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.

Enhancement of mesenchymal stem cells' chondrogenic potential by type II collagen-based bioscaffolds.

BACKGROUND: Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration. METHODS AND RESULTS: In the present study, we evaluated the role of type II collagen, as the main matrix macromolecule in the cartilage matrix, to promote chondrogenic differentiation in two MSC in vitro culture systems. The chondrogenic differentiation of human Wharton's jelly- and dental pulp-derived MSCs was investigated over a 24-day culture period on type II collagen coating to improve the binding affinity of MSCs. Functional assays, demonstrated that type II collagen promoted chondrogenic differentiation in both MSCs tested, which was confirmed through gene and protein analysis of major chondrogenic markers. CONCLUSIONS: Our data support that type II collagen contributes as a natural bioscaffold enhancing chondrogenesis in both MSC models, thus enhancing the commitment of MSC-based therapeutic approaches in regenerative medicine to target OA and bring therapy closer to the clinical use.

Inhibition of Cancer Cell Proliferation and Bacterial Growth by Silver(I) Complexes Bearing a CH3-Substituted Thiadiazole-Based Thioamide.

Ag(I) coordination compounds have recently attracted much attention as antiproliferative and antibacterial agents against a wide range of cancer cell lines and pathogens. The bioactivity potential of these complexes depends on their structural characteristics and the nature of their ligands. Herein, we present a series of four Ag(I) coordination compounds bearing as ligands the CH3-substituted thiadiazole-based thioamide 5-methyl-1,3,4-thiadiazole-2-thiol (mtdztH) and phosphines, i.e., [AgCl(mtdztH)(PPh3)2] (1), [Ag(mtdzt)(PPh3)3] (2), [AgCl(mtdztH)(xantphos)] (3), and [AgmtdztH)(dppe)(NO3)]n (4), where xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and dppe = 1,2-bis(diphenylphosphino)ethane, and the assessment of their in vitro antibacterial and anti-cancer efficiency. Among them, diphosphine-containing compounds 3 and 4 were found to exhibit broad-spectrum antibacterial activity characteristics against both Gram-(+) and Gram-(-) bacterial strains, showing high in vitro bioactivity with IC50 values as low as 4.6 µΜ. In vitro cytotoxicity studies against human ovarian, pancreatic, lung, and prostate cancer cell lines revealed the strong cytotoxic potential of 2 and 4, with IC50 values in the range of 3.1-24.0 µΜ, while 3 and 4 maintained the normal fibroblast cells' viability at relatively higher levels. Assessment of these results, in combination with those obtained for analogous Ag(I) complexes bearing similar heterocyclic thioamides, suggest the pivotal role of the substituent groups of the thioamide heterocyclic ring in the antibacterial and anti-cancer efficacy of the respective Ag(I) complexes. Compounds 1-4 exhibited moderate in vitro antioxidant capacity for free radicals scavenging, as well as reasonably strong ability to interact with calf-thymus DNA, suggesting the likely implication of these properties in their bioactivity mechanisms. Complementary insights into the possible mechanism of their anti-cancer activity were provided by molecular docking calculations, exploring their ability to bind to the overexpressed fibroblast growth factor receptor 1 (FGFR1), affecting cancer cells' functionalities.

Oxidative stress, DNA damage, and mutagenicity induced by the extractable organic matter of airborne particulates on bacterial models.

The biological activity induced by the extractable organic matter (EOM) of size-segregated airborne Particulate Matter (PM) from two urban sites, urban traffic (UT) and urban background (UB), was assessed by using bacterial assays. The Gram-negative Escherichia coli (E. coli) coliform bacterium was used to measure the intracellular formation of Reactive Oxygen Species (ROS) by employing the Nitroblue tetrazolium (NBT) reduction assay and the lipid peroxidation by malondialdehyde (MDA) measurement. To the best of our knowledge, this is the first study using E. coli for assessing the bioactivity of ambient air in term of oxidative mechanism studies. E. coli BL21 cells were further used for DNA damage assessment by employing the reporter (ß-galactosidase) gene expression assay. The bacterial strain S. typhimurium TA100 was used to assess the mutagenic potential of PM by employing the well-known mutation assay (Ames test). Four PM size fractions were assessed for bioactivity, specifically the quasi-ultrafine mode (<0.49 µm), the upper accumulation mode (0.49-0.97 µm), the upper fine mode (0.97-3 µm), and the coarse mode (>3.0 µm). The EOM of each PM sample included three organic fractions of successively increased polarity: the non-polar organic fraction (NPOF), the moderately polar organic fraction (MPOF), and the polar organic fraction (POF). The toxicological endpoints induced by each organic fraction were correlated with the concentrations of various organic chemical components determined in previous studies in an attempt to identify the chemical classes involved.

Investigating the entire course of telithromycin binding to Escherichia coli ribosomes.

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

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.

Inhibition of PERK Kinase, an Orchestrator of the Unfolded Protein Response (UPR), Significantly Reduces Apoptosis and Inflammation of Lung Epithelial Cells Triggered by SARS-CoV-2 ORF3a Protein.

SARS-CoV-2 ORF3a accessory protein was found to be involved in virus release, immunomodulation and exhibited a pro-apoptotic character. In order to unravel a potential ORF3a-induced apoptotic and inflammatory death mechanism, lung epithelial cells (A549) were transfected with in vitro synthesized ORF3a mRNA. The protein's dynamic involvement as "stress factor" for the endoplasmic reticulum, causing the activation of PERK kinase and other UPR-involved proteins and therefore the upregulation of their signaling pathway executioners (ATF6, XBP-1s, PERK, phospho eIF2a, ATF4, CHOP, GADD34), has been clearly demonstrated. Furthermore, the overexpression of BAX and BH3-only pro-apoptotic protein PUMA, the upregulation of Bcl-2 family genes (BAX, BAK, BID, BAD), the reduced expression of Bcl-2 in mRNA and protein levels, and lastly, the cleavage of PARP-1 and caspase family members (caspase-3,-8 and -9) indicate that ORF3a displays its apoptotic character through the mitochondrial pathway of apoptosis. Moreover, the upregulation of NFκB, phosphorylation of p65 and IκΒα and the elevated expression of pro-inflammatory cytokines (IL-1b, IL-6, IL-8 and IL-18) in transfected cells with ORF3a mRNA indicate that this protein causes the inflammatory response through NFκB activation and therefore triggers lung injury. An intriguing finding of our study is that upon treatment of the ORF3a-transfected cells with GSK2606414, a selective PERK inhibitor, both complications (apoptosis and inflammatory response) were neutralized, and cell survival was favored, whereas treatment of transfected cells with z-VAD (a pan-caspase inhibitor) despite inhibiting cell death, could not ameliorate the inflammatory response of transfected A549 cells. Given the above, we point out that PERK kinase is a "master tactician" and its activation constitutes the main stimulus for the emergence of ORF3a apoptotic and inflammatory nature and therefore could serve as potential target for developing novel therapeutic approaches against COVID-19.

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.

A Novel Drastic Peptide Genetically Adapted to Biomimetic Scaffolds "Delivers" Osteogenic Signals to Human Mesenchymal Stem Cells.

This work describes the design, preparation, and deep investigation of "intelligent nanobiomaterials" that fulfill the safety rules and aim to serve as "signal deliverers" for osteogenesis, harboring a specific peptide that promotes and enhances osteogenesis at the end of their hydrogel fibers. The de novo synthesized protein fibers, besides their mechanical properties owed to their protein constituents from elastin, silk fibroin and mussel-foot adhesive protein-1 as well as to cell-attachment peptides from extracellular matrix glycoproteins, incorporate the Bone Morphogenetic Protein-2 (BMP2) peptide (AISMLYLDEN) that, according to our studies, serves as "signal deliverer" for osteogenesis. The osteogenetic capacity of the biomaterial has been evidenced by investigating the osteogenic marker genes ALP, RUNX2, Osteocalcin, COL1A1, BMPR1A, and BMPR2, which were increased drastically in cells cultured on scaffold-BMP2 for 21 days, even in the absence of osteogenesis medium. In addition, the induction of phosphorylation of intracellular Smad-1/5 and Erk-1/2 proteins clearly supported the osteogenetic capacity of the biomaterial.

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.

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.

Silver(I) complexes bearing heterocyclic thioamide ligands with NH2 and CF3 substituents: effect of ligand group substitution on antibacterial and anticancer properties.

In recent years, there has been an increasing interest in the study of Ag(I) coordination compounds as potent antibacterial and anticancer agents. Herein, a series of Ag(I) complexes bearing phosphines and heterocyclic thioamide ligands with highly electronegative NH2- and CF3-group substituents, i.e. [AgCl(atdztH)(xantphos)] (1), [Ag(µ-atdztH)(DPEphos)]2(NO3)2 (2), [Ag(atdzt)(PPh3)3] (3), [Ag(µ-atdzt)(DPEphos)]2 (4), and [Ag(µ-mtft)(DPEphos)]2 (5), where atdztH = 5-amino-1,3,4-thiadiazole-2-thiol, mtftH = 4-methyl-5-(trifluoromethyl)-1,2,4-triazol-3-thiol, xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and DPEphos = bis(2-diphenylphosphino-phenyl)ether, were synthesized, and their in vitro antibacterial and anticancer properties were evaluated. Complexes 1-4 bearing the NH2-substituted thioamide exhibited moderate-to-high activity against S. aureus, B. subtilis, B. cereus and E. coli bacterial strains. A high antiproliferative activity was also observed for 1-3 against SKOV-3, Hup-T3, DMS114 and PC3 cancer cell lines (IC50 = 4.0-11.7 µM), as well as some degree of selectivity against MRC-5 normal cells. Interestingly, 5 bearing the CF3-substituted thioamide is completely inactive in all bioactivity studies. Binding of 1-3 to drug-carrier proteins BSA and HSA is reasonably strong for their uptake and subsequent release to possible target sites. The three complexes show a significant in vitro antioxidant ability for scavenging free radicals, suggesting likely implication of this property in the mechanism of their bioactivity, but a low potential to destroy the double-strand structure of CT-DNA by intercalation. Complementary insights into possible bioactivity mechanisms were provided by molecular docking calculations, exploring the ability of complexes to bind to bacterial DNA gyrase, and to the overexpressed in the aforementioned cancer cells Fibroblast Growth Factor Receptor 1, affecting their functionalities.

Biocompatible silver(I) complexes with heterocyclic thioamide ligands for selective killing of cancer cells and high antimicrobial activity - A combined in vitro and in silico study.

A series of heteroleptic Ag(I) complexes bearing 4,6-dimethyl-2-pyrimidinethiol (dmp2SH), i.e., [AgCl(dmp2SH)(PPh3)2] (1), [Ag(dmp2SH)(PPh3)2]NO3 (2), [Ag(dmp2SΗ)(xantphos)]NO3 (3), [Ag(µ-dmp2S)(PPh3)]2 (4), [Ag(dmp2S)(xantphos)] (5), [Ag(µ-dmp2S)(DPEphos)]2 (6) (xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and DPEPhos = bis[(2-diphenylphosphino)phenyl]ether) were synthesized. The complexes display systematic variation of particular structural characteristics which were proved to have a significant impact on their in vitro cytotoxicity and antimicrobial properties. A moderate-to-high potential for bacteria growth inhibition was observed for all complexes, with 2, 3 and 5 being particularly effective against Gram-(+) bacteria (IC50 = 1.6-4.5 µM). The three complexes exhibit high in vitro cytotoxicity against HeLa and MCF-7 cancer cells (IC50 = 0.32-3.00 µΜ), suggesting the importance of coordination unsaturation and cationic charge for effective bioactivity. A very low cytotoxicity against HDFa normal cells was observed, revealing a high degree of selectivity (selectivity index ~10) and, hence, biocompatibility. Fluorescence microscopy using 2 showed effective targeting on the membrane of the HeLa cancer cells, subsequently inducing cell death. Binding of the complexes to serum albumin proteins is reasonably strong for potential uptake and subsequent release to target sites. A moderate in vitro antioxidant capacity for free radicals scavenging was observed and a low potential to destroy the double-strand structure of calf-thymus DNA by intercalation, suggesting likely implication of these properties in the bioactivity mechanisms of these complexes. Further insight into possible mechanisms of bioactivity was obtained by molecular modeling calculations, by exploring their ability to act as potential inhibitors of DNA-gyrase, human estrogen receptor alpha, human cyclin-dependent kinase 6, and human papillomavirus E6 oncoprotein.

Biopharmaceutics 4.0, Advanced Pre-Clinical Development of mRNA-Encoded Monoclonal Antibodies to Immunosuppressed Murine Models.

Administration of mRNA against SARS-CoV-2 has demonstrated sufficient efficacy, tolerability and clinical potential to disrupt the vaccination field. A multiple-arm, cohort randomized, mixed blind, placebo-controlled study was designed to investigate the in vivo expression of mRNA antibodies to immunosuppressed murine models to conduct efficacy, safety and bioavailability evaluation. Enabling 4.0 tools we reduced animal sacrifice, while interventions were designed compliant to HARRP and SPIRIT engagement: (a) Randomization, blinding; (b) pharmaceutical grade formulation, monitoring; (c) biochemical and histological analysis; and (d) theoretic, statistical analysis. Risk assessment molded the study orientations, according to the ARRIVE guidelines. The primary target of this protocol is the validation of the research hypothesis that autologous translation of Trastuzumab by in vitro transcribed mRNA-encoded antibodies to immunosuppressed animal models, is non-inferior to classical treatments. The secondary target is the comparative pharmacokinetic assessment of the novel scheme, between immunodeficient and healthy subjects. Herein, the debut clinical protocol, investigating the pharmacokinetic/pharmacodynamic impact of mRNA vaccination to immunodeficient organisms. Our design, contributes novel methodology to guide the preclinical development of RNA antibody modalities by resolving efficacy, tolerability and dose regime adjustment for special populations that are incapable of humoral defense.

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.

De Novo Synthesis of Elastin-like Polypeptides (ELPs): An Applied Overview on the Current Experimental Techniques.

Elastin-like polypeptides (ELPs) are protein-based biopolymers genetically produced from polypeptides composed of a repeating pentapeptide sequence V-P-G-X-G. The inherent properties of recombinant ELPs, such as smart nature, controlled sequence complexity, physicochemical properties, and biocompatibility, make these polymers suitable for use in nanobiotechnological applications, as biofunctionalized scaffolds for tissue-engineering purposes and drug delivery. In this work, we report the design and synthesis of two elastomeric self-assembling polypeptides (ELPs) that mimic the endogenous human tropoelastin. Using molecular biology techniques, two artificial genes that encode two ELP concatemers of approximate molecular mass 60 kDa, one of them carrying biotin-binding peptide motifs, were constructed. These motifs could facilitate biofunctionalization of the ELPs through tethering biotinylated factors, such as growth factors. The ELPs were heterologously overexpressed in E. coli and subsequently purified in two steps: a nonchromatographic technique by organic solvent extraction, followed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The characterization of the biochemical properties and biocompatibility of ELPs was also performed in this study. The ELP carrying the biotin-binding motifs was tested for its capability to bind biotin, and indeed, it was observed that it can bind biotinylated proteins specifically. Additionally, results concerning the cytotoxicity of the ELPs exhibited excellent compatibility of the ELPs with mammalian cells in vitro. We anticipate that these ELPs can be used as components of a scaffold that mimics the extracellular matrix (ECM) for the regeneration of endogenously highly elastic tissues.

The C-terminal region of HPNAP activates neutrophils and promotes their adhesion to endothelial cells.

Entire Helicobacter Pylori Neutrophil Activated Protein (HPNAP) and its truncated forms NH(2)-terminal region HPNAP(1-57) and C-terminal region HPNAP(58-144) after cloning into pET29c vector, purification and removal of LPS traces were subjected to human neutrophil activation. Our results revealed that the C-terminal region of HPNAP is indispensable for human neutrophil stimulation and their further adhesion to endothelial cells - a step necessary to H. pylori inflammation - in a ratio equal to that exhibited by the entire protein. In addition, experiments concerning the implication of Arabino-Galactan-Proteins (AGPs) derived from Chios Mastic Gum (CMG), the natural resin of the plant Pistacia lentiscus var. Chia revealed the inhibition of neutrophil activation and therefore their adhesion to endothelial cells, in vitro. Both, the involvement of HPNAP C-terminal region in stimulation-adhesion of neutrophils to endothelial cells as well as the inhibition of this process by AGPs have to be further investigated and may be exploited in a future anti-inflammatory therapy for H. pylori patients.