Search

A new dual-functional implant based on gellan-xanthan hydrogel with calcium-magnesium silicate ceramic diopside and recombinant lysostaphin and bone morphogenetic protein 2 (BMP-2)-ray is developed. In this composite, BMP-2 is immobilized on microparticles of diopside while lysostaphin is mixed directly into the hydrogel, providing sustained release of BMP-2 to allow gradual bone formation and rapid release of lysostaphin to eliminate infection immediately after implantation. Introduction of diopside of up to 3% (w/v) has a negligible effect on the mechanical properties of the hydrogel but provides a high sorption capacity for BMP-2. The hydrogels show good biocompatibility and antibacterial activity. Lysostaphin released from the implants over a 3 h period efficiently kills planktonic cells and completely destroys 24 h pre-formed biofilms of Staphylococcus aureus. Furthermore, in vivo experiments in a mouse model of critically-sized cranial defects infected with S. aureus show a complete lack of osteogenesis when implants contain only BMP-2, whereas, in the presence of lysostaphin, complete closure of the defect with newly formed mineralized bone tissue is observed. Thus, the new implantable gellan-xanthan hydrogel with diopside and recombinant lysostaphin and BMP-2 shows both osteogenic and antibacterial properties and represents a promising material for the treatment and/or prevention of osteomyelitis after bone trauma., (© 2024 Wiley‐VCH GmbH.)

Phylogenetic inference based on protein sequence alignment is a widely used procedure. Numerous phylogenetic algorithms have been developed, most of which have many parameters and options. Choosing a program, options, and parameters can be a nontrivial task. No benchmark for comparison of phylogenetic programs on real protein sequences was publicly available. We have developed PhyloBench, a benchmark for evaluating the quality of phylogenetic inference, and used it to test a number of popular phylogenetic programs. PhyloBench is based on natural, not simulated, protein sequences of orthologous evolutionary domains. The measure of accuracy of an inferred tree is its distance to the corresponding species tree. A number of tree-to-tree distance measures were tested. The most reliable results were obtained using the Robinson-Foulds distance. Our results confirmed recent findings that distance methods are more accurate than maximum likelihood (ML) and maximum parsimony. We tested the bayesian program MrBayes on natural protein sequences and found that, on our datasets, it performs better than ML, but worse than distance methods. Of the methods we tested, the Balanced Minimum Evolution method implemented in FastME yielded the best results on our material. Alignments and reference species trees are available at https://mouse.belozersky.msu.ru/tools/phylobench/ together with a web-interface that allows for a semi-automatic comparison of a user's method with a number of popular programs., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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 Na 2 HPO 4 , Ca(OH) 2 , (NH 2 ) 2 CO, Na 2 SiO 3 , CuSO 4 , 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 Cu 2+ 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.

High efficiency of hybrid implants based on calcium-magnesium silicate ceramic, diopside, as a carrier of recombinant BMP-2 and xenogenic demineralized bone matrix (DBM) as a scaffold for bone tissue regeneration was demonstrated previously using the model of critical size cranial defects in mice. In order to investigate the possibility of using these implants for growing autologous bone tissue using in vivo bioreactor principle in the patient's own body, effectiveness of ectopic osteogenesis induced by them in intramuscular implantation in mice was studied. At the dose of 7 μg of BMP-2 per implant, dense agglomeration of cells, probably skeletal muscle satellite precursor cells, was observed one week after implantation with areas of intense chondrogenesis, initial stage of indirect osteogenesis, around the implants. After 12 weeks, a dense bone capsule of trabecular structure was formed covered with periosteum and mature bone marrow located in the spaces between the trabeculae. The capsule volume was about 8-10 times the volume of the original implant. There were practically no signs of inflammation and foreign body reaction. Microcomputed tomography data showed significant increase of the relative bone volume, number of trabeculae, and bone tissue density in the group of mice with BMP-2-containing implant in comparison with the group without BMP-2. Considering that DBM can be obtained in practically unlimited quantities with required size and shape, and that BMP-2 is obtained by synthesis in E. coli cells and is relatively inexpensive, further development of the in vivo bioreactor model based on the hybrid implants constructed from BMP-2, diopside, and xenogenic DBM seems promising.

Lysostaphin is a zinc-dependent endopeptidase that is effective against both antibiotic-sensitive and antibiotic-resistant strains of Staphylococcus aureus. Lysostaphin is typically purified on cation-exchange or metal-chelate affinity resins, and there are data indicating potential influence of the chromatographic resin on the lysostaphin activity. In this study, we systematically investigated the impact of the resin used to purify the recombinant lysostaphin on its activity. To this end, recombinant lysostaphin with an additional histidine tag at the C-terminus was purified using a cation-exchange resin, three types of nickel-chelate resins with different strength of metal ion binding, or a zinc-chelate resin. Lysostaphin samples purified on the cation-exchange resin (WorkBeads 40S), the nickel-chelate resin with a strong nickel ion binding (WorkBeads NiMAC), and the zinc-chelate resin (WorkBeads NTA with immobilized zinc ions) had equal activity. On the contrary, the activity of lysostaphin preparations purified on nickel-chelate resins with medium (WorkBeads Ni-NTA) and relatively weak (WorkBeads Ni-IDA) nickel ion binding was significantly reduced. The decrease in activity can be explained by the interaction of lysostaphin with the nickel ions leached from the resin and is caused by either the exchange of the zinc ion in the lysostaphin active center with a nickel ion from the resin, or binding of an additional ion that inhibits the enzymatic activity. Removal of the metal ions from the active site of lysostaphin and subsequent incorporation of the native zinc ions lead to complete restoration of the activity of the enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Some restriction-modification systems contain two DNA methyltransferases. In the present work, we have classified such systems according to the families of catalytic domains present in the restriction endonucleases and both DNA methyltransferases. Evolution of the restriction-modification systems containing an endonuclease with a NOV_C family domain and two DNA methyltransferases, both with DNA_methylase family domains, was investigated in detail. Phylogenetic tree of DNA methyltransferases from the systems of this class consists of two clades of the same size. Two DNA methyltransferases of each restriction-modification system of this class belong to the different clades. This indicates independent evolution of the two methyltransferases. We detected multiple cross-species horizontal transfers of the systems as a whole, as well as the cases of gene transfer between the systems.

Background: Diopside-based ceramic is a perspective biocompatible material with numerous potential applications in the field of bone prosthetics. Implantable devices and materials are often prone to colonization and biofilm formation by pathogens such as Staphylococcus aureus , which in the case of bone grafting leads to osteomyelitis, an infectious bone and bone marrow injury. To lower the risk of bacterial colonization, implanted materials can be impregnated with antimicrobials. In this work, we loaded the antibacterial enzyme lysostaphin on diopside powder and studied the antibacterial and antibiofilm properties of such material to probe the utility of this approach for diopside-based prosthetic materials., Methods: Diopside powder was synthesized by the solid-state method, lysostaphin was loaded on diopside by adsorption, the release of lysostaphin from diopside was monitored by ELISA, and antibacterial and anti-biofilm activity was assessed by standard microbiological procedures., Results and Conclusions: Lysostaphin released from diopside powder showed high antibacterial activity against planktonic bacteria and effectively destroyed 24-h staphylococcal biofilms. Diopside-based materials possess a potential for the development of antibacterial bone grafting materials.

a e-mail: sas@belozersky.msu.ru Protein phylogeny is usually reconstructed basing on a multiple alignment of amino acid sequences. One of the problems of such alignments is the presence of regions with different degree of conservation, including those with a questionable quality of the alignment. This problem is often solved by filtering the alignment columns with a special software developed for this purpose. In this work, we investigated various approaches to the phylogeny reconstruction using proteins with two evolutionary domains as examples. The sequences of such proteins are inherently heterogeneous in the degree of conservation due to the presence of both evolutionary domains and linkers between them, as well as the N- and C-termini. It is shown that filtering the alignment columns on average improves the quality of reconstruction only when using the full-length sequences and only for eukaryotic proteins. Limiting the alignment to the evolutionary domains with rejection of less conserved linkers and terminal sequences on average worsened the quality of phylogenetic reconstruction.

Calcium-magnesium silicate ceramics, diopside, is a promising material for use in bone plastics, but until now the possibility of its use as a carrier of recombinant bone morphogenetic protein-2 (BMP-2) has not been studied, as well as the features of reparative osteogenesis mediated by the materials based on diopside with BMP-2. Powder of calcium-magnesium silicate ceramics was obtained by solid-state synthesis using biowaste - rice husks and egg shells - as source components. Main phase of the obtained ceramics was diopside. The obtained particles were irregularly shaped with an average size of about 2.3 μm and ~20% porosity; average pore size was about 24 nm, which allowed the material to be classified as mesoporous. Diopside powder adsorbs more than 150 μg of recombinant BMP-2 per milligram, which exceeds binding capacity of hydroxyapatite, a calcium-phosphate ceramic often used in hybrid implants, by more than 3 times. In vitro release kinetics of BMP-2 was characterized by a burst release in the first 2 days and a sustained release of approximately 0.4 to 0.5% of the loaded protein over the following 7 days. In vivo experiments were performed with a mouse model of cranial defects of critical size with implantation of a suspension of diopside powder with/without BMP-2 in hyaluronic acid incorporated into the disks of demineralized bone matrix with 73-90% volume porosity and macropore size from 50 to 650 μm. Dynamics of neoosteogenesis and bone tissue remodeling was investigated histologically at the time points of 12, 21, 48, and 63 days. Diopside particles were evenly spread in the matrix and caused minimal foreign body reaction. In the presence of BMP-2 by the day 63 significant foci of newly formed bone tissue were formed in the implant pores with bone marrow areas, moreover, large areas of demineralized bone matrix in the implant center and maternal bone at the edges were involved in the remodeling. Diopside could be considered as a promising material for introduction into hybrid implants as an effective carrier of BMP-2.

Peptidoglycan-degrading enzymes are a group of proteins intensively studied as novel antibacterials, with some of them having reached pre-clinical and clinical stages of research. Many peptidoglycan-degrading enzymes have modular organization and consist of a catalytic and a cell wall binding domain. This property has been exploited in enzyme engineering efforts, and many new peptidoglycan-degrading enzymes were generated through domain exchange. However, rational combination of domains from different enzymes is still challenging since relative contribution of every domain to the cumulative bacteriolytic activity is not yet clearly understood. In this work, we investigated the influence of ionic strength and pH on the catalytic efficiency and cell binding of peptidoglycan-degrading enzyme lysostaphin and how this influence is reflected in the lysostaphin bacteriolytic activity. Contrary to generally accepted view, lysostaphin domains are not completely independent and their combination within one protein leads to increased bacteriolytic activity with increasing NaCl concentration, despite both catalysis and cell binding being inhibited by NaCl. This effect is likely mediated by changes in conformation of bacterial cell wall peptidoglycan rather than the physical inter-domain interaction. KEY POINTS: • NaCl enhances bacteriolytic activity of lysostaphin but not of its catalytic domain. • Catalytic activity and cell binding of lysostaphin are inhibited by NaCl. • Peptidoglycan conformation likely affects lysostaphin bacteriolytic activity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Osteoplastic materials PLA/PCL/HA and PHB/HA and scaffolds with a highly porous structure based on them with potential applications in regenerative medicine have been obtained by solvent casting with thermopressing and salt leaching for PLA-based samples and solid-state mixing with subsequent thermopressing and salt leaching for PHB-based samples. The scaffolds were characterized by SEM-EDX, DSC, FTIR spectroscopy, mechanical tests in compression, measurement of the contact angle, in vitro studies, including loading by recombinant BMP-2 and EPO and their release kinetics, and in vivo studies on a model of regeneration of critical-sized cranial defects in mice. Biomimetic scaffolds with micropores sizes ranged from 300 to 500 μm and volume porosity of 70% imitate trabecular bone's structure and have increased hydrophilicity to achieve osteoconductive properties. Mechanical characteristics correspond to native trabecular bone. Elastic modulus - key mechanical characteristics of bone implants - showed the values of 0.15 ± 0.04 and 0.18 ± 0.08 GPa for PLA/PCL/HA and PHB/HA scaffolds, respectively. Both materials have high biocompatibility and can be used together with recombinant proteins BMP-2 and EPO. Introduction of BMP-2 leads to induction of new bone formation, introduction of EPO results in increased angiogenesis in the implantation area. The obtained scaffolds with recombinant proteins can be used as bone implants for reconstruction of defects of lightly or non-loaded bones., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.

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.

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus . Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

The major problem in bone tissue engineering is the development of scaffolds which can simultaneously meet the requirements of porous structure, as well as have the ability to guide the regeneration of damaged tissue by biological fixation. Composites containing biodegradable matrix and bioactive filler are the new hope in this research field. Herein we employed a simple and facile solvent casting particulate-leaching method for producing polylactide acid/hydroxyapatite (PLA/HA) composites at room temperature. FT-IR analysis confirmed the existence of necessary functional groups associated with the PLA/HA composite, whereas energy-dispersive X-ray (EDX) spectra indicated the uniform distribution of hydroxyapatite particles in the polymer matrix. The beehive-like surface morphology of the composites revealed the presence of macropores, ranged from 300 to 400 μm, whereas the thickness of the pores was noticed to be 1-2 μm. The total porosity of the scaffolds, calculated by hydrostatic weighing, was found to be 79%. The water contact angle of pure PLA was decreased from 83.6 ± 1.91° to 62.4 ± 4.17° due to the addition of hydroxyapatite in the polymer matrix. Thus, the wettability of the polymeric biomaterial could be increased by preparing their composites with hydroxyapatite. The adhesion of multipotent mesenchymal stromal cells over the surface of PLA/HA scaffolds was 3.2 times ( p = 0.03) higher than the pure PLA sample. Subcutaneous implantation in mice demonstrated a good tolerance of all tested porous scaffolds and widespread ingrowth of tissue into the implant pores. HA-containing scaffolds showed a less pronounced inflammatory response after two weeks of implantation compared to pure PLA. These observations suggest that PLA/HA composites have enormous potential for hard tissue engineering and restoring maxillofacial defects.

The spread of bacterial strains resistant to commonly used antibiotics urges the development of novel antibacterial compounds. Ideally, these novel antimicrobials should be less prone to the development of resistance. Peptidoglycan-degrading enzymes are a promising class of compounds with a fundamentally different mode of action compared to traditionally used antibiotics. The difference in the mechanism of action implies differences both in the mechanisms of resistance and the chances of its emergence. To critically assess the potential of resistance development to peptidoglycan-degrading enzymes, we review the available evidence for the development of resistance to these enzymes in vitro , along with the known mechanisms of resistance to lysozyme, bacteriocins, autolysins, and phage endolysins. We conclude that genetic determinants of resistance to peptidoglycan-degrading enzymes are unlikely to readily emerge de novo. However, resistance to these enzymes would probably spread by the horizontal transfer between intrinsically resistant and susceptible species. Finally, we speculate that the higher cost of the therapeutics based on peptidoglycan degrading enzymes compared to classical antibiotics might result in less misuse, which in turn would lead to lower selective pressure, making these antibacterials less prone to resistance development.

A promising direction for the replacement of expanded bone defects is the development of bioimplants based on synthetic biocompatible materials impregnated with growth factors that stimulate bone remodeling. Novel biomimetic highly porous ultra-high molecular weight polyethylene (UHMWPE)/40% hydroxyapatite (HA) scaffold for reconstructive surgery with the porosity of 85 ± 1% vol. and a diameter of pores in the range of 50-800 μm was developed. The manufacturing process allowed the formation of trabecular-like architecture without additional solvents and thermo-oxidative degradation. Biomimetic UHMWPE/HA scaffold was biocompatible and provided effective tissue ingrowth on a model of critical-sized cranial defects in mice. The combined use of UHMWPE/HA with Bone Morphogenetic Protein-2 (BMP-2) demonstrated intensive mineralized bone formation as early as 3 weeks after surgery. The addition of erythropoietin (EPO) significantly enhanced angiogenesis in newly formed tissues. The effect of EPO of bacterial origin on bone tissue defect healing was demonstrated for the first time. The developed biomimetic highly porous UHMWPE/HA scaffold can be used separately or in combination with rhBMP-2 and EPO for reconstructive surgery to solve the problems associated with difference between implant architecture and trabecular bone, low osteointegration and bioinertness., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 CaCO 3 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.

Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.

The increasing prevalence of antibiotic-resistant strains of pathogenic bacteria is a major healthcare problem. Antibacterial lysins are enzymes that cleave the peptidoglycan of the bacterial cell wall. These proteins hold potential as a supplement or an alternative to traditional antibiotics since they are active against antibiotic resistant strains. However, antibacterial lysins are rapidly eliminated from the systemic circulation, which limits their application. Dimerization of an anti-pneumococcal lysin Cpl-1 has been demonstrated to decrease the clearance rate of this protein in mice. In the present work, we constructed a dimer of an anti-staphylococcal lysin lysostaphin by fusing it with an anti-parallel α-helical dimerization domain. Lysostaphin dimer had a more favorable pharmacokinetic profile with increased terminal half-life and area under the curve (AUC) values compared to monomeric lysostaphin. However, the staphylolytic activity of dimerized lysostaphin was decreased. This decrease in activity was likely caused by the dimerization; since the catalytic efficacy of lysostaphin dimer towards pentaglycine peptide was unaltered. Our results demonstrate that, although dimerization is indeed beneficial for the pharmacokinetics of antibacterial lysins, this approach might not be suitable for all lysins, as it can negatively affect the lysin activity.

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., (© 2017 John Wiley & Sons A/S.)

Listeria monocytogenes is a causative agent of foodborne infection in humans and animals. The virulence factor InlB interacts with mammalian receptor c-Met via its internalin domain to provide L. monocytogenes invasion in non-professional phagocytes. Naturally occurring InlB internalin domain variants form four subclusters on the maximal likelihood tree. Four variants belonging to distinct subclusters were cloned into the vector carrying 3΄ and 5΄-flanking sequences to restore full length inlB and expressed in the L. monocytogenes strain EGDeΔinlB. The substitutions Val132Ile, Thr117Ala and Ile138Leu, Thr251Met/Ser were specific for variants 13, 14 and 1, respectively, the variant 9 carried Ser73Asn, Ile91Val, Leu164Pro, Met251Ser/Thr substitutions. All InlB variants improved invasion of the parental strain in murine colon carcinoma C26 cells with 4.6-fold difference between the most and least effective variants (variants 14 and 13, respectively, P < 0.05). Bacterial loads in livers of intragastrically infected mice were 258, 149 and 92 times higher for variant 14, 13 and 1 carrying strains, respectively, than for EGDeΔinlB (P < 0.01). In contrast, the variant 9 did not noticeably improve infection comparatively to the parental strain. Overall, obtained results demonstrated that naturally occurred InlB internalin domain variants differed in their ability to support intragastric infection in mice., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

A comparative analysis of all available structures of complexes of TATA-box binding proteins (TBPs) with DNA is performed. Conserved features of DNA-protein interaction are described, including nine amino acid residues that form conserved hydrogen bonds, 13 residues participating in formation of two conserved hydrophobic clusters at DNA-protein interface, and four conserved water-mediated contacts. Partial symmetry of conserved contacts reflects quasi-symmetry of TBP structure.

Palindromes are frequently underrepresented in prokaryotic genomes. Palindromic 5[Formula: see text]-GATC-3[Formula: see text] site is a recognition site of different Restriction-Modification (R-M) systems, as well as solitary methyltransferase Dam. Classical GATC-specific R-M systems methylate GATC and cleave unmethylated GATC. On the contrary, methyl-directed Type II restriction endonucleases cleave methylated GATC. Methylation of GATC by Dam methyltransferase is involved in the regulation of different cellular processes. The diversity of functions of GATC-recognizing proteins makes GATC sequence a good model for studying the reasons of palindrome avoidance in prokaryotic genomes. In this work, the influence of R-M systems and solitary proteins on the GATC site avoidance is described by a mathematical model. GATC avoidance is strongly associated with the presence of alternate (methyl-directed or classical Type II R-M system) genes in different strains of the same species, as we have shown for Streptococcus pneumoniae, Neisseria meningitidis, Eubacterium rectale, and Moraxella catarrhalis. We hypothesize that GATC avoidance can result from a DNA exchange between strains with different methylation status of GATC site within the process of natural transformation. If this hypothesis is correct, the GATC avoidance is a sign of a DNA exchange between bacteria with different methylation status in a mixed population.

The recent upgrade of nucleic acid-protein interaction database (NPIDB, http://npidb.belozersky.msu.ru/) includes a newly elaborated classification of complexes of protein domains with double-stranded DNA and a classification of families of related complexes. Our classifications are based on contacting structural elements of both DNA: the major groove, the minor groove and the backbone; and protein: helices, beta-strands and unstructured segments. We took into account both hydrogen bonds and hydrophobic interaction. The analyzed material contains 1942 structures of protein domains from 748 PDB entries. We have identified 97 interaction modes of individual protein domain-DNA complexes and 17 DNA-protein interaction classes of protein domain families. We analyzed the sources of diversity of DNA-protein interaction modes in different complexes of one protein domain family. The observed interaction mode is sometimes influenced by artifacts of crystallization or diversity in secondary structure assignment. The interaction classes of domain families are more stable and thus possess more biological sense than a classification of single complexes. Integration of the classification into NPIDB allows the user to browse the database according to the interacting structural elements of DNA and protein molecules. For each family, we present average DNA shape parameters in contact zones with domains of the family., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Avoidance of palindromic recognition sites of Type II restriction-modification (R-M) systems was shown for many R-M systems in dozens of prokaryotic genomes. However the phenomenon has not been investigated systematically for all presently available genomes and annotated R-M systems. We have studied all known recognition sites in thousands of prokaryotic genomes and found factors that influence their avoidance., Results: Only Type II R-M systems consisting of independently acting endonuclease and methyltransferase (called 'orthodox' here) cause avoidance of their sites, both palindromic and asymmetric, in corresponding prokaryotic genomes; the avoidance takes place for ~ 50 % of 1774 studied cases. It is known that prokaryotes can acquire and lose R-M systems. Thus it is possible to talk about the lifespan of an R-M system in a genome. We have shown that the recognition site avoidance correlates with the lifespan of R-M systems. The sites of orthodox R-M systems that are encoded in host genomes for a long time are avoided more often (up to 100 % in certain cohorts) than the sites of recently acquired ones. We also found cases of site avoidance in absence of the corresponding R-M systems in the genome. An analysis of closely related bacteria shows that such avoidance can be a trace of lost R-M systems. Sites of Type I, IIС/G, IIM, III, and IV R-M systems are not avoided in vast majority of cases., Conclusions: The avoidance of orthodox Type II R-M system recognition sites in prokaryotic genomes is a widespread phenomenon. Presence of an R-M system without an underrepresentation of its site may indicate that the R-M system was acquired recently. At the same time, a significant underrepresentation of a site may be a sign of presence of the corresponding R-M system in this organism or in its ancestors for a long time. The drastic difference between site avoidance for orthodox Type II R-M systems and R-M systems of other types can be explained by a higher rate of specificity changes or a less self-toxicity of the latter.

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) consists of a methyltransferase domain (residues 72-379) and an N-terminal region (residues 1-71) which regulates transcription in SsoII restriction-modification system. Small-angle X-ray scattering (SAXS) is employed here to study the low resolution structure of M.SsoII and its complex with DNA containing the methylation site. The shapes reconstructed ab initio from the SAXS data reveal two distinct protein domains of unequal size. The larger domain matches the crystallographic structure of a homologous DNA methyltransferase HhaI (M.HhaI), and the cleft in this domain is occupied by DNA in the model of the complex reconstructed from the SAXS data. This larger domain can thus be identified as the methyltransferase domain whereas the other domain represents the N-terminal region. Homology modeling of the M.SsoII structure is performed by using the model of M.HhaI for the methyltransferase domain and representing the N-terminal region either as a flexible chain of dummy residues or as a rigid structure of a homologous protein (phage 434 repressor) connected to the methyltransferase domain by a short flexible linker. Both models are compatible with the SAXS data and demonstrate high mobility of the N-terminal region. The linker flexibility might play an important role in the function of M.SsoII as a transcription factor.

Release factor eRF1 plays a key role in the termination of protein synthesis in eukaryotes. The eRF1 consists of three domains (N, M and C) that perform unique roles in termination. Previous studies of eRF1 point mutants and standard/variant code eRF1 chimeras unequivocally demonstrated a direct involvement of the highly conserved N-domain motifs (NIKS, YxCxxxF and GTx) in stop codon recognition. In the current study, we extend this work by investigating the role of the 41 invariant and conserved N-domain residues in stop codon decoding by human eRF1. Using a combination of the conservative and non-conservative amino acid substitutions, we measured the functional activity of >80 mutant eRF1s in an in vitro reconstituted eukaryotic translation system and selected 15 amino acid residues essential for recognition of different stop codon nucleotides. Furthermore, toe-print analyses provide evidence of a conformational rearrangement of ribosomal complexes that occurs during binding of eRF1 to messenger RNA and reflects stop codon decoding activity of eRF1. Based on our experimental data and molecular modelling of the N-domain at the ribosomal A site, we propose a two-step model of stop codon decoding in the eukaryotic ribosome.

The Nucleic acid-Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid-Protein Interaction DataBase is an upgrade of the version published in 2007. The improvements include a new web interface, new tools for calculation of intermolecular interactions, a classification of SCOP families that contains DNA-binding protein domains and data on conserved water molecules on the DNA-protein interface.

Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.

LAGLIDADG family of homing endonucleases are rare-cutting enzymes which recognize long target sequences and are of great interest in genome engineering. Despite advances in homing endonuclease engineering, effective methods of broadening the range of cleaved sequences are still lacking. Here, we present a study of conserved structural features of LAGLIDADG homing endonucleases that might aid further development of such methods. The protein-DNA interface of LAGLIDADG homing endonucleases differs considerably with the particular nuclease, and the analysis of conserved protein-DNA interactions could not identify any residues crucial for DNA binding and common to most nucleases of the family. For the homing endonuclease PI-SceI, a comparison of structural and experimental data derived from literature helped to identify 23 residues that are likely to be important for DNA binding. Analysis of the LAGLIDADG domain dimerization interface allowed the choosing of six positions that contribute to dimerization specificity most, while comparison of 446 sequences of LAGLIDADG endonucleases revealed groups of residues in these positions that appear to be most favorable for dimerization.

Standard Affymetrix technology evaluates gene expression by measuring the intensity of mRNA hybridization with a panel of the 25-mer oligonucleotide probes, and summarizing the probe signal intensities by a robust average method. However, in many cases, signal intensity of the probe does not correlate with gene expression. This could be due to the hybridization of the probe to a transcript of another gene, mapping of the probe to an intron, alternative splicing, single nucleotide polymorphisms and other reasons. We have developed a database, PLANdbAffy (available at http://affymetrix2.bioinf.fbb.msu.ru), that contains the results of the alignment of probe sequences from five Affymetrix expression microarrays to the human genome. We have determined the probes matching the transcript-coding regions in the correct orientation. For each such probe alignment region, we determined the mRNA and EST sequences that contain the probe sequence. In the textual part of the database interface we summarize the data on the sequences that cover the probe alignment region and SNPs that are located inside it. The graphical part of our database interface is implemented as custom tracks to the UCSC genome browser that allows one to utilize all the data that are offered by UCSC browser.

Water molecules immobilized on a protein or DNA surface are known to play an important role in intramolecular and intermolecular interactions. Comparative analysis of related three-dimensional (3D) structures allows to predict the locations of such water molecules on the protein surface. We have developed and implemented the algorithm WLAKE detecting "conserved" water molecules, i.e. those located in almost the same positions in a set of superimposed structures of related proteins or macromolecular complexes. The problem is reduced to finding maximal cliques in a certain graph. Despite exponential algorithm complexity, the program works appropriately fast for dozens of superimposed structures. WLAKE was used to predict functionally significant water molecules in enzyme active sites (transketolases) as well as in intermolecular (ETS-DNA complexes) and intramolecular (thiol-disulfide interchange protein) interactions. The program is available online at http://monkey.belozersky.msu.ru/~evgeniy/wLake/wLake.html.

Unlabelled: The database NPIDB (Nucleic Acids-Protein Interaction DataBase) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from PDB (1834 complexes in July 2007). It is organized as a collection of files in PDB format and is equipped with a web-interface and a set of tools for extracting biologically meaningful characteristics of complexes. The content of the database is weekly updated., Availability: http://monkey.belozersky.msu.ru/NPIDB/

The conserved hydrophobic core is an important feature of a family of protein domains. We suggest a procedure for finding and the analysis of conserved hydrophobic cores. The procedure is based on using an original program called CluD (http://monkey.belozersky.msu.ru/CluD/cgi-bin/hftri.pl). Conserved hydrophobic cores of several families including homeodomains and interlock-containing domains are described. Hydrophobic clusters on some protein-DNA and protein-protein interfaces were also analyzed.

Crystals of the (cytosine-5)-DNA methyltransferase NlaX from Neisseria lactamica (molecular weight 36.5 kDa) have been grown at 291 K using 2.5 M NaCl as precipitant. The crystals diffract to 3.0 A resolution at 100 K. The crystals belong to space group P321, with unit-cell parameters a = 121.98, b = 121.98, c = 56.71 A. There is one molecule in the asymmetric unit and the solvent content is estimated to be 62.1% by volume.