Osteoconductive, Osteogenic, and Antipathogenic Plasma Electrolytic Oxidation Coatings on Titanium Implants with BMP-2.

We report a one-pot plasma electrolytic oxidation (PEO) strategy for forming a multi-element oxide layer on the titanium surface using complex electrolytes containing Na2HPO4, Ca(OH)2, (NH2)2CO, Na2SiO3, CuSO4, and KOH compounds. For even better bone implant ingrowth, PEO coatings were additionally loaded with bone morphogenetic protein-2 (BMP-2). The samples were tested in vivo in a mouse craniotomy model. Tests for bactericidal and fungicidal activity were carried out using clinically isolated multi-drug-resistant Escherichia coli (E. coli) K261, E. coli U20, methicillin-resistant Staphylococcus aureus (S. aureus) CSA154 bacterial strains, and Neurospora crassa (N. crassa) and Candida albicans (C. albicans) D2528/20 fungi. The PEO-Cu coating effectively inactivated both Gram-positive and Gram-negative bacteria at low concentrations of Cu2+ ions: minimal bactericidal concentration for E. coli and N. crassa (99.9999%) and minimal inhibitory concentration (99.0%) for S. aureus were 5 ppm. For all studied bacterial and fungal strains, PEO-Cu coating completely prevented the formation of bacterial and fungal biofilms. PEO and PEO-Cu coatings demonstrated bone remodeling and moderate osteoconductivity in vivo, while BMP-2 significantly enhanced osteoconduction and osteogenesis. The obtained results are encouraging and indicate that Ti-based materials with PEO coatings loaded with BMP-2 can be widely used in customized medicine as implants for orthopedics and cranio-maxillofacial surgery.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Development of Optimized Strategies for Growth Factor Incorporation onto Electrospun Fibrous Scaffolds To Promote Prolonged Release.

Growth factor incorporation in biomedical constructs for their local delivery enables specific pharmacological effects such as the induction of cell growth and differentiation. This has enabled a promising way to improve the tissue regeneration process. However, it remains challenging to identify an appropriate approach that provides effective growth factor loading into biomedical constructs with their following release kinetics in a prolonged manner. In the present work, we performed a systematic study, which explores the optimal strategy of growth factor incorporation into sub-micrometric-sized CaCO3 core-shell particles (CSPs) and hollow silica particles (SiPs). These carriers were immobilized onto the surface of the polymer scaffolds based on polyhydroxybutyrate (PHB) with and without reduced graphene oxide (rGO) in its structure to examine the functionality of incorporated growth factors. Bone morphogenetic protein-2 (BMP-2) and ErythroPOietin (EPO) as growth factor models were included into CSPs and SiPs using different entrapping strategies, namely, physical adsorption, coprecipitation technique, and freezing-induced loading method. It was shown that the loading efficiency, release characteristics, and bioactivity of incorporated growth factors strongly depend on the chosen strategy of their incorporation into delivery systems. Overall, we demonstrated that the combination of scaffolds with drug delivery systems containing growth factors has great potential in the field of tissue regeneration compared with individual scaffolds.

Identification of chlamydial T3SS inhibitors through virtual screening against T3SS ATPase.

Chlamydia trachomatis is a widespread sexually transmitted pathogen that resides within a special vacuole inside host cells. Although acute infection can be treated with antibiotics, chlamydia can enter persistent state, leading to chronic infection that is difficult to cure. Thus, novel anti-chlamydial compounds active against persistent chlamydia are required. Chlamydiae rely upon type III secretion system (T3SS) to inject effector proteins into host cell cytoplasm, and T3SS inhibitors are viewed as promising compounds for treatment of chlamydial infections. C. trachomatis ATPase SctN is an important T3SS component and has not been targeted before. We thus used virtual screening against homology modeled SctN structure to search for SctN inhibitors. Selected compounds were tested for their ability to inhibit chlamydial survival and development within eukaryotic cells, and for the ability to suppress normal T3SS functioning. We identified two compounds that were able to block normal protein translocation through T3SS and inhibit chlamydial survival within eukaryotic cells in 50-100 µm concentrations. These two novel T3SS inhibitors also possessed relatively low toxicity toward eukaryotic cells. A small series of derivatives was further synthesized for the most active of two inhibitors to probe SAR properties.

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

BACKGROUND: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. METHODS: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. RESULTS: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. CONCLUSIONS: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.