Polyclonal aptamer libraries as binding entities on a graphene FET based biosensor for the discrimination of apo- and holo-retinol binding protein 4.

Oligonucleotide DNA aptamers represent an emergently important class of binding entities towards as different analytes as small molecules or even whole cells. Without requiring the canonical isolation of individual aptamers following the SELEX process, the focused polyclonal libraries prepared by this in vitro evolution and selection can directly be used to label their dedicated targets and to serve as binding molecules on surfaces. Here we report the first instance of a sensor able to discriminate between loaded and unloaded retinol-binding protein 4 (RBP4), an important biomarker for the prediction of diabetes and kidney disease. The sensor relies on two aptamer libraries tuned such that they discriminate between the protein isoforms, requiring no further sample labelling to detect RBP4 in both states. The evolution, binding properties of the libraries and the functionalization of graphene FET sensor chips are presented as well as the functionality of the resulting biosensor.

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood.

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.

FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila.

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

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.

Diffusion-controlled release of the theranostic protein-photosensitizer Azulitox from composite of Fmoc-Phenylalanine Fibrils encapsulated with BSA hydrogels.

Hydrogels offer a promising potential for the encapsulation and regulated release of drugs due to their biocompatibility and their tunable properties as materials. Only a limited number of systems and procedures enable the encapsulation of sensitive proteins. N-terminally fmoc-protected phenylalanine has been shown to self-assemble into a transparent, stable hydrogel It can be considered a supergelator due to the low amount of monomers necessary for hydrogelation (0.1% w/v), making it a good candidate for the encapsulation and stabilization of sensitive proteins. However, application options for this hydrogel are rather limited to those of many other fibril-based materials due to its intrinsic lack of mechanical strength and high susceptibility to changes in environmental conditions. Here, we demonstrate that the stability of a fibrillary system and the resulting release of the protein-photosensitizer Azulitox can be increased by combining the hydrogel with a tightly cross-linked BSA hydrogel. Azulitox is known to display cell-penetrating properties, anti-proliferative activity and has a distinctive fluorescence. Confocal microscopy and fluorescence measurements verified the maintenance of all essential functions of the encapsulated protein. In contrast, the combination of fibrillary and protein hydrogel resulted in a significant stabilization of the matrix and an adjustable release pattern for encapsulated protein.

Antimicrobial Activity of Cyclic-Monomeric and Dimeric Derivatives of the Snail-Derived Peptide Cm-p5 against Viral and Multidrug-Resistant Bacterial Strains.

Cm-p5 is a snail-derived antimicrobial peptide, which demonstrated antifungal activity against the pathogenic strains of Candida albicans. Previously we synthetized a cyclic monomer as well as a parallel and an antiparallel dimer of Cm-p5 with improved antifungal activity. Considering the alarming increase of microbial resistance to conventional antibiotics, here we evaluated the antimicrobial activity of these derivatives against multiresistant and problematic bacteria and against important viral agents. The three peptides showed a moderate activity against Pseudomonas aeruginosa, Klebsiella pneumoniae Extended Spectrum ß-Lactamase (ESBL), and Streptococcus agalactiae, with MIC values > 100 µg/mL. They exerted a considerable activity with MIC values between 25-50 µg/mL against Acinetobacter baumanii and Enterococcus faecium. In addition, the two dimers showed a moderate activity against Pseudomonas aeruginosa PA14. The three Cm-p5 derivatives inhibited a virulent extracellular strain of Mycobacterium tuberculosis, in a dose-dependent manner. Moreover, they inhibited Herpes Simplex Virus 2 (HSV-2) infection in a concentration-dependent manner, but had no effect on infection by the Zika Virus (ZIKV) or pseudoparticles of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). At concentrations of >100 µg/mL, the three new Cm-p5 derivatives showed toxicity on different eukaryotic cells tested. Considering a certain cell toxicity but a potential interesting activity against the multiresistant strains of bacteria and HSV-2, our compounds require future structural optimization.

Antimicrobial Peptides Pom-1 and Pom-2 from Pomacea poeyana Are Active against Candidaauris, C. parapsilosis and C. albicans Biofilms.

Recently two peptides isolated from the Cuban freshwater snail Pomacea poeyana (Pilsbry, 1927) were described to have antimicrobial activity against bacterial pathogens. Here we show considerable activities of Pom-1 and Pom-2 to reduce the viability of C. albicans, C. parapsilosis and the less common species C. auris measured as the decrease of metabolic activity in the resazurin reduction assay for planktonic cells. Although these activities were low, Pom-1 and Pom-2 turned out to be highly potent inhibitors of biofilm formation for the three Candida species tested. Whereas Pom-1 was slightly more active against C. albicans and C. parapsilosis as representatives of the more common Candida species Pom-2 showed no preference and was fully active also against biofilms of the more uncommon species C. auris. Pom-1 and Pom-2 may represent promising lead structures for the development of a classical peptide optimization strategy with the realistic aim to further increase antibiofilm properties and other pharmacologic parameters and to generate finally the first antifungal drug with a pronounced dedication against Candida biofilms.

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.

New Antibacterial Peptides from the Freshwater Mollusk Pomacea poeyana (Pilsbry, 1927).

Antimicrobial peptides (AMPs) are biomolecules with antimicrobial activity against a broad group of pathogens. In the past few decades, AMPs have represented an important alternative for the treatment of infectious diseases. Their isolation from natural sources has been widely investigated. In this sense, mollusks are promising organisms for the identification of AMPs given that their immune system mainly relies on innate response. In this report, we characterized the peptide fraction of the Cuban freshwater snail Pomacea poeyana (Pilsbry, 1927) and identified 37 different peptides by nanoLC-ESI-MS-MS technology. From these peptide sequences, using bioinformatic prediction tools, we discovered two potential antimicrobial peptides named Pom-1 (KCAGSIAWAIGSGLFGGAKLIKIKKYIAELGGLQ) and Pom-2 (KEIERAGQRIRDAIISAAPAVETLAQAQKIIKGG). Database search revealed that Pom-1 is a fragment of Closticin 574 previously isolated from the bacteria Clostridium tyrobutyrium, and Pom-2 is a fragment of cecropin D-like peptide first isolated from Galleria mellonella hemolymph. These sequences were chemically synthesized and evaluated against different human pathogens. Interestingly, structural predictions of both peptides in the presence of micelles showed models that comprise two alpha helices joined by a short loop. The CD spectra analysis of Pom-1 and Pom-2 in water showed for both structures a high random coil content, a certain content of α-helix and a low ß-sheet content. Like other described AMPs displaying a disordered structure in water, the peptides may adopt a helical conformation in presence of bacterial membranes. In antimicrobial assays, Pom-1 demonstrated high activity against the Gram-negative bacteria Pseudomonas aeruginosa and moderate activity against Klebsiella pneumoniae and Listeria monocytogenes. Neither of the two peptides showed antifungal action. Pom-1 moderately inhibits Zika Virus infection but slightly enhances HIV-1 infectivion in vitro. The evaluation of cell toxicity on primary human macrophages did not show toxicity on THP-1 cells, although slight overall toxicity was observed in high concentrations of Pom-1. We assume that both peptides may play a key role in innate defense of P. poeyana and represent promising antimicrobial candidates for humans.

Derivates of the Antifungal Peptide Cm-p5 Inhibit Development of Candida auris Biofilms In Vitro.

Growth in biofilms as a fascinating and complex microbial lifestyle has become widely accepted as one of the key features of pathogenic microbes, to successfully express their full virulence potential and environmental persistence. This also increases the threat posed by Candida auris, which has a high intrinsic ability to persist on abiotic surfaces including those of surgical instruments and medical tubing. In a previous study, cyclic and helical-stabilized analogues of the antifungal peptide Cm-p5 were designed and synthetized, and proved to have increased activities against C. albicans and C. parapsilosis, but not against planktonic C. auris cells cultivated in suspension cultures. Here, we demonstrate, initially, that these derivatives, however, exhibited semi-inhibitory concentrations between 10-21 µg/mL toward C. auris biofilms. Maturated biofilms were also arrested between 71-97%. These novel biofilm inhibitors may open urgently needed new routes for the development of novel drugs and treatments for the next stage of fight against C. auris.

The Diversity of a Polyclonal FluCell-SELEX Library Outperforms Individual Aptamers as Emerging Diagnostic Tools for the Identification of Carbapenem Resistant Pseudomonas aeruginosa.

Textbook procedures require the use of individual aptamers enriched in SELEX libraries which are subsequently chemically synthesized after their biochemical characterization. Here we show that this reduction of the available sequence space of large libraries and thus the diversity of binding molecules reduces the labelling efficiency and fidelity of selected single aptamers towards different strains of the human pathogen Pseudomonas aeruginosa compared to a polyclonal aptamer library enriched by a whole-cell-SELEX involving fluorescent aptamers. The library outperformed single aptamers in reliable and specific targeting of different clinically relevant strains, allowed to inhibit virulence associated cellular functions and identification of bound cell surface targets by aptamer based affinity purification and mass spectrometry. The stunning ease of this FluCell-SELEX and the convincing performance of the P. aeruginosa specific library may pave the way towards generally new and efficient diagnostic techniques based on polyclonal aptamer libraries not only in clinical microbiology.

A Cerberus-Inspired Anti-Infective Multicomponent Gatekeeper Hydrogel against Infections with the Emerging "Superbug" Yeast Candida auris.

The pathogenic yeast Candida auris has received increasing attention due to its ability to cause fatal infections, its resistance toward important fungicides, and its ability to persist on surfaces including medical devices in hospitals. To brace health care systems for this considerable risk, alternative therapeutic approaches such as antifungal peptides are urgently needed. In clinical wound care, a significant focus has been directed toward novel surgical (wound) dressings as first defense lines against C. auris. Inspired by Cerberus the Greek mythological "hound of Hades" that prevents the living from entering and the dead from leaving hell, the preparation of a gatekeeper hybrid hydrogel is reported featuring lectin-mediated high-affinity immobilization of C. auris cells from a collagen gel as a model substratum in combination with a release of an antifungal peptide drug to kill the trapped cells. The vision is an efficient and safe two-layer medical composite hydrogel for the treatment of severe wound infections that typically occur in hospitals. Providing this new armament to the repertoire of possibilities for wound care in critical (intensive care) units may open new routes to shield and defend patients from infections and clinical facilities from spreading and invasion of C. auris and probably other fungal pathogens.

Design of a Helical-Stabilized, Cyclic, and Nontoxic Analogue of the Peptide Cm-p5 with Improved Antifungal Activity.

Following the information obtained by a rational design study, a cyclic and helical-stabilized analogue of the peptide Cm-p5 was synthetized. The cyclic monomer showed an increased activity in vitro against Candida albicans and Candida parapsilosis, compared to Cm-p5. Initially, 14 mutants of Cm-p5 were synthesized following a rational design to improve the antifungal activity and pharmacological properties. Antimicrobial testing showed that the activity was lost in each of these 14 analogues, suggesting, as a main conclusion, that a Glu-His salt bridge could stabilize Cm-p5 helical conformation during the interaction with the plasma membrane. A derivative, obtained by substitution of Glu and His for Cys, was synthesized and oxidized with the generation of a cyclic monomer with improved antifungal activity. In addition, two dimers were generated during the oxidation procedure, a parallel and antiparallel one. The dimers showed a helical secondary structure in water, whereas the cyclic monomer only showed this conformation in SDS. Molecular dynamic simulations confirmed the helical stabilizations for all of them, therefore indicating the possible essential role of the Glu-His salt bridge. In addition, the antiparallel dimer showed a moderate activity against Pseudomonas aeruginosa and a significant activity against Listeria monocytogenes. Neither the cyclic monomer nor the dimers were toxic against macrophages or THP-1 human cells. Due to its increased capacity for fungal control compared to fluconazole, its low cytotoxicity, together with a stabilized α-helix and disulfide bridges, that may advance its metabolic stability, and in vivo activity, the new cyclic Cm-p5 monomer represents a potential systemic antifungal therapeutic candidate.

Lectin-Functionalized Composite Hydrogels for "Capture-and-Killing" of Carbapenem-Resistant Pseudomonas aeruginosa.

Infections with multiresistant pathogens are a leading cause for mortality worldwide. Just recently, the World Health Organization (WHO) increased the threat rating for multiresistant Pseudomonas aeruginosa to the highest possible level. With this background, it is crucial to develop novel materials and procedures in the fight against multiresistant pathogens. In this study, we present a novel antimicrobial material, which could find applications as a wound dressing or antimicrobial coating. Lectins are multivalent sugar-binding proteins, which can be found in a variety of plants and bacteria, where they are associated with biofilm formation. By immobilizing lectin B on a protein-based hydrogel surface, we provided the hydrogel with the ability to immobilize ("catch") pathogens upon contact. Furthermore, another hydrogel layer was added which inhibits biofilm formation and releases a highly potent antimicrobial peptide to eradicate microorganisms ("kill"). The composite hydrogel showed a high antimicrobial activity against the reference strain Pseudomonas aeruginosa PAO1 as well as against a carbapenem-resistant clinical isolate (multiresistant Gram-negative class 4) and may thus represent a novel material to develop a new type of antimicrobial wound dressings to prevent infections with this problematic pathogen of burn or other large wounds.

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 µm in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Lectin-mediated reversible immobilization of human cells into a glycosylated macroporous protein hydrogel as a cell culture matrix.

3D cell culture is a helpful approach to study cell-cell interaction in a native-like environment, but is often limited due the challenge of retrieving cells from the material. In this study, we present the use of recombinant lectin B, a sugar-binding protein with four binding cavities, to enable reversible cell integration into a macroporous protein hydrogel matrix. By functionalizing hydrogel precursors with saccharose, lectin B can both bind to sugar moieties on the cellular surface as well as to the modified hydrogel network. Confocal microscopy and flow cytometry analysis revealed cells to be integrated into the network and to adhere and proliferate. Furthermore, the specificity and reversibility was investigated by using a recombinantly produced yellow fluorescent - lectin B fusion protein and a variety of sugars with diverging affinities for lectin B at different concentrations and elution times. Cells could be eluted within minutes by addition of L-fucose to the cell-loaded hydrogels to make cells available for further analysis.

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.

Evaluation of methods for pore generation and their influence on physio-chemical properties of a protein based hydrogel.

Different methods to create and manipulate pore sizes in hydrogel fabrication are available, but systematic studies are normally conducted with hydrogels made of synthetic chemical compounds as backbones. In this study, a hydrogel made of natural and abundant protein in combination with different, well-available techniques was used to produce different architectures within the hydrogel matrix. Pore sizes and distribution are compared and resulting hydrogel properties like swelling ratio, resistance towards external stimuli and enzymatic degradation were investigated. Porous hydrogels were functionalized and two cancer cell lines were successfully adhered onto the material. With simple methods, pores with a radius between 10 and 80 µm and channels of 25 µm radius with a length of several hundreds of µm could be created and analyzed with laser scanning confocal microscopy and electron microscopy respectively. Furthermore, the influence of different methods on swelling ratio, enzymatic degradation and pH and temperature resistance was observed.