Fusing Peptide Epitopes for Advanced Multiplex Serological Testing for SARS-CoV-2 Antibody Detection.

The tragic COVID-19 pandemic, which has seen a total of 655 million cases worldwide and a death toll of over 6.6 million seems finally tailing off. Even so, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise, the severity of which cannot be predicted in advance. This is concerning for the maintenance and stability of public health, since immune evasion and increased transmissibility may arise. Therefore, it is crucial to continue monitoring antibody responses to SARS-CoV-2 in the general population. As a complement to polymerase chain reaction tests, multiplex immunoassays are elegant tools that use individual protein or peptide antigens simultaneously to provide a high level of sensitivity and specificity. To further improve these aspects of SARS-CoV-2 antibody detection, as well as accuracy, we have developed an advanced serological peptide-based multiplex assay using antigen-fused peptide epitopes derived from both the spike and the nucleocapsid proteins. The significance of the epitopes selected for antibody detection has been verified by in silico molecular docking simulations between the peptide epitopes and reported SARS-CoV-2 antibodies. Peptides can be more easily and quickly modified and synthesized than full length proteins and can, therefore, be used in a more cost-effective manner. Three different fusion-epitope peptides (FEPs) were synthesized and tested by enzyme-linked immunosorbent assay (ELISA). A total of 145 blood serum samples were used, compromising 110 COVID-19 serum samples from COVID-19 patients and 35 negative control serum samples taken from COVID-19-free individuals before the outbreak. Interestingly, our data demonstrate that the sensitivity, specificity, and accuracy of the results for the FEP antigens are higher than for single peptide epitopes or mixtures of single peptide epitopes. Our FEP concept can be applied to different multiplex immunoassays testing not only for SARS-CoV-2 but also for various other pathogens. A significantly improved peptide-based serological assay may support the development of commercial point-of-care tests, such as lateral-flow-assays.

Selectively Positioned Catechol Moiety Supports Ultrashort Self-Assembling Peptide Hydrogel Adhesion for Coral Restoration.

Coral reef survival is threatened globally. One way to restore this delicate ecosystem is to enhance coral growth by the controlled propagation of coral fragments. To be sustainable, this technique requires the use of biocompatible underwater adhesives. Hydrogels based on rationally designed ultrashort self-assembling peptides (USP) are of great interest for various biological and environmental applications, due to their biocompatibility and tunable mechanical properties. Implementing superior adhesion properties to the USP hydrogel compounds is crucial in both water and high ionic strength solutions and is relevant in medical and marine environmental applications such as coral regeneration. Some marine animals secrete large quantities of the aminoacids dopa and lysine to enhance their adhesion to wet surfaces. Therefore, the addition of catechol moieties to the USP sequence containing lysine (IIZK) should improve the adhesive properties of USP hydrogels. However, it is challenging to place the catechol moiety (Do) within the USP sequence at an optimal position without compromising the hydrogel self-assembly process and mechanical properties. Here, we demonstrate that, among three USP hydrogels, DoIIZK is the least adhesive and that the adhesiveness of the IIZDoK hydrogel is compromised by its poor mechanical properties. The best adhesion outcome was achieved using the IIZKDo hydrogel, the only one to show equally sound adhesive and mechanical properties. A mechanistic understanding of this outcome is presented here. This property was confirmed by the successful gluing of coral fragments by means of IIZKDo hydrogel that are still thriving after more than three years since the deployment. The validated biocompatibility of this underwater hydrogel glue suggests that it could be advantageously implemented for other applications, such as surgical interventions.

Fabrication of a three-dimensional bone marrow niche-like acute myeloid Leukemia disease model by an automated and controlled process using a robotic multicellular bioprinting system.

BACKGROUND: Acute myeloid leukemia (AML) is a hematological malignancy that remains a therapeutic challenge due to the high incidence of disease relapse. To better understand resistance mechanisms and identify novel therapies, robust preclinical models mimicking the bone marrow (BM) microenvironment are needed. This study aimed to achieve an automated fabrication process of a three-dimensional (3D) AML disease model that recapitulates the 3D spatial structure of the BM microenvironment and applies to drug screening and investigational studies. METHODS: To build this model, we investigated a unique class of tetramer peptides with an innate ability to self-assemble into stable hydrogel. An automated robotic bioprinting process was established to fabricate a 3D BM (niche-like) multicellular AML disease model comprised of leukemia cells and the BM's stromal and endothelial cellular fractions. In addition, monoculture and dual-culture models were also fabricated. Leukemia cell compatibility, functionalities (in vitro and in vivo), and drug assessment studies using our model were performed. In addition, RNAseq and gene expression analysis using TaqMan arrays were also performed on 3D cultured stromal cells and primary leukemia cells. RESULTS: The selected peptide hydrogel formed a highly porous network of nanofibers with mechanical properties similar to the BM extracellular matrix. The robotic bioprinter and the novel quadruple coaxial nozzle enabled the automated fabrication of a 3D BM niche-like AML disease model with controlled deposition of multiple cell types into the model. This model supported the viability and growth of primary leukemic, endothelial, and stromal cells and recapitulated cell-cell and cell-ECM interactions. In addition, AML cells in our model possessed quiescent characteristics with improved chemoresistance attributes, resembling more the native conditions as indicated by our in vivo results. Moreover, the whole transcriptome data demonstrated the effect of 3D culture on enhancing BM niche cell characteristics. We identified molecular pathways upregulated in AML cells in our 3D model that might contribute to AML drug resistance and disease relapse. CONCLUSIONS: Our results demonstrate the importance of developing 3D biomimicry models that closely recapitulate the in vivo conditions to gain deeper insights into drug resistance mechanisms and novel therapy development. These models can also improve personalized medicine by testing patient-specific treatments.

Evaluation of Potential Peptide-Based Inhibitors against SARS-CoV-2 and Variants of Concern.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has greatly affected all aspect of life. Although several vaccines and pharmaceuticals have been developed against SARS-CoV-2, the emergence of mutated variants has raised several concerns. The angiotensin-converting enzyme (ACE2) receptor cell entry mechanism of this virus has not changed despite the vast mutation in emerging variants. Inhibiting the spike protein by which the virus identifies the host ACE2 receptor is a promising therapeutic countermeasure to keep pace with rapidly emerging variants. Here, we synthesized two ACE2-derived peptides, P1 and P25, to target and potentially inhibit SARS-CoV-2 cell entry. These peptides were evaluated in vitro using pseudoviruses that contained the SARS-CoV-2 original spike protein, the Delta-mutated spike protein, or the Omicron spike protein. An in silico investigation was also done for these peptides to evaluate the interaction of the synthesized peptides and the SARS-CoV-2 variants. The P25 peptide showed a promising inhibition potency against the tested pseudoviruses and an even higher inhibition against the Omicron variant. The IC50 of the Omicron variant was 60.8 µM, while the IC50s of the SARS-CoV-2 original strain and the Delta variant were 455.2 µM and 546.4 µM, respectively. The in silico experiments also showed that the amino acid composition design and structure of P25 boosted the interaction with the spike protein. These findings suggest that ACE2-derived peptides, such as P25, have the potential to inhibit SARS-CoV-2 cell entry in vitro. However, further in vivo studies are needed to confirm their therapeutic efficacy against emerging variants.

Dipeptide-Based Photoreactive Instant Glue for Environmental and Biomedical Applications.

Nature-inspired smart materials offer numerous advantages over environmental friendliness and efficiency. Emulating the excellent adhesive properties of mussels foot proteins, where the lysine is in close proximity with the 3,4-dihydroxy-l-phenylalanine (DOPA), we report the synthesis of a novel photocurable peptide-based adhesive consisting exclusively of these two amino acids. Our adhesive is a highly concentrated aqueous solution of a monomer, a cross-linker, and a photoinitiator. Lap-shear adhesion measurements on plastic and glass surfaces and comparison with different types of commercial adhesives showed that the adhesive strength of our glue is comparable when applied in air and superior when used underwater. No toxicity of our adhesive was observed when the cytocompatibility on human dermal fibroblast cells was assessed. Preliminary experiments with various tissues and coral fragments showed that our adhesive could be applied to wound healing and coral reef restoration. Given the convenience of the facile synthesis, biocompatibility, ease of application underwater, and high adhesive strength, we expect that our adhesive may find application, but not limited, to the biomedical and environmental field.

The Impact of Mechanical Cues on the Metabolomic and Transcriptomic Profiles of Human Dermal Fibroblasts Cultured in Ultrashort Self-Assembling Peptide 3D Scaffolds.

Cells' interactions with their microenvironment influence their morphological features and regulate crucial cellular functions including proliferation, differentiation, metabolism, and gene expression. Most biological data available are based on in vitro two-dimensional (2D) cellular models, which fail to recapitulate the three-dimensional (3D) in vivo systems. This can be attributed to the lack of cell-matrix interaction and the limitless access to nutrients and oxygen, in contrast to in vivo systems. Despite the emergence of a plethora of 3D matrices to address this challenge, there are few reports offering a proper characterization of these matrices or studying how the cell-matrix interaction influences cellular metabolism in correlation with gene expression. In this study, two tetrameric ultrashort self-assembling peptide sequences, FFIK and FIIK, were used to create in vitro 3D models using well-described human dermal fibroblast cells. The peptide sequences are derived from naturally occurring amino acids that are capable of self-assembling into stable hydrogels without UV or chemical cross-linking. Our results showed that 2D cultured fibroblasts exhibited distinct metabolic and transcriptomic profiles compared to 3D cultured cells. The observed changes in the metabolomic and transcriptomic profiles were closely interconnected and influenced several important metabolic pathways including the TCA cycle, glycolysis, MAPK signaling cascades, and hemostasis. Data provided here may lead to clearer insights into the influence of the surrounding microenvironment on human dermal fibroblast metabolic patterns and molecular mechanisms, underscoring the importance of utilizing efficient 3D in vitro models to study such complex mechanisms.

3D bioprinting of ultrashort self-assembling peptides to engineer scaffolds with different matrix stiffness for chondrogenesis.

62Articular cartilage is a nonvascularized and poorly cellularized tissue with a low self-repair capacity. Therefore, damage to this tissue due to trauma or degenerative joint diseases such as osteoarthritis needs a high-end medical intervention. However, such interventions are costly, have limited healing capacity, and could impair patients' quality of life. In this regard, tissue engineering and three-dimensional (3D) bioprinting hold great potential. However, identifying suitable bioinks that are biocompatible, with the desired mechanical stiffness, and can be used under physiological conditions is still a challenge. In this study, we developed two tetrameric self-assembling ultrashort peptide bioinks that are chemically well-defined and can spontaneously form nanofibrous hydrogels under physiological conditions. The printability of the two ultrashort peptides was demonstrated; different shape constructs were printed with high shape fidelity and stability. Furthermore, the developed ultrashort peptide bioinks gave rise to constructs with different mechanical properties that could be used to guide stem cell differentiation toward specific lineages. Both ultrashort peptide bioinks demonstrated high biocompatibility and supported the chondrogenic differentiation of human mesenchymal stem cells. Additionally, the gene expression analysis of differentiated stem cells with the ultrashort peptide bioinks revealed articular cartilage extracellular matrix formation preference. Based on the different mechanical stiffness of the two ultrashort peptide bioinks, they can be used to fabricate cartilage tissue with different cartilaginous zones, including the articular and calcified cartilage zones, which are essential for engineered tissue integration.

Peptide nanogels as a scaffold for fabricating dermal grafts and 3D vascularized skin models.

Millions of people worldwide suffer from skin injuries, which create significant problems in their lives and are costly to cure. Tissue engineering is a promising approach that aims to fabricate functional organs using biocompatible scaffolds. We designed ultrashort tetrameric peptides with promising properties required for skin tissue engineering. Our work aimed to test the efficacy of these scaffolds for the fabrication of dermal grafts and 3D vascularized skin tissue models. We found that the direct contact of keratinocytes and fibroblasts enhanced the proliferation of the keratinocytes. Moreover, the expression levels of TGF-ß1, b-FGF, IL-6, and IL-1α is correlated with the growth of the fibroblasts and keratinocytes in the co-culture. Furthermore, we successfully produced a 3D vascularized skin co-culture model using these peptide scaffolds. We believe that the described results represent an advancement in the fabrication of skin tissue equivalent, thereby providing the opportunity to rebuild missing, failing, or damaged parts.

Scaffolds from Self-Assembling Tetrapeptides Support 3D Spreading, Osteogenic Differentiation, and Angiogenesis of Mesenchymal Stem Cells.

The apparent rise of bone disorders demands advanced treatment protocols involving tissue engineering. Here, we describe self-assembling tetrapeptide scaffolds for the growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The rationally designed peptides are synthetic amphiphilic self-assembling peptides composed of four amino acids that are nontoxic. These tetrapeptides can quickly solidify to nanofibrous hydrogels that resemble the extracellular matrix and provide a three-dimensional (3D) environment for cells with suitable mechanical properties. Furthermore, we can easily tune the stiffness of these peptide hydrogels by just increasing the peptide concentration, thus providing a wide range of peptide hydrogels with different stiffnesses for 3D cell culture applications. Since successful bone regeneration requires both osteogenesis and vascularization, our scaffold was found to be able to promote angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. The results presented suggest that ultrashort peptide hydrogels are promising candidates for applications in bone tissue engineering.

Ultrashort Peptide Bioinks Support Automated Printing of Large-Scale Constructs Assuring Long-Term Survival of Printed Tissue Constructs.

We report about rationally designed ultrashort peptide bioinks, overcoming severe limitations in current bioprinting procedures. Bioprinting is increasingly relevant in tissue engineering, regenerative and personalized medicine due to its ability to fabricate complex tissue scaffolds through an automated deposition process. Printing stable large-scale constructs with high shape fidelity and enabling long-term cell survival are major challenges that most existing bioinks are unable to solve. Additionally, they require chemical or UV-cross-linking for the structure-solidifying process which compromises the encapsulated cells, resulting in restricted structure complexity and low cell viability. Using ultrashort peptide bioinks as ideal bodylike but synthetic material, we demonstrate an instant solidifying cell-embedding printing process via a sophisticated extrusion procedure under true physiological conditions and at cost-effective low bioink concentrations. Our printed large-scale cell constructs and the chondrogenic differentiation of printed mesenchymal stem cells point to the strong potential of the peptide bioinks for automated complex tissue fabrication.

Self-assembling tetrameric peptides allow in situ 3D bioprinting under physiological conditions.

We have developed an in situ bioprinting method that allows the printing of cells under true physiological conditions by applying self-assembling ultrashort peptides as bioinks. This method avoids cell stressing methods, such as UV-treatment, chemical crosslinking and viscous bioink printing methods. We further demonstrate that different nanomaterials can easily be synthesized or incorporated in the 3D bioprinted peptide scaffolds which opens up the possibility of functionalized 3D scaffolds.