Statistical Analysis and Tokenization of Epitopes to Construct Artificial Neoepitope Libraries.

Epitopes are specific regions on an antigen's surface that the immune system recognizes. Epitopes are usually protein regions on foreign immune-stimulating entities such as viruses and bacteria, and in some cases, endogenous proteins may act as antigens. Identifying epitopes is crucial for accelerating the development of vaccines and immunotherapies. However, mapping epitopes in pathogen proteomes is challenging using conventional methods. Screening artificial neoepitope libraries against antibodies can overcome this issue. Here, we applied conventional sequence analysis and methods inspired in natural language processing to reveal specific sequence patterns in the linear epitopes deposited in the Immune Epitope Database (www.iedb.org) that can serve as building blocks for the design of universal epitope libraries. Our results reveal that amino acid frequency in annotated linear epitopes differs from that in the human proteome. Aromatic residues are overrepresented, while the presence of cysteines is practically null in epitopes. Byte pair encoding tokenization shows high frequencies of tryptophan in tokens of 5, 6, and 7 amino acids, corroborating the findings of the conventional sequence analysis. These results can be applied to reduce the diversity of linear epitope libraries by orders of magnitude.

Engineered Repeat Protein Hybrids: The New Horizon for Biologic Medicines and Diagnostic Tools.

ConspectusThe last decades have witnessed unprecedented scientific breakthroughs in all the fields of knowledge, from basic sciences to translational research, resulting in the drastic improvement of the lifespan and overall quality of life. However, despite these great advances, the treatment and diagnosis of some diseases remain a challenge. Inspired by nature, scientists have been exploring biomolecules and their derivatives as novel therapeutic/diagnostic agents. Among biomolecules, proteins raise much interest due to their high versatility, biocompatibility, and biodegradability.Protein binders (binders) are proteins that bind other proteins, in certain cases, inhibiting or modulating their action. Given their therapeutic potential, binders are emerging as the next generation of biopharmaceuticals. The most well-known example of binders are antibodies, and inspired by them researchers have developed alternative binders using protein design approaches. Protein design can be based on naturally occurring proteins in which, by means of rational design or combinatorial approaches, new binding interfaces can be engineered to obtain specific functions or based on de novo proteins emerging from state-of-the-art computational methodologies.Among the novel designed proteins, a class of engineered repeat proteins, the consensus tetratricopeptide repeat (CTPR) proteins, stand out due to their stability and robustness. The CTPR unit is a helix-turn-helix motif constituted of 34 amino acids, of which only 8 are essential to ensure correct folding of the structure. The small number of conserved residues of CTPR proteins leaves plenty of freedom for functional mutations, making them a base scaffold that can be easily and reproducibly tailored to endow desired functions to the protein. For example, the introduction of metal-binding residues (e.g., histidines, cysteines) drives the coordination of metal ions and the subsequent formation of nanomaterials. Additionally, the CTPR unit can be conjugated with other peptides/proteins or repeated in tandem to encode larger CTPR proteins with superhelical structures. These properties allow for the design of both binder and nanomaterial-coordination modules as well as their combination within the same molecule, making the CTPR proteins, as we have demonstrated in several recent examples, the ideal platform to develop protein-nanomaterial hybrids. Generally, the fusion of two distinct materials exploits the best properties of each; however, in protein-nanomaterial hybrids, the fusion takes on a new dimension as new properties arise.These hybrids have ushered the use of protein-based nanomaterials as biopharmaceuticals beyond their original therapeutic scope and paved the way for their use as theranostic agents. Despite several reports of protein-stabilized nanomaterials found in the literature, these systems offer limited control in the synthesis and properties of the grown nanomaterials, as the protein acts just as a stabilizing agent with no significant functional contribution. Therefore, the rational design of protein-based nanomaterials as true theranostic agents is still incipient. In this context, CTPR proteins have emerged as promising scaffolds to hold simultaneously therapeutic and diagnostic functions through protein engineering, as it has been recently demonstrated in pioneering in vitro and in vivo examples.

Engineering conductive protein films through nanoscale self-assembly and gold nanoparticles doping.

Protein-based materials are usually considered as insulators, although conductivity has been recently shown in proteins. This fact opens the door to develop new biocompatible conductive materials. While there are emerging efforts in this area, there is an open challenge related to the limited conductivity of protein-based systems. This work shows a novel approach to tune the charge transport properties of protein-based materials by using electron-dense AuNPs. Two strategies are combined in a unique way to generate the conductive solid films: (1) the controlled self-assembly of a protein building block; (2) the templating of AuNPs by the engineered building block. This bottom-up approach allows controlling the structure of the films and the distribution of the AuNPs within, leading to enhanced conductivity. This work illustrates a promising strategy for the development of effective hybrid protein-based bioelectrical materials.

Biomarker sensing platforms based on fluorescent metal nanoclusters.

Metal nanoclusters (NCs) and their unique properties are increasing in importance and their applications are covering a wide range of areas. Their remarkable fluorescence properties and easy synthesis procedure and the possibility of functionalizing them for the detection of specific targets, such as biomarkers, make them a very interesting biosensing tool. Nowadays the detection of biomarkers related to different diseases is critical. In this context, NCs scaffolded within an appropriate molecule can be used to detect and quantify biomarkers through specific interactions and fluorescence properties of the NCs. These methods include analytical detection and biolocalization using imaging techniques. This review covers a selection of recent strategies to detect biomarkers related to diverse diseases (from infectious, inflammatory, or tumour origin) using fluorescent nanoclusters.

Engineering multifunctional metal/protein hybrid nanomaterials as tools for therapeutic intervention and high-sensitivity detection.

Protein-based hybrid nanomaterials have recently emerged as promising platforms to fabricate tailored multifunctional biologics for biotechnological and biomedical applications. This work shows a simple, modular, and versatile strategy to design custom protein hybrid nanomaterials. This approach combines for the first time the engineering of a therapeutic protein module with the engineering of a nanomaterial-stabilizing module within the same molecule, resulting in a multifunctional hybrid nanocomposite unachievable through conventional material synthesis methodologies. As the first proof of concept, a multifunctional system was designed ad hoc for the therapeutic intervention and monitoring of myocardial fibrosis. This hybrid nanomaterial combines a designed Hsp90 inhibitory domain and a metal nanocluster stabilizing module resulting in a biologic drug labelled with a metal nanocluster. The engineered nanomaterial actively reduced myocardial fibrosis and heart hypertrophy in an animal model of cardiac remodeling. In addition to the therapeutic effect, the metal nanocluster allowed for in vitro, ex vivo, and in vivo detection and imaging of the fibrotic disease under study. This study evidences the potential of combining protein engineering and protein-directed nanomaterial engineering approaches to design custom nanomaterials as theranostic tools, opening up unexplored routes to date for the next generation of advanced nanomaterials in medicine.

Design and fabrication of biocompatible wrinkled hydrogel films with selective antibiofouling properties.

In this article, we explored the selective antibiofouling capacity acquired by functional wrinkled hydrogel films via a fine tuning of their chemical structure through the gradual insertion of hydrophobic radical groups in their network. The hydrogel consists of three main components: hydroxyethyl methacrylate (HEMA, amphiphilic monomer), trifluoroethyl methacrylate (TFMA, hydrophobic monomer), and poly(ethylene glycol) diacrylate (PEGDA, hydrophilic crosslinking agent). Interestingly, the manipulation of the chemical composition affects both, surface morphology and physicochemical characteristics of the patterns, inducing transitions between different surface microstructures, i.e. from wrinkles to creases, to folds, and to crumples. Contact angle measurements show that the insertion of TFMA produces a slight decrease in surface wettability, remaining however highly hydrophilic. By using confocal Raman spectroscopy, important information about wrinkle formation mechanism could be obtained. The procedure presented in this article involves two consecutive thermal and photopolymerization steps, generating a "pseudo" two-layer system, which contracts at different extents when is exposed to external stimuli, leading to the formation of wrinkled surfaces. Finally, bacterial and cellular adhesion/proliferation studies were carried out, evidencing that the amount of TFMA included clearly reduce the bacterial adhesion while mammalian cells are able to still proliferate.

Sensors Based on Metal Nanoclusters Stabilized on Designed Proteins.

Among all new nanomaterials, metal nanoclusters (NCs) have attracted special attention due to their interesting optical properties, among others. Metal NCs have been recently studied and used as sensors for different analytes. However, there is a need to explore the potential of these new sensors in a systematic manner and to develop new systems to broaden the possibilities that sensing offers to the industry. In this work, we show the potential use of repeat protein scaffolds as versatile templates for the synthesis and stabilization of various metal NCs, specifically Au, Ag, and CuNCs. The resulting protein-metal NCs hybrids are evaluated as sensors for different stimuli such as temperature, ions, or reactive oxygen species (ROS). Among the three protein-metal NCs, all performed nicely as temperature sensors, AuNCs responded to metal ions, and AgNCs were able to detect ROS.

Detection of amyloid fibrils in Parkinson's disease using plasmonic chirality.

Amyloid fibrils, which are closely associated with various neurodegenerative diseases, are the final products in many protein aggregation pathways. The identification of fibrils at low concentration is, therefore, pivotal in disease diagnosis and development of therapeutic strategies. We report a methodology for the specific identification of amyloid fibrils using chiroptical effects in plasmonic nanoparticles. The formation of amyloid fibrils based on α-synuclein was probed using gold nanorods, which showed no apparent interaction with monomeric proteins but effective adsorption onto fibril structures via noncovalent interactions. The amyloid structure drives a helical nanorod arrangement, resulting in intense optical activity at the surface plasmon resonance wavelengths. This sensing technique was successfully applied to human brain homogenates of patients affected by Parkinson's disease, wherein protein fibrils related to the disease were identified through chiral signals from Au nanorods in the visible and near IR, whereas healthy brain samples did not exhibit any meaningful optical activity. The technique was additionally extended to the specific detection of infectious amyloids formed by prion proteins, thereby confirming the wide potential of the technique. The intense chiral response driven by strong dipolar coupling in helical Au nanorod arrangements allowed us to detect amyloid fibrils down to nanomolar concentrations.

Smart pH-Responsive Antimicrobial Hydrogel Scaffolds Prepared by Additive Manufacturing.

We report on the fabrication of 3D printed pH-responsive and antimicrobial hydrogels with a micrometer-scale resolution achieved by stereolithography (SLA) 3D printing. The preparation of the hydrogels was optimized by selecting the most appropriate difunctional polyethylene glycol dimethacrylates (testing cross-linking agents with chain lengths ranging from 2 up to 14 units ethylene glycol) and introducing acrylic acid (AA) as a monofunctional monomer. As a result of the incorporation of AA, the hydrogels described are able to reversibly swell and shrink upon environmental changes on the pH, and the swelling extent is directly related to the amount of AA and can be thus finely tuned. More interestingly, upon optimization of the UV penetration depth employing a photoabsorber (Sudan I), a reliable procedure for the fabrication of 3D objects with a high model accuracy is shown. Finally, the antimicrobial properties of all of the hydrogels were demonstrated using Staphylococcus aureus as a bacterial model. We found that even those hydrogels with a low amount of AA monomeric units presented excellent antimicrobial properties against S. aureus.

Preparation of Biodegradable Cationic Polycarbonates and Hydrogels through the Direct Polymerization of Quaternized Cyclic Carbonates.

Polymers exhibiting both antimicrobial and biodegradable properties are of great interest for next generation materials in healthcare. Among those, cationic polycarbonates are one of the most promising classes of materials because of their biodegradability, low toxicity, and biocompatibility. They are typically prepared by a chemical postmodification after the polymer has been synthesized. The main problem with the latter is the challenges of ensuring and verifying complete quaternization within the polymer structure. Herein, we report the first example of synthesizing and polymerizing charged aliphatic cyclic carbonates with three different alkane pendant groups (N-methyl, N-butyl, and N-hexyl) by ring-opening polymerization (ROP). These charged eight-membered cyclic carbonates displayed extraordinary reactivity and were even polymerizable in polar solvents (e.g., DMSO) and in catalyst free conditions that are generally unobtainable for other ring opening polymerization processes. A computational study was carried out and the findings were in agreement with the experimental data in regards to the dramatic increase in reactivity of the charged monomer over their neutral analogs. Furthermore, a series of hydrogels were prepared using the different charged eight-membered cyclic carbonates, and we found it to have a significant impact on the hydrogels' ability to swell and degrade in water. Finally, the hydrogels demonstrated antibacterial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). These materials could be ideal candidates for biologically relevant applications where cationic structure is required.

Escitalopram/reboxetine combination in depressed patients with substance use disorder.

Acting pharmacologically on different transmitter systems has been suggested to have some advantages in patients with substance abuse and may possibly address a larger spectrum of symptoms. One major drawback of using antidepressants addressing several neurotransmitters is that the relative activities on the different neurotransmitters cannot individually be adjusted. Combining antidepressants targeting different neurotransmitter systems may allow adapting the effect on each neurotransmitter system corresponding to patients' response and tolerance. Three cases of patients presenting a substance use disorder with comorbid major depression episodes are presented, who were treated with a reboxetine/escitalopram combination and who showed a rapid response of their depressive syndrome.