Aptamers as Novel Binding Molecules on an Antimicrobial Peptide-Armored Composite Hydrogel Wound Dressing for Specific Removal and Efficient Eradication of Pseudomonas aeruginosa.

Here we present for the first time a potential wound dressing material implementing aptamers as binding entities to remove pathogenic cells from newly contaminated surfaces of wound matrix-mimicking collagen gels. The model pathogen in this study was the Gram-negative opportunistic bacterium Pseudomonas aeruginosa, which represents a considerable health threat in hospital environments as a cause of severe infections of burn or post-surgery wounds. A two-layered hydrogel composite material was constructed based on an established eight-membered focused anti-P. aeruginosa polyclonal aptamer library, which was chemically crosslinked to the material surface to form a trapping zone for efficient binding of the pathogen. A drug-loaded zone of the composite released the C14R antimicrobial peptide to deliver it directly to the bound pathogenic cells. We demonstrate that this material combining aptamer-mediated affinity and peptide-dependent pathogen eradication can quantitatively remove bacterial cells from the "wound" surface, and we show that the surface-trapped bacteria are completely killed. The drug delivery function of the composite thus represents an extra safeguarding property and thus probably one of the most important additional advances of a next-generation or smart wound dressing ensuring the complete removal and/or eradication of the pathogen of a freshly infected wound.

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots.

In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes.

Azulitox-A Pseudomonas aeruginosa P28-Derived Cancer-Cell-Specific Protein Photosensitizer.

Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating peptide P28. The photosensitizer domain (EcFbFP) was derived from a bacterial blue-light receptor, which belongs to the family of light-oxygen-voltage proteins and produces reactive oxygen species (ROS) upon excitation. P28 is derived from the cupredoxin protein azurin that is known to specifically penetrate cancer cells and bind to the tumor suppressor protein p53. We show that the P28 domain specifically directs and translocates the fused photosensitizer into cancer cells. Under blue-light illumination, Azulitox significantly induced cytotoxicity. Compared to the extracellular application of EcFbFP, Azulitox caused death to about 90% of cells, as monitored by flow cytometry, which also directly correlated with the amount of ROS produced in the cells. Azulitox may open new avenues toward targeted polypeptide-photosensitizer-based photodynamic therapies with reduced systemic toxicity compared to conventional photosensitizers.

Correction to: Monitoring dynamic release of intracellular hydrogen peroxide through a microelectrode based enzymatic biosensor.

The authors would like to call the reader's attention to the fact that unfortunately Alberto Pasquarelli's and Kay-Eberhard Gottschalk's affiliations were wrong in the original publication.

Monitoring dynamic release of intracellular hydrogen peroxide through a microelectrode based enzymatic biosensor.

A high sensitive and selective hydrogen peroxide (H2O2) biosensor was fabricated on the basis of reduced hemoglobin (Hb) and single-walled carbon nanotubes (SWCNTs) for detecting the release of H2O2 from living HepG2 cancer cells in the process of the in situ biosynthesis of ZnO quantum. The modification of carbon fiber microelectrode (CFME) was carried out by physical adsorption. By the scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS), the dense cover of surface and successful immobilization were characterized. Electrochemical investigation demonstrates that the as-prepared modified microelectrode showed a quasi-reversible process toward the reduction of H2O2, which exhibited a linear range from 0.51 to 10.6 µM, with a limit of detection of 0.23 µM. This microelectrode biosensor was applied for the quantification of the change of H2O2 concentration released from HepG2 cells through the in situ biosynthesis of ZnO quantum dots, which was further confirmed by the fluorescence staining.

Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles.

In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion.

Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.

Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100 µm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150 µM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.

Peripheral monocytes are functionally altered and invade the CNS in ALS patients.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease affecting primarily the upper and lower motor neurons. A common feature of all ALS cases is a well-characterized neuroinflammatory reaction within the central nervous system (CNS). However, much less is known about the role of the peripheral immune system and its interplay with CNS resident immune cells in motor neuron degeneration. Here, we characterized peripheral monocytes in both temporal and spatial dimensions of ALS pathogenesis. We found the circulating monocytes to be deregulated in ALS regarding subtype constitution, function and gene expression. Moreover, we show that CNS infiltration of peripheral monocytes correlates with improved motor neuron survival in a genetic ALS mouse model. Furthermore, application of human immunoglobulins or fusion proteins containing only the human Fc, but not the Fab antibody fragment, increased CNS invasion of peripheral monocytes and delayed the disease onset. Our results underline the importance of peripheral monocytes in ALS pathogenesis and are in agreement with a protective role of monocytes in the early phase of the disease. The possibility to boost this beneficial function of peripheral monocytes by application of human immunoglobulins should be evaluated in clinical trials.

Ageing-induced shrinkage of intervessel pit membranes in xylem of Clematis vitalba modifies its mechanical properties as revealed by atomic force microscopy.

Bordered pit membranes of angiosperm xylem are anisotropic, mesoporous media between neighbouring conduits, with a key role in long distance water transport. Yet, their mechanical properties are poorly understood. Here, we aim to quantify the stiffness of intervessel pit membranes over various growing seasons. By applying an AFM-based indentation technique "Quantitative Imaging" we measured the effective elastic modulus (E effective) of intervessel pit membranes of Clematis vitalba in dependence of size, age, and hydration state. The indentation-deformation behaviour was analysed with a non-linear membrane model, and paired with magnetic resonance imaging to visualise sap-filled and embolised vessels, while geometrical data of bordered pits were obtained using electron microscopy. E effective was transformed to the geometrically independent apparent elastic modulus E apparent and to aspiration pressure P b. The material stiffness (E apparent) of fresh pit membranes was with 57 MPa considerably lower than previously suggested. The estimated pressure for pit membrane aspiration was 2.20+28 MPa. Pit membranes from older growth rings were shrunken, had a higher material stiffness and a lower aspiration pressure than current year ones, suggesting an irreversible, mechanical ageing process. This study provides an experimental-stiffness analysis of hydrated intervessel pit membranes in their native state. The estimated aspiration pressure suggests that membranes are not deflected under normal field conditions. Although absolute values should be interpreted carefully, our data suggest that pit membrane shrinkage implies increasing material stiffness, and highlight the dynamic changes of pit membrane mechanics and their complex, functional behaviour for fluid transport.

Cell shape and tension alter focal adhesion structure.

Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How tension influences the structure of focal adhesions is not well understood. Here, we analyse the effect of tension on the location of key focal adhesion proteins, namely vinculin, paxillin and actin. We use micropatterning on gold surfaces to manipulate the cell shape, to create focal adhesions at specific cell areas, and to perform metal-induced energy transfer (MIET) measurements on the patterned cells. MIET resolves the different protein locations with respect to the gold surface with nanometer accuracy. Further, we use drugs influencing the cellular motor protein myosin or mechanosensitive ion channels to get deeper insight into focal adhesions at different tension states. We show here that in particular actin is affected by the rationally tuned force balance. Blocking mechanosensitive ion channels has a particularly high influence on the actin and focal adhesion architecture, resulting in larger focal adhesions with elevated paxillin and vinculin and strongly lowered actin stress fibres. Our results can be explained by a balance of adhesion tension with cellular tension together with ion channel-controlled focal adhesion homeostasis, where high cellular tension leads to an elevation of vinculin and actin, while high adhesion tension lowers these proteins.

Remodeling of the focal adhesion complex by hydrogen-peroxide-induced senescence.

Cellular senescence is a phenotype characterized by cessation of cell division, which can be caused by exhaustive replication or environmental stress. It is involved in age-related pathophysiological conditions and affects both the cellular cytoskeleton and the prime cellular mechanosensors, focal adhesion complexes. While the size of focal adhesions increases during senescence, it is unknown if and how this is accompanied by a remodeling of the internal focal adhesion structure. Our study uses metal-induced energy transfer to study the axial dimension of focal adhesion proteins from oxidative-stress-induced senescent cells with nanometer precision, and compares these to unstressed cells. We influenced cytoskeletal tension and the functioning of mechanosensitive ion channels using drugs and studied the combined effect of senescence and drug intervention on the focal adhesion structure. We found that H2O2-induced restructuring of the focal adhesion complex indicates a loss of tension and altered talin complexation. Mass spectroscopy-based proteomics confirmed the differential regulation of several cytoskeletal proteins induced by H2O2 treatment.

Influence of ROCK Pathway Manipulation on the Actin Cytoskeleton Height.

The actin cytoskeleton with its dynamic properties serves as the driving force for the movement and division of cells and gives the cell shape and structure. Disorders in the actin cytoskeleton occur in many diseases. Deeper understanding of its regulation is essential in order to better understand these biochemical processes. In our study, we use metal-induced energy transfer (MIET) as a tool to quantitatively examine the rarely considered third dimension of the actin cytoskeleton with nanometer accuracy. In particular, we investigate the influence of different drugs acting on the ROCK pathway on the three-dimensional actin organization. We find that cells treated with inhibitors have a lower actin height to the substrate while treatment with a stimulator for the ROCK pathway increases the actin height to the substrate, while the height of the membrane remains unchanged. This reveals the precise tuning of adhesion and cytoskeleton tension, which leads to a rich three-dimensional structural behaviour of the actin cytoskeleton. This finetuning is differentially affected by either inhibition or stimulation. The high axial resolution shows the importance of the precise finetuning of the actin cytoskeleton and the disturbed regulation of the ROCK pathway has a significant impact on the actin behavior in the z dimension.

A Polyclonal SELEX Aptamer Library Allows Differentiation of Candida albicans, C. auris and C. parapsilosis Cells from Human Dermal Fibroblasts.

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples.

Albumin Microspheres as "Trans-Ferry-Beads" for Easy Cell Passaging in Cell Culture Technology.

Protein hydrogels represent ideal materials for advanced cell culture applications, including 3D-cultivation of even fastidious cells. Key properties of fully functional and, at the same time, economically successful cell culture materials are excellent biocompatibility and advanced fabrication processes allowing their easy production even on a large scale based on affordable compounds. Chemical crosslinking of bovine serum albumin (BSA) with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) in a water-in-oil emulsion with isoparaffinic oil as the continuous phase and sorbitan monooleate as surfactant generates micro-meter-scale spherical particles. They allow a significant simplification of an indispensable and laborious step in traditional cell culture workflows. This cell passaging (or splitting) to fresh culture vessels/flasks conventionally requires harsh trypsinization, which can be omitted by using the "trans-ferry-beads" presented here. When added to different pre-cultivated adherent cell lines, the beads are efficiently boarded by cells as passengers and can be easily transferred afterward for the embarkment of novel flasks. After this procedure, cells are perfectly viable and show normal growth behavior. Thus, the trans-ferry-beads not only may become extremely affordable as a final product but also may generally replace trypsinization in conventional cell culture, thereby opening new routes for the establishment of optimized and resource-efficient workflows in biological and medical cell culture laboratories.

The conformations of amino acids on a gold(111) surface.

The interactions of amino acids with inorganic surfaces are of interest for biologists and biotechnologists alike. However, the structural determinants of peptide-surface interactions have remained elusive, but are important for a structural understanding of the interactions of biomolecules with gold surfaces. Molecular dynamics simulations are a tool to analyze structures of amino acids on surfaces. However, such an approach is challenging due to lacking parameterization for many surfaces and the polarizability of metal surfaces. Herein, we report DFT calculations of amino acid fragments in vacuo and molecular dynamics simulations of the interaction of all amino acids with a gold(111) surface in explicit solvent, using the recently introduced polarizable gold force field GolP. We describe preferred orientations of the amino acids on the metal surface. We find that all amino acids preferably interact with the gold surface at least partially with their backbone, underlining an unfolding propensity of gold surfaces.

Interaction of amino acids with the Au(111) surface: adsorption free energies from molecular dynamics simulations.

Interactions of proteins with inorganic surfaces are of high importance in biological events and in modern biotechnological applications. Therefore, peptides have been engineered to recognize inorganic surfaces with high specificity. However, the underlying interactions are still not well understood. Here, we investigated the adsorption of amino acids as protein building blocks onto a Au(111) surface. In particular, using molecular dynamics simulations, we calculated the potential of mean force between all the 20 amino acids and the gold surface. We found a strong dependence of the binding affinities on the chemical character of the amino acids. Additionally, the interaction free energy is correlated with the propensity of amino acids to form beta-sheets, hinting at design principles for gold binding peptides and induction of beta-sheet formation near surfaces.

Micropatterning of Cells on Gold Surfaces for Biophysical Applications.

We developed a reproducible micropatterning method to manipulate and normalize cell shape and cell-cell separation on gold. We used methoxy polyethylene glycol thiol (PEG-SH) to create a self-assembled monolayer that can be oxidized at desired shapes through a photomask with deep UV light. The oxidized PEG can be coated with extracellular matrix proteins and seeded with cells adopting the pre-defined shape. The developed and analyzed surfaces can be used in a wide range of biophysical applications.

The effect of different force applications on the protein-protein complex Barnase-Barstar.

Steered molecular dynamics simulations are a tool to examine the energy landscape of protein-protein complexes by applying external forces. Here, we analyze the influence of the velocity and geometry of the probing forces on a protein complex using this tool. With steered molecular dynamics, we probe the stability of the protein-protein complex Barnase-Barstar. The individual proteins are mechanically labile. The Barnase-Barstar binding site is more stable than the folds of the individual proteins. By using different force protocols, we observe a variety of responses of the system to the applied tension.

The viscoelasticity of membrane tethers and its importance for cell adhesion.

Cell adhesion mechanically couples cells to surfaces. The durability of individual bonds between the adhesive receptors and their ligands in the presence of forces determines the cellular adhesion strength. For adhesive receptors such as integrins, it is a common paradigm that the cell regulates its adhesion strength by altering the affinity state of the receptors. However, the probability distribution of rupture forces is dependent not only on the affinity of individual receptor-ligand bonds but also on the mechanical compliance of the cellular anchorage of the receptor. Hence, by altering the anchorage, the cell can regulate its adhesion strength without changing the affinity of the receptor. Here, we analyze the anchorage of the integrin VLA-4 with its ligand VCAM-1. For this purpose, we develop a model based on the Kelvin body, which allows one to quantify the mechanical properties of the adhesive receptor's anchorage using atomic force microscopy on living cells. As we demonstrate, the measured force curves give valuable insight into the mechanics of the cellular anchorage of the receptor, which is described by the tether stiffness, the membrane rigidity, and the membrane viscosity. The measurements relate to a tether stiffness of k(t) = 1.6 microN/m, an initial membrane rigidity of k(i) = 260 microN/m, and a viscosity of mu = 5.9 microN x s/m. Integrins exist in different activation states. When activating the integrin with Mg(2+), we observe altered viscoelastic parameters of k(t) = 0.9 microN/m, k(i) = 190 microN/m, and mu = 6.0 microN x s/m. Based on our model, we postulate that anchorage-related effects are common regulating mechanisms for cellular adhesion beyond affinity regulation.

Mechanical regulation of cell adhesion.

Cellular adhesion against external forces is governed by both the equilibrium affinity of the involved receptor-ligand bonds and the mechanics of the cell. Certain receptors like integrins change their affinity as well as the mechanics of their anchorage to tune the adhesiveness. Whereas in the last few years the focus of integrin research has lain on the affinity regulation of the adhesion receptors, more recently the importance of cellular mechanics became apparent. Here, we focus on different aspects of the mechanical regulation of the cellular adhesiveness.