranes with etched pores. The chemotaxis of neutrophils is shown across the barrier in presence of inflammatory response that is naturally impeded in confined regions. Conventional requirement of 3 µm or larger pore size can lead to barrier disruption due to epithelial/endothelial cell invasion. Despite high porosity, the interconnected BM mimic prevents barrier disruption and allows neutrophil transmigration, thereby demonstrating the physiological relevance of the thin nanofibrous meshes. It is envisioned that these bipolar cultured barriers would contribute to an organ-level in vitro model for pathological disease, environmental pollutants, and nanotoxicology. © 2021 The Authors. Advanced Biology published by Wiley-VCH GmbH
Recycling and Separation of Homogeneous Catalyst from Aqueous Multicomponent Mixture by Organic Solvent Nanofiltration
Organic solvent nanofiltration (OSN) has evolved to an established recycling method for homogeneous catalysts. However, commercial availability has not circumvented the need for classification and the scoping of possible applications for specific solvent mixtures. Therefore, Evonik’s DuraMem® 300 was assessed for the recycling of magnesium triflate at two transmembrane pressures from a mixture of ethanol, ethyl acetate and water. Catalyst retention up to 98% and permeability of up to 4.44·10−1∙L∙bar−1∙m−2∙h−1 were possible when less than 25% ethyl acetate was in the mixture. The retention of some of the components in the ternary mixture was observed while others were enriched, making the membrane also suitable for fractioning thereof.
Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase
Cellulases are industrially important enzymes, e.g., in the production of bioethanol, in pulp and paper industry, feedstock, and textile. Thermostability is often a prerequisite for high process stability and improving thermostability without affecting specific activities at lower temperatures is challenging and often time-consuming. Protein engineering strategies that combine experimental and computational are emerging in order to reduce experimental screening efforts and speed up enzyme engineering campaigns. Constraint Network Analysis (CNA) is a promising computational method that identifies beneficial positions in enzymes to improve thermostability. In this study, we compare CNA and directed evolution in the identification of beneficial positions in order to evaluate the potential of CNA in protein engineering campaigns (e.g., in the identification phase of KnowVolution). We engineered the industrially relevant endoglucanase EGLII from Penicillium verruculosum towards increased thermostability. From the CNA approach, six variants were obtained with an up to 2-fold improvement in thermostability. The overall experimental burden was reduced to 40% utilizing the CNA method in comparison to directed evolution. On a variant level, the success rate was similar for both strategies, with 0.27% and 0.18% improved variants in the epPCR and CNA-guided library, respectively. In essence, CNA is an effective method for identification of positions that improve thermostability.
All-Conjugated Polymer Core-Shell and Core-Shell-Shell Particles with Tunable Emission Profiles and White Light Emission
Future applications of conjugated polymer particles (CPP) in medicine, organic photonics, and optoelectronics greatly depend on high performance and precisely adjustable optical properties of the particles. To meet these criteria, current particle systems often combine conjugated polymers with inorganic particles in core-shell geometries, extending the possible optical characteristics of CPP. However, current conjugated polymer particles are restricted to a single polymer phase composed of a distinct polymer or a polymer blend. Here, a synthetic toolbox is presented that enables the synthesis of monodisperse core-shell and core-shell-shell particles, which consist entirely of conjugated polymers but of different types in the core and the shells. Seeded and fed-batch dispersion polymerizations based on Suzuki-Miyaura-type cross-coupling are investigated. The different approaches allow accurate control over the created interface between the conjugated polymer phases and thus also over the energy transfer phenomena between them. This approach opens up completely new synthetic freedom for fine tuning of the optical properties of CPP, enabling, for example, the synthesis of individual white light-emitting particles.
A scalable bubble-free membrane aerator for biosurfactant production
The bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production.
Wetting-Induced Polyelectrolyte Pore Bridging
Active layers of ion separation membranes often consist of charged layers that retain ions based on electrostatic repulsion. Conventional fabrication of these layers, such as polyelectrolyte deposition, can in some cases lead to excess coating to prevent defects in the active layer. This excess deposition increases the overall membrane transport resistance. The study at hand presents a manufacturing procedure for controlled polyelectrolyte complexation in and on porous supports by support wetting control. Pre-wetting of the microfiltration membrane support, or even supports with larger pore sizes, leads to ternary phase boundaries of the support, the coating solution, and the pre-wetting agent. At these phase boundaries, polyelectrolytes can be complexated to form partially freestanding selective structures bridging the pores. This polyelectrolyte complex formation control allows the production of membranes with evenly distributed polyelectrolyte layers, providing (1) fewer coating steps needed for defect-free active layers, (2) larger support diameters that can be bridged, and (3) a precise position control of the formed polyelectrolyte multilayers. We further analyze the formed structures regarding their position, composition, and diffusion dialysis performance.
Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways
Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.g., local dehydration or competitive inhibition, and indirect effects, e.g., structural perturbations or disturbed catalytic site integrity. However, the molecular origin of indirect effects has largely remained elusive. Here we show by multi-μs long molecular dynamics simulations, free energy computations, and rigidity analyses that aIL favorably interact with specific residues of Bacillus subtilis Lipase A (BsLipA) and modify the local structural stability of this model enzyme by inducing long-range perturbations of noncovalent interactions. The perturbations percolate over neighboring residues and eventually affect the catalytic site and the buried protein core. Validation against a complete experimental site saturation mutagenesis library of BsLipA (3620 variants) reveals that the residues of the perturbation pathways are distinguished sequence positions where substitutions highly likely yield significantly improved residual activity. Our results demonstrate that identifying these perturbation pathways and specific IL ion-residue interactions there effectively predicts focused variant libraries with improved aIL tolerance.
Unraveling the Mechanism and Kinetics of Binding of an LCI-eGFP-Polymer for Antifouling Coatings
The ability of proteins to adsorb irreversibly onto surfaces opens new possibilities to functionalize biological interfaces. Herein, the mechanism and kinetics of adsorption of protein-polymer macromolecules with the ability to equip surfaces with antifouling properties are investigated. These macromolecules consist of the liquid chromatography peak I peptide from which antifouling polymer brushes are grafted using single electron transfer-living radical polymerization. Surface plasmon resonance spectroscopy reveals an adsorption mechanism that follows a Langmuir-type of binding with a strong binding affinity to gold. X-ray reflectivity supports this by proving that the binding occurs exclusively by the peptide. However, the lateral organization at the surface is directed by the cylindrical eGFP. The antifouling functionality of the unimolecular coatings is confirmed by contact with blood plasma. All coatings reduce the fouling from blood plasma by 8894% with only minor effect of the degree of polymerization for the studied range (DP between 101 and 932). The excellent antifouling properties, combined with the ease of polymerization and the straightforward coating procedure make this a very promising antifouling concept for a multiplicity of applications.
Molecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept
In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1-linker-mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.
Bioactive Nanogels Mimicking the Antithrombogenic Nitric Oxide-Release Function of the Endothelium
Nitric oxide (NO) plays a significant role in controlling the physiology and pathophysiology of the body, including the endothelial antiplatelet function and therefore, antithrombogenic property of the blood vessels. This property of NO can be exploited to prevent thrombus formation on artificial surfaces like extracorporeal membrane oxygenators, which when come into contact with blood lead to protein adsorption and thereby platelet activation causing thrombus formation. However, NO is extremely reactive and has a very short biological half-life in blood, so only endogenous generation of NO from the blood contacting material can result into a stable and kinetically controllable local delivery of NO. In this regards, highly hydrophilic bioactive nanogels are presented which can endogenously generate NO in blood plasma from endogenous NO-donors thereby maintaining a physiological NO flux. It is shown that NO releasing nanogels could initiate cGMP-dependent protein kinase signaling followed by phosphorylation of vasodilator-stimulated phosphoprotein in platelets. This prevents platelet activation and aggregation even in presence of highly potent platelet activators like thrombin, adenosine 5′-diphosphate, and U46619 (thromboxane A2 mimetic).