Insights into the Activation of Unfolded Protein Response Mechanism during Coronavirus Infection.

Coronaviruses represent a significant class of viruses that affect both animals and humans. Their replication cycle is strongly associated with the endoplasmic reticulum (ER), which, upon virus invasion, triggers ER stress responses. The activation of the unfolded protein response (UPR) within infected cells is performed from three transmembrane receptors, IRE1, PERK, and ATF6, and results in a reduction in protein production, a boost in the ER's ability to fold proteins properly, and the initiation of ER-associated degradation (ERAD) to remove misfolded or unfolded proteins. However, in cases of prolonged and severe ER stress, the UPR can also instigate apoptotic cell death and inflammation. Herein, we discuss the ER-triggered host responses after coronavirus infection, as well as the pharmaceutical targeting of the UPR as a potential antiviral strategy.

In Vitro Chondrogenesis Induction by Short Peptides of the Carboxy-Terminal Domain of Transforming Growth Factor ß1.

Τransforming growth factor ß1 (TGF-ß1) comprises a key regulator protein in many cellular processes, including in vivo chondrogenesis. The treatment of human dental pulp stem cells, separately, with Leu83-Ser112 (C-terminal domain of TGF-ß1), as well as two very short peptides, namely, 90-YYVGRKPK-97 (peptide 8) and 91-YVGRKP-96 (peptide 6) remarkably enhanced the chondrogenic differentiation capacity in comparison to their full-length mature TGF-ß1 counterpart either in monolayer cultures or 3D scaffolds. In 3D scaffolds, the reduction of the elastic modulus and viscous modulus verified the production of different amounts and types of ECM components. Molecular dynamics simulations suggested a mode of the peptides' binding to the receptor complex TßRII-ALK5 and provided a possible structural explanation for their role in inducing chondrogenesis, along with endogenous TGF-ß1. Further experiments clearly verified the aforementioned hypothesis, indicating the signal transduction pathway and the involvement of TßRII-ALK5 receptor complex. Real-time PCR experiments and Western blot analysis showed that peptides favor the ERK1/2 and Smad2 pathways, leading to an articular, extracellular matrix formation, while TGF-ß1 also favors the Smad1/5/8 pathway which leads to the expression of the metalloproteinases ADAMTS-5 and MMP13 and, therefore, to a hypertrophic chondrocyte phenotype. Taken together, the two short peptides, and, mainly, peptide 8, could be delivered with a scaffold to induce in vivo chondrogenesis in damaged articular cartilage, constituting, thus, an alternative therapeutic approach for osteoarthritis.

Fabrication of a Smart Fibrous Biomaterial That Harbors an Active TGF-ß1 Peptide: A Promising Approach for Cartilage Regeneration.

The regeneration of articular cartilage remains a serious problem in various pathological conditions such as osteoarthritis, due to the tissue's low self-healing capacity. The latest therapeutic approaches focus on the construction of biomaterials that induce cartilage repair. This research describes the design, synthesis, and investigation of a safe, "smart", fibrous scaffold containing a genetically incorporated active peptide for chondrogenic induction. While possessing specific sequences and the respective mechanical properties from natural fibrous proteins, the fibers also incorporate a Transforming Growth Factor-ß1 (TGF-ß1)-derived peptide (YYVGRKPK) that can promote chondrogenesis. The scaffold formed stable porous networks with shear-thinning properties at 37 °C, as shown by SEM imaging and rheological characterization, and were proven to be non-toxic to human dental pulp stem cells (hDPSCs). Its chondrogenic capacity was evidenced by a strong increase in the expression of specific chondrogenesis gene markers SOX9, COL2, ACAN, TGFBR1A, and TGFBR2 in cells cultured on "scaffold-TGFß1" for 21 days and by increased phosphorylation of intracellular signaling proteins Smad-2 and Erk-1/2. Additionally, intense staining of glycosaminoglycans was observed in these cells. According to our results, "scaffold-TGFß1" is proposed for clinical studies as a safe, injectable treatment for cartilage degeneration.

Freeze-Drying Process for the Fabrication of Collagen-Based Sponges as Medical Devices in Biomedical Engineering.

This paper presents a systematic review of a key sector of the much promising and rapidly evolving field of biomedical engineering, specifically on the fabrication of three-dimensional open, porous collagen-based medical devices, using the prominent freeze-drying process. Collagen and its derivatives are the most popular biopolymers in this field, as they constitute the main components of the extracellular matrix, and therefore exhibit desirable properties, such as biocompatibility and biodegradability, for in vivo applications. For this reason, freeze-dried collagen-based sponges with a wide variety of attributes can be produced and have already led to a wide range of successful commercial medical devices, chiefly for dental, orthopedic, hemostatic, and neuronal applications. However, collagen sponges display some vulnerabilities in other key properties, such as low mechanical strength and poor control of their internal architecture, and therefore many studies focus on the settlement of these defects, either by tampering with the steps of the freeze-drying process or by combining collagen with other additives. Furthermore, freeze drying is still considered a high-cost and time-consuming process that is often used in a non-optimized manner. By applying an interdisciplinary approach and combining advances in other technological fields, such as in statistical analysis, implementing the Design of Experiments, and Artificial Intelligence, the opportunity arises to further evolve this process in a sustainable and strategic manner, and optimize the resulting products as well as create new opportunities in this field.

ssDNA-Modified Gold Nanoparticles as a Tool to Detect miRNA Biomarkers in Osteoarthritis.

Recently, miRNAs have been established as promising, specific biomarkers for the diagnosis of many diseases, including osteoarthritis. Herein, we report a ssDNA-based detection method for miRNAs implicated in osteoarthritis, specifically, miR-93 and miR-223. In this study, gold nanoparticles (AuNPs) were modified with oligonucleotide ssDNA to detect miRNAs circulating in the blood in healthy subjects and patients suffering from osteoarthritis. The detection method was based on the colorimetric and spectrophotometric assessment of biofunctionalized AuNPs upon interaction with the target and their subsequent aggregation. Results showed that these methods could be used to detect easily and rapidly miR-93 but not miR-223 in osteoarthritic patients, and they could potentially be used as a diagnostic tool for blood biomarkers. Visual-based detection as well as spectroscopic methods are simple, rapid, and label-free, due to which they can be used as a diagnostic tool.

SpAD Biofunctionalized Cellulose Acetate Scaffolds Inhibit Staphylococcus aureus Adherence in a Coordinating Function with the von Willebrand A1 Domain (vWF A1).

Staphylococcus aureus is one of the major pathogens causing and spreading hospital acquired infections. Since it is highly resistant to new generation antibiotics, novel strategies have to be developed such as the construction of biofunctionalized non-adherent surfaces that will prevent its tethering and subsequent spread in the hospital environment. In this frame, the domain D of protein A (SpAD) of S. aureus has been immobilized onto cellulose acetate scaffolds by using the streptavidin/biotin interaction, in order to study its interaction with the A1 domain of von Willebrand factor (vWF A1), a protein essential for hemostasis, found in human plasma. Subsequently, the biofunctionalized cellulose acetate scaffolds were incubated with S. aureus in the presence and absence of vWF A1 at different time periods and their potential to inhibit S. aureus growth was studied with scanning electron microscopy (SEM). The SpAD biofunctionalized scaffolds perceptibly ameliorated the non-adherent properties of the material, and in particular, the interaction between SpAD and vWF A1 effectively inhibited the growth of S. aureus. Thus, the exhibition of significant non-adherent properties of scaffolds addresses their potential use for covering medical equipment, implants, and stents.

Effect of a Bone Morphogenetic Protein-2-derived peptide on the expression of tumor marker ZNF217 in osteoblasts and MCF-7 cells.

Zinc Finger Protein 217 (ZNF217), a transcription factor and oncogene product, has been found to dysregulate Bone Morphogenetic Protein (BMP) signaling and induce invasion in breast tumors. In this study, the effect of BMP-2 or an active BMP-2 peptide, AISMLYLDEN, on the expression of ZNF217, BMP4 and CDK-inhibitor p21 gene, CDKN1A, was investigated in MCF-7 breast cancer cells. In parallel, the entire protein (BMP-2) as well as the aforementioned peptide were investigated in hDPSCs during osteogenic differentiation. The treatment of MCF-7 cancer cells with different concentrations of peptide AISMLYLDEN showed that the addition of 22.6 ng/ml was more effective in comparison to the other used concentrations. In particular, 48 h after treatment, CDKN1A and BMP4 mRNA levels were substantially increased in contrast to ZNF217 mRNA levels which were decreased. These results are strongly supported by BrdU assay that clearly indicated inhibition of cancer cell proliferation. Taken together, these results open ways for a concurrent use, at appropriate concentrations, of the peptide AISMLYLDEN during conventional therapeutic treatment in breast tumors with a metastatic tendency to the bones. Regarding the effect of the entire protein as well as its peptide on hDPSCs differentiation into osteocytes, the mRNA levels of osteocalcin, an osteogenic marker, showed that the peptide enhanced osteogenesis at a higher degree in comparison to the entire BMP-2 without however altering ZNF217, CDKN1A and BMP4 expression levels, which remained as expected of non-cancer cells.

Aza-Reversine Promotes Reprogramming of Lung (MRC-5) and Differentiation of Mesenchymal Cells into Osteoblasts.

Reversine or 2-(4-morpholinoanilino)-N6-cyclohexyladenine was originally identified as a small organic molecule that induces dedifferentiation of lineage-committed mouse myoblasts, C2C12, and redirects them into lipocytes or osteoblasts under lineage-specific conditions (LISCs). Further, it was proven that this small molecule can induce cell cycle arrest and apoptosis and thus selectively lead cancer cells to cell death. Further studies demonstrated that reversine, and more specifically the C2 position of the purine ring, can tolerate a wide range of substitutions without activity loss. In this study, a piperazine analog of reversine, also known as aza-reversine, and a biotinylated derivative of aza-reversine were synthesized, and their potential medical applications were investigated by transforming the endoderm originates fetal lung cells (MRC-5) into the mesoderm originated osteoblasts and by differentiating mesenchymal cells into osteoblasts. Moreover, the reprogramming capacity of aza-reversine and biotinylated aza-reversine was investigated against MRC-5 cells and mesenchymal cells after the immobilization on PMMA/HEMA polymeric surfaces. The results showed that both aza-reversine and the biofunctionalized, biotinylated analog induced the reprogramming of MRC-5 cells to a more primitive, pluripotent state and can further transform them into osteoblasts under osteogenic culture conditions. These molecules also induced the differentiation of dental and adipose mesenchymal cells to osteoblasts. Thus, the possibility to load a small molecule with useful "information" for delivering that into specific cell targets opens new therapeutic personalized applications.

Specific amino acids from the broad C-terminal region of BMP-2 are crucial for osteogenesis.

The shortest functional domains of growth factor Bone Morphogenetic Protein 2 (BMP-2) that are dynamical implicated in osteogenesis have been investigated and well characterized. In particular, the broad C-terminal region expanding from Val63 to Arg114 as well as its shorter sequence 86-AISMLYLDEN-95 exhibited the highest osteogenic ability for regeneration and reconstruction of bone tissue. In addition, the amino acids Ser88 and Leu90 have been identified as crucial for receptor binding and osteogenic efficacy. Furthermore, the above-mentioned domains in contrary to full length BMP-2 protein signal mainly through the Smad pathway as it is evidenced by phosphorylation decrease of Extracellular-signal-Regulated Kinase (ERK1/2). Taking together, our results are significant for clinical applications regarding the generation of biomaterials and healing of orthopedic fractures.

Graphene-Wrapped Copper Nanoparticles: An Antimicrobial and Biocompatible Nanomaterial with Valuable Properties for Medical Uses.

The great demand for antibacterial, biocompatible, and easily manufactured nanostructures has led to the design and development of graphene-wrapped copper nanoparticles (CuNPs) supported on Si wafers. In this study, we investigated the antibacterial properties of graphene/CuNPs nanostructures against Gram-positive and Gram-negative bacteria. Additional experiments regarding graphene/CuNPs nanostructures behavior against mouse fibroblast cell line L929 indicated their biocompatibility and consequently render them as model biomaterials for medical uses. Biofunctionalization of graphene/CuNPs nanostructures with a high-molecular-weight protein (green fluorescent protein), which retains its functionality after a "tight binding" on the nanostructure's surface, opens the way for attaching and other proteins, or biomolecules of great biological interest, to prepare novel biomaterials.