De novo design of a polycarbonate hydrolase.

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha /\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha /\beta $-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. With the help of Rosetta an active site catalysing the hydrolysis of polycarbonate is introduced into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in Escherichia coli. Thermal analysis suggests that the design has a melting temperature of >95$^{\circ }$C. Activity toward polycarbonate was confirmed using atomic force spectroscopy (AFM), proving the successful design of a PCase.

Amperometric Biosensor for Quantitative Measurement Using Sandwich Immunoassays.

State-of-the-art clinical detection methods typically involve standard immunoassay methods, requiring specialized equipment and trained personnel. This impedes their use in the Point-of-Care (PoC) environment, where ease of operation, portability, and cost efficiency are prioritized. Small, robust electrochemical biosensors provide a means with which to analyze biomarkers in biological fluids in PoC environments. Optimized sensing surfaces, immobilization strategies, and efficient reporter systems are key to improving biosensor detection systems. The signal transduction and general performance of electrochemical sensors are determined by surface properties that link the sensing element to the biological sample. We analyzed the surface characteristics of screen-printed and thin-film electrodes using scanning electron microscopy and atomic force microscopy. An enzyme-linked immunosorbent assay (ELISA) was adapted for use in an electrochemical sensor. The robustness and reproducibility of the developed electrochemical immunosensor were investigated by detecting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in urine. The sensor showed a detection limit of 1 ng/mL, a linear range of 3.5-80 ng/mL, and a CV% of 8%. The results demonstrate that the developed platform technology is suitable for immunoassay-based sensors on either screen-printed or thin-film gold electrodes.

Biochemical Characterization and NMR Study of a PET-Hydrolyzing Cutinase from Fusarium solani pisi.

In recent years, the drawbacks of plastics have become evident, with plastic pollution becoming a major environmental issue. There is an urgent need to find solutions to efficiently manage plastic waste by using novel recycling methods. Biocatalytic recycling of plastics by using enzyme-catalyzed hydrolysis is one such solution that has gained interest, in particular for recycling poly(ethylene terephthalate) (PET). To provide insights into PET hydrolysis by cutinases, we have here characterized the kinetics of a PET-hydrolyzing cutinase from Fusarium solani pisi (FsC) at different pH values, mapped the interaction between FsC and the PET analogue BHET by using NMR spectroscopy, and monitored product release directly and in real time by using time-resolved NMR experiments. We found that primarily aliphatic side chains around the active site participate in the interaction with BHET and that pH conditions and a mutation around the active site (L182A) can be used to tune the relative amounts of degradation products. Moreover, we propose that the low catalytic performance of FsC on PET is caused by poor substrate binding combined with slow MHET hydrolysis. Overall, our results provide insights into obstacles that preclude efficient PET hydrolysis by FsC and suggest future approaches for overcoming these obstacles and generating efficient PET-hydrolyzing enzymes.

Melt Electrospinning of PET and Composite PET-Aerogel Fibers: An Experimental and Modeling Study.

Increasingly advanced applications of polymer fibers are driving the demand for new, high-performance fiber types. One way to produce polymer fibers is by electrospinning from polymer solutions and melts. Polymer melt electrospinning produces fibers with small diameters through solvent-free processing and has applications within different fields, ranging from textile and construction, to the biotech and pharmaceutical industries. Modeling of the electrospinning process has been mainly limited to simulations of geometry-dependent electric field distributions. The associated large change in viscosity upon fiber formation and elongation is a key issue governing the electrospinning process, apart from other environmental factors. This paper investigates the melt electrospinning of aerogel-containing fibers and proposes a logistic viscosity model approach with parametric ramping in a finite element method (FEM) simulation. The formation of melt electrospun fibers is studied with regard to the spinning temperature and the distance to the collector. The formation of PET-Aerogel composite fibers by pneumatic transport is demonstrated, and the critical parameter is found to be the temperature of the gas phase. The experimental results form the basis for the electrospinning model, which is shown to reproduce the trend for the fiber diameter, both for polymer as well as polymer-aerogel composites.

Comparative study of antibacterial properties of polystyrene films with TiO x and Cu nanoparticles fabricated using cluster beam technique.

Background: Antibacterial materials are of high importance for medicine, and for the production and conservation of food. Among these materials, polymer films with metal nanoparticles (NPs) are of considerable interest for many practical applications. Results: The paper describes a novel approach for the formation of bactericidal polymer thin films (polystyrene in this case), produced by spin-coating, with Ti and Cu NPs deposited from cluster beams. Ti NPs are treated in three different ways in order to study different approaches for oxidation and, thus, efficiency in formation of the particles with semiconducting properties required for the catalytic formation of reactive oxygen species. Cu NPs are used as deposited. Partial NP embedding into polystyrene is realised in a controllable manner using thermal annealing in order to improve surface adhesion and make the particles resistant against wash-out. The formed composite films with TiO x and Cu species are tested as bactericidal media using E.coli bacteria as model microorganisms. Conclusion: The obtained results show considerable efficiency in destroying the bacteria and a good possibility of multiple re-use of the same composite films making the suggested approach attractive for the cases requiring reusable polymer-based antibacterial media.

Formation of Conductive DNA-Based Nanowires via Conjugation of dsDNA with Cationic Peptide.

A novel conductive DNA-based nanomaterial, DNA-peptide wire, composed of a DNA core and a peripheral peptide layer, is presented. The electrical conductivity of the wire is found to be at least three orders in magnitude higher than that of native double-stranded DNA (dsDNA). High conductivity of the wires along with a better resistance to mechanical deformations caused by interactions between the substrate and electrode surface make them appealing for a wide variety of nanoelectronic and biosensor applications.

Atomic Force Microscopy Study of the Interactions of Indolicidin with Model Membranes and DNA.

The cell membrane is the first barrier and quite often the primary target that antimicrobial peptides (AMPs) have to destroy or penetrate to fulfill their mission. Upon penetrating through the membrane, the peptides can further attack intracellular targets, in particular DNA. Studying the interaction of an antimicrobial peptide with a cell membrane and DNA holds keys to understanding its killing mechanisms. Commonly, these interactions are studied by using optical or scanning electron microscopy and appropriately labeled peptides. However, labeling can significantly affect the hydrophobicity, conformation, and size of the peptide, hence altering the interaction significantly. Here, we describe the use of atomic force microscopy (AFM) for a label-free study of the interactions of peptides with model membranes under physiological conditions and DNA as a possible intracellular target.

The Physical Properties and Self-Assembly Potential of the RFFFR Peptide.

The self-assembly of fibers from peptides has attracted a tremendous amount of attention due to its many applications, such as in drug-delivery systems, in tissue engineering, and in electronic devices. Recently, the self-assembly potential of the designer peptide RFFFR has been reported. Here it is experimentally verified that the peptide forms fibers that are entangled and form solid spheres without water inside. Upon dilution below the critical fiber concentration, the fibers untangle and become totally separated prior to dissolution. These structures readily bind thioflavin T, resulting in a characteristic change in fluorescent properties consistent with ß-sheet-rich amyloid structures with aromatic/hydrophobic grooves. The circular dichroism spectroscopy data are dominated by a π→π* transition, thus indicating that the fibers are stabilized by π-stacking. Contrary to what was expected, the dissolution of the spheres/fibers results in increasing fluorescence anisotropy over time. This is explained in terms of HomoFRET between phenylalanine residues with a T-shaped π-stacking mode, which was determined in another study to be the dominant mode through atomistic simulations and semiempirical calculations. Kelvin probe force microscopy measurements indicate that the spheres and fibers have a conductivity comparable to that of gold. Hence, these self-assembled structures might be applicable in organic solid-state electronic devices. The dissolution properties of the spheres further suggest that they might be used as drug-delivery systems.

Large-scale fabrication of achiral plasmonic metamaterials with giant chiroptical response.

A variety of extrinsic chiral metamaterials were fabricated by a combination of self-ordering anodic oxidation of aluminum foil, nanoimprint lithography and glancing angle deposition. All of these techniques are scalable and pose a significant improvement to standard metamaterial fabrication techniques. Different interpore distances and glancing angle depositions enable the plasmonic resonance wavelength to be tunable in the range from UVA to IR. These extrinsic chiral metamaterials only exhibit significant chiroptical response at non-normal angles of incidence. This intrinsic property enables the probing of both enantoimeric structures on the same sample, by inverting the tilt of the sample relative to the normal angle. In biosensor applications this allows for more precise, cheap and commercialized devices. As a proof of concept two different molecules were used to probe the sensitivity of the metamaterials. These proved the applicability to sense proteins through non-specific adsorption on the metamaterial surface or through functionalized surfaces to increase the sensing sensitivity. Besides increasing the sensing sensitivity, these metamaterials may also be commercialized and find applications in surface-enhanced IR spectroscopy, terahertz generation and terahertz circular dichroism spectroscopy.

A computational study of the self-assembly of the RFFFR peptide.

The ß-amyloid peptide sequence, LVFFA, inspired the investigation of the fiber formation potential of the RFFFR peptide. The self-assembly was studied in silico by coarse grained-, atomistic molecular dynamics simulations and semi-empirical quantum mechanical calculations. The fiber formation was found to occur according to a three step process starting with the emergence of small aggregates that join together and form fiber segments that eventually form one continuous fiber. From a series of simulations the critical fiber concentration was determined to be in the interval between 70 mM and 100 mM. To obtain more structural information of the stable fiber, the final coarse grained configuration was backtransformed to atomistic detail. Based on this structure a 10 ns atomistic simulation was performed, which suggests that the fiber is stabilized by hydrogen bonds and water mediated hydrogen bonds. These stabilizing bonds are, however, reduced by competitive protein-water hydrogen bonds. Hence, π-stacking is suspected to play a larger role in fiber stabilization. The π-stacking of intermolecular Phe residues are found to favor a T-shaped stacking mode, while intramolecular π-stacking interactions assume a broad variety of modes from the parallel displaced mode to the T-shaped stacking mode and modes in between, with equal probability. Selected snapshots from the atomistic simulation were geometry optimized using semi-empirical quantum mechanical methods to validate the fiber stability and π-stacking configuration. An average Cα-RMSD was determined to be 2.68 Å. These findings indicate that the fiber may be used as a novel model system for the study of amyloid fibers or self-assembled conductive biowires, respectively.

Engineering a Robust Photovoltaic Device with Quantum Dots and Bacteriorhodopsin.

We present a route toward a radical improvement in solar cell efficiency using resonant energy transfer and sensitization of semiconductor metal oxides with a light-harvesting quantum dot (QD)/bacteriorhodopsin (bR) layer designed by protein engineering. The specific aims of our approach are (1) controlled engineering of highly ordered bR/QD complexes; (2) replacement of the liquid electrolyte by a thin layer of gold; (3) highly oriented deposition of bR/QD complexes on a gold layer; and (4) use of the Forster resonance energy transfer coupling between bR and QDs to achieve an efficient absorbing layer for dye-sensitized solar cells. This proposed approach is based on the unique optical characteristics of QDs, on the photovoltaic properties of bR, and on state-of-the-art nanobioengineering technologies. It permits spatial and optical coupling together with control of hybrid material components on the bionanoscale. This method paves the way to the development of the solid-state photovoltaic device with the efficiency increased to practical levels.

Fouling of enhanced biological phosphorus removal-membrane bioreactors by humic-like substances.

Fouling by free extracellular polymeric substances was studied in an enhanced biological phosphorus removal-membrane bioreactor. It was demonstrated that the free extracellular polymeric substances, primarily consisting of humic-like substances, were adsorbed to the membrane used in the enhanced biological phosphorus removal-membrane bioreactor plant. Infrared analyses indicated the presence of the humic-like substances on the membrane's active surface after filtration of the free extracellular polymeric substances suspension. Scanning electron microscopy showed the presence of a gel layer on the membrane surface after filtration of the free extracellular polymeric substances suspension. The gel layer caused a significant decline in water flux. This layer was not entirely removed by a backwashing, and the membrane's water flux could not be re-established. The membrane used in the enhanced biological phosphorus removal-membrane bioreactor plant showed infrared spectra similar to that fouled by the free extracellular polymeric substances suspension in the laboratory. Thus, the results of this study show the importance of humic-like substances in irreversible fouling of enhanced biological phosphorus removal-membrane bioreactor systems.

Water condensation: a multiscale phenomenon.

The condensation of water is a phenomenon occurring in multiple situations in everyday life, e.g., when fog is formed or when dew forms on the grass or on windows. This means that this phenomenon plays an important role within the different fields of science including meteorology, building physics, and chemistry. In this review we address condensation models and simulations with the main focus on heterogeneous condensation of water. The condensation process is, at first, described from a thermodynamic viewpoint where the nucleation step is described by the classical nucleation theory. Further, we address the shortcomings of the thermodynamic theory in describing the nucleation and emphasize the importance of nanoscale effects. This leads to the description of condensation from a molecular viewpoint. Also presented is how the nucleation can be simulated by use of molecular models, and how the condensation process is simulated on the macroscale using computational fluid dynamics. Finally, examples of hybrid models combining molecular and macroscale models for the simulation of condensation on a surface are presented.

Study of the tryptophan-terbium FRET pair coupled to silver nanoprisms for biosensing applications.

Plasmonic coupling between fluorophores and metal surfaces has become a focal point of optical research during the last two decades, however, the interactions of FRET couples with metal surfaces remain relatively unexplored. In this study, interactions of the tryptophan-Tb(3+) FRET pair with silver nanoprisms for potential biosensor development have been investigated. For this purpose an engineered lanthanide binding peptide (LBTtrp) containing tryptophan as the sensitizer for bound lanthanide ions (Tb(3+)) as well as a trypsin cleavage site was synthesized. The modified LBTtrp peptide contained two N-terminal cysteine residues to provide a stronger coupling to the silver nanoprisms (~6 nm high, ~50 nm wide). This study investigated the interaction between tryptophan, chelated Tb(3+) ions, and silver nanoprisms in solution using fluorescence and transient absorption spectroscopy. We have found that Tb(3+) luminescence decreases upon binding of the LBTtrp-Tb(3+) to silver nanoprisms and increases upon trypsin cleavage. The transient absorption spectroscopy measurements showed a significant decrease in the lifetime of the excited singlet state of tryptophan upon Tb(3+) chelation, while coupling to the silver nanoprisms did not show a significant effect on tryptophan. The results obtained in this work demonstrate a first proof of concept for a new sensitive optical biosensor in solution.

Patterned polymeric surfaces to study the influence of nanotopography on the growth and differentiation of mesenchymal stem cells.

The implementation of micro- and nanotechnologies to biomaterials constitutes a unique platform to improve our understanding on microenvironmental regulation of stem cell functions. In the recent years, various methods have been developed for the fabrication of micro- and nanopatterned polymeric culture substrates, and many of these novel surfaces are opening possibilities for new applications. Here, we provide procedures for creating nanoscale topographic features on films of poly(lactic acid), a biodegradable polymer frequently used for the fabrication of tissue engineering scaffolds. In addition, we provide methods to assess the growth and differentiation of mesenchymal stem cells cultured on the substrates.

Overexpression of NRPS4 leads to increased surface hydrophobicity in Fusarium graminearum.

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.

Patterned poly(lactic acid) films support growth and spontaneous multilineage gene expression of adipose-derived stem cells.

Conventional culture surfaces do not provide optimal environmental cues for expansion or differentiation of adult stem cells. Aiming to increase the efficiency of the in vitro culture conditions, biocompatible and biodegradable biomaterials such as poly(lactic acid) (PLA) have been proposed to engineer the stem cell microenvironment. In this study, we explored the feasibility of using PLA substrates to control the responses of adipose-derived stem cells (ASCs). The substrates consisted of flat and patterned PLA films fabricated by casting a chloroform-PLA solution on a glass surface. Patterning was achieved through the condensation of nano-sized water droplets during chloroform evaporation, which resulted in films displaying irregularly distributed circular indentations with a mean diameter of 248±65 nm. Both types of PLA substrates were assessed for protein adsorption using fibronectin and in vitro cell culturing. Tissue-culture polystyrene (TCPS) plates were used as control surfaces. The experiments demonstrated that the patterned PLA substrates had a significantly higher fibronectin adsorption capacity when compared with the flat counterparts. For the entire duration of the culture period, there was no significant difference in cell growth rate on the PLA surfaces with respect to TCPS despite signs of reduced adhesion. In addition, the semi-quantitative real-time RT-PCR analysis of a set of 14 lineage-specific genes revealed that the PLA-related transcriptional activity significantly surpassed that of TCPS. Remarkably, when assessing the effect of patterning, the patterned films proved superior regarding the activation of genes involved in the skeletal myogenic, cardiomyogenic, chondrogenic, and adipogenic pathways. Taken together, our data provide evidence that the surface patterning can exert such an influence on the stem cell microenvironment that the differentiation process can be effectively modulated. Consequently, the patterned PLA surfaces could potentially be used as a platform for localized delivery and engraftment of stem cells.

PH-dependent self-assembly of the short Surfactant-like peptide KA6.

Self-assembly of amphiphilic peptides designed during the last ten years by different research groups lead to a large variety of 3D-structures that already found applications in e.g., for stabilization of large protein complexes, cell culturing systems etc. We present synthesis and characterization of a novel amphiphilic peptide KA6 that exhibits clear charge separation controllable by the pH of the environment. The self-assembly in this system is largely governed by electrostatic interaction, thus a change in pH will not only lead to a change in critical micellar concentration (CMC) of the peptide but also to the changes in micellar structure as revealed by atomic force microscopy (AFM) and circular dichroism (CD) study. At basic pH the micellar structure inverts exposing the opposite end of the peptide chain to the solution. This interesting phenomenon could provide basis for novel pH sensitive materials including drug delivery and controlled release systems.

Towards control of aggregational behaviour of alpha-lactalbumin at acidic pH.

alpha-Lactalbumin (alpha-La) undergoes considerable structural changes upon loss of bound Ca2+ at acidic pH, leaving alpha-La in a molten globule structure. Using fluorescence the present work provides more insight into the structural transition of alpha-La at acidic pH leading to protein aggregation, most likely caused by a combination of hydrophobic and electrostatic interactions. The rate of aggregation is determined by the protein concentration and temperature applied. Availability of Ca2+ stabilises the protein, and thus prevent aggregation at pH values as low as pH 2.9. In contrast, presence of Cu2+ induces a destabilisation of the protein, which can be explained by a binding to the Zn2+ binding site in alpha-La, possibly resulting in structural alterations of the protein. In general, presence of anions destabilize alpha-La at pH values below pI, with SO4(2-) exhibiting the strongest effect on the protein stability, thus correlating well with the Hofmeister series. At more acidic pH values far from pI, alpha-La becomes more stable towards ion induced aggregation, since higher ion activity is required to efficiently screen the charges on the protein surface. The results presented in this paper provide detailed knowledge on the external parameters leading to aggregation of alpha-La at acidic pH, thus permitting rational design of the aggregation process.