Marina, Valeriya I

Alferova V.A., Zotova P.A., Baranova A.A., Guglya E.B., Belozerova O.A., Pipiya S.O., Kudzhaev A.M., Logunov S.E., Prokopenko Y.A., Marenkova E.A., Marina V.I., Novikova E.A., Komarova E.S., Starodumova I.P., Bueva O.V., et. al.

Puromycin (Puro) is a natural aminonucleoside antibiotic that inhibits protein synthesis by its incorporation into elongating peptide chains. The unique mechanism of Puro finds diverse applications in molecular biology, including the selection of genetically engineered cell lines, in situ protein synthesis monitoring, and studying ribosome functions. However, the key step of Puro biosynthesis remains enigmatic. In this work, pur6-guided genome mining is carried out to explore the natural diversity of Puro-like antibiotics. The diversity of biosynthetic gene cluster (BGC) architectures suggests the existence of distinct structural analogs of puromycin encoded by pur-like clusters. Moreover, the presence of tRNACys in some BGCs, i.e., cst-like clusters, leads us to the hypothesis that Pur6 utilizes aminoacylated tRNA as an activated peptidyl precursor, resulting in cysteine-based analogs. Detailed metabolomic analysis of Streptomyces sp. VKM Ac-502 containing cst-like BGC revealed the production of a cysteinyl-based analog of Puro—cystocin (Cst). Similar to puromycin, cystocin inhibits both prokaryotic and eukaryotic translation by the same mechanism. Aminonucleoside N-acetyltransferase CstC inactivated Cst, mediating antibiotic resistance in genetically modified bacteria and human cells. The substrate specificity of CstC originated from the steric hindrance of its active site. We believe that novel aminonucleosides and their inactivating enzymes can be developed through the directed evolution of the discovered biosynthetic machinery.

Panteleev P.V., Pichkur E.B., Kruglikov R.N., Paleskava A., Shulenina O.V., Bolosov I.A., Bogdanov I.V., Safronova V.N., Balandin S.V., Marina V.I., Kombarova T.I., Korobova O.V., Shamova O.V., Myasnikov A.G., Borzilov A.I., et. al.

AbstractThe antimicrobial resistance crisis along with challenges of antimicrobial discovery revealed the vital necessity to develop new antibiotics. Many of the animal proline-rich antimicrobial peptides (PrAMPs) inhibit the process of bacterial translation. Genome projects allowed to identify immune-related genes encoding animal host defense peptides. Here, using genome mining approach, we discovered a family of proline-rich cathelicidins, named rumicidins. The genes encoding these peptides are widespread among ruminant mammals. Biochemical studies indicated that rumicidins effectively inhibited the elongation stage of bacterial translation. The cryo-EM structure of the Escherichia coli 70S ribosome in complex with one of the representatives of the family revealed that the binding site of rumicidins span the ribosomal A-site cleft and the nascent peptide exit tunnel interacting with its constriction point by the conservative Trp23-Phe24 dyad. Bacterial resistance to rumicidins is mediated by knockout of the SbmA transporter or modification of the MacAB-TolC efflux pump. A wide spectrum of antibacterial activity, a high efficacy in the animal infection model, and lack of adverse effects towards human cells in vitro make rumicidins promising molecular scaffolds for development of ribosome-targeting antibiotics.

Tolicheva O.A., Bidzhieva M.S., Kasatskiy P.S., Marina V.I., Sergiev P.V., Konevega A.L., Paleskava A.

The development of simple, readily available, and sensitive methods for studying bacterial protein synthesis is an important fundamental and applied task. We recently demonstrated the possibility of visualizing short BODIPY-labeled phenylalanine- and leucine-containing peptides using urea-polyacrylamide gel electrophoresis under denaturing conditions. In this work, we expand the range of test sequences and included peptides with valine, lysine, and the modified amino acid εNH2-DOTA-lysine. The described method can also be used to study the mechanism of translation inhibition, as shown by elaboration of the specific action of the antibiotics etamycin A and viomycin.

Klementieva N.V., Lunev E.A., Shmidt A.A., Loseva E.M., Savchenko I.M., Svetlova E.A., Galkin I.I., Polikarpova A.V., Usachev E.V., Vassilieva S.G., Marina V.I., Dzhenkova M.A., Romanova A.D., Agutin A.V., Timakova A.A., et. al.

Marina V.I., Bidzhieva M., Tereshchenkov A.G., Orekhov D., Sagitova V.E., Sumbatyan N.V., Tashlitsky V.N., Ferberg A.S., Maviza T.P., Kasatsky P., Tolicheva O., Paleskava A., Polshakov V.I., Osterman I.A., Dontsova O.A., et. al.

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1 to 7 amino acids, BODIPY-labelled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labelled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

Pigareva V.A., Paltsev O.S., Marina V.I., Lukianov D.A., Moiseenko A.V., Shchelkunov N.M., Fedyanin A.A., Sybachin A.V.

Biocidal coatings are of great interest to the healthcare system. In this work, the biocidal activity of coatings based on a complex biocide containing polymer and inorganic active antibacterial components was studied. Silver oxide was distributed in a matrix of a positively charged interpolyelectrolyte complex (IPEC) of polydiallyldimethylammonium chloride (PDADMAC) and sodium polystyrene sulfonate (PSS) using ultrasonic dispersion, forming nanoparticles with an average size of 5–6 nm. The formed nanoparticles in the matrix are not subject to agglomeration and changes in morphology during storage. It was found that the inclusion of silver oxide in a positively charged IPEC allows a more than 4-fold increase in the effectiveness of the complex biocide against E. coli K12 in comparison with the biocidal effect of PDADMAC and IPEC. Polycation, IPEC, and the IPEC/Ag2O ternary complex form coatings on the glass surface due to electrostatic adsorption. Adhesive and cohesive forces in the resulting coatings were studied with micron-scale coatings using dynamometry. It was found that the stability of the coating is determined primarily by adhesive interactions. At the macro level, it is not possible to reliably identify the role of IPEC formation in adhesion. On the other hand, use of the optical tweezers method makes it possible to analyze macromolecules at the submicron scale and to evaluate the multiple increase in adhesive forces when forming a coating from IPEC compared to coatings from PDADMAC. Thus, the application of ternary IPEC/Ag2O complexes makes it possible to obtain coatings with increased antibacterial action and improved adhesive characteristics.

Pigareva V.A., Marina V.I., Bolshakova A.V., Berkovich A.K., Kuznetsova O.A., Semenova A.A., Yushina Y.K., Bataeva D.S., Grudistova M.A., Sybachin A.V.

Positively charged polyelectrolytes hold significant potential as materials for creating antibacterial coatings. We examined the physicochemical and mechanical properties of the macromolecules in water solutions and in coatings for the series of branched polyethyleneimine (PEI) and linear polydiallyldimethylammonium chloride (PDADMAC) with different molecular weights. The microbiological study was conducted to analyze the biocidal activity of the polycation solutions and coatings towards foodborne bacteria. While the moisture saturation of the polycationic coatings and biocidal activity did not significantly depend on the chemical nature of charged groups or the molecular weight or architecture of macromolecules, the lowering of the molecular weight of polymers resulted in the loss of cohesive forces in the coatings and to a dramatic loss of stability when being washed off with water. The diffusion coefficient (D0) of macromolecules was identified as a key parameter for the wash-off mechanism. Films formed by molecules with a D0 below 1 × 10−7 cm2/s demonstrated a high resistance to wash-off procedures. We demonstrated that PEI and PDADMAC samples with high molecular weights showed high antimicrobial activity towards L. monocytogenes. Our results highlight the importance of macromolecule characteristics in the development of new biocidal coatings based on polycations.

Pigareva V.A., Bol’shakova A.V., Marina V.I., Sybachin A.V.

Polycation-based coatings represent a promising class of protective antimicrobial coatings. Water-soluble complexes of poly(diallyldimethylammonium chloride) (PDADMAC) with sodium polyacrylate (PANa) have been studied by turbidimetry. It has been shown that the addition of the polyanion (12 mol %) to the polycation leads to the formation of an interpolyelectrolyte complex (IPEC) stable with respect to phase separation in water-salt media with salt concentrations as high as 0.1–0.2 M. In contrast to the traditional method of obtaining coatings from IPEC by layer-by-layer deposition, we have studied the preparation of the coatings directly from a solution of water-soluble IPEC on a hydrophilic glass surface and a surface of more hydrophobic polycarbonate. It has been found that the formation of IPEC makes it possible to increase the resistance of the coating to wash-off with water compared to the individual PDADMAC coating on both types of substrates.

Shirokikh I.G., Osterman I.A., Lukianov D.A., Marina V.I., Biryukov M.V., Belozerova O.A., Guglya E.B., Shirokikh A.A., Nazarova Y.I., Bokov N.A., Zakalyukina Y.V.

Pigareva V.A., Marina V.I., Sybachin A.V.

Biocidal compositions based on interpolyelectrolyte complexes and a low molecular weight antibiotic can become a promising material for creating biocidal coatings, as they combine wash-off resistance and dual biocidal action due to the biocide and the polycation. Molecular mass characteristics of polymers play an essential role in the physics and mechanical properties of the coatings. In this work, the properties of polydiallyldimethylammonium chloride (PDADMAC) coatings of various molecular weights are investigated and assumptions are made about the optimal molecular weight needed to create antibacterial compositions. To study the resistance to washing off and moisture saturation of the coatings, the gravimetric method was used, and the adhesive properties of the coatings were studied by dynamometry. It has been established that an increase in molecular weight affects the wash-off resistance of coatings, but does not affect moisture absorption and adhesion mechanics of coatings. All samples of PDADMAC were demonstrated to exhibit the same antibacterial activity. Thus, when developing systems for creating antibacterial coatings, it must be taken into account that in order to create stable coatings, the requirement to use PDADMAC with a high degree of polymerization is necessary for the coating desorption control during wash off-but not mandatory for the control of mechanical and antibacterial properties of the coating.

Paranjpe M., Marina V., Grachev A., Maviza T., Tolicheva O., Paleskava A., Osterman I., Sergiev P., Konevega A., Polikanov Y., Gagnon M.

Abstract Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.

Zakalyukina Y.V., Pavlov N.A., Lukianov D.A., Marina V.I., Belozerova O.A., Tashlitsky V.N., Guglya E.B., Osterman I.A., Biryukov M.V.

There are several well-studied examples of protective symbiosis between insect host and symbiotic actinobacteria, producing antimicrobial metabolites to inhibit host pathogens. These mutualistic relationships are best described for some wasps and leaf-cutting ants, while a huge variety of insect species still remain poorly explored. For the first time, we isolated actinobacteria from the harvester ant Messor structor and evaluated the isolates’ potential as antimicrobial producers. All isolates could be divided into two morphotypes of single and mycelial cells. We found that the most common mycelial morphotype was observed among soldiers and least common among larvae in the studied laboratory colony. The representative of this morphotype was identified as Streptomyces globisporus subsp. globisporus 4-3 by a polyphasic approach. It was established using a E. coli JW5503 pDualRep2 system that crude broths of mycelial isolates inhibited protein synthesis in reporter strains, but it did not disrupt the in vitro synthesis of proteins in cell-free extracts. An active compound was extracted, purified and identified as albomycin δ2. The pronounced ability of albomycin to inhibit the growth of entomopathogens suggests that Streptomyces globisporus subsp. globisporus may be involved in defensive symbiosis with the Messor structor ant against infections.

Pigareva V.A., Stepanova D.A., Bolshakova A.V., Marina V.I., Osterman I.A., Sybachin A.V.

Hyperbranched Kaustamin as a commercially available cationic flocculant adsorbs on a glass surface with formation of the polymer film. Kaustamin has antimicrobial effect on Gram-negative and Gram-positive bacteria, its film on glass keeps integrity and shape after absorption of water from air, which make the polymer appropriate as an antibacterial cover.

Lunev E.A., Shmidt A.A., Vassilieva S.G., Savchenko I.M., Loginov V.A., Marina V.I., Egorova T.V., Bardina M.V.

GNAO1 encephalopathy is an orphan genetic disease associated with early infantile epilepsy, impaired motor control, and severe developmental delay. The disorder is caused by mutations in the GNAO1 gene, leading to dysfunction of the encoded protein Gαo1. There is no cure for this disease, and symptomatic therapy is ineffective. Phenotypic heterogeneity highlights the need for a personalized approach for treating patients with a specific clinical variant of GNAO1 and requires the study of the disease mechanism in animal and cell models. Towards this aim, we developed an approach for modeling GNAO1 encephalopathy and testing gene therapy drugs in primary neurons derived from healthy mice. We optimized the delivery of transgenes to Gαo1-expressing neurons using recombinant adeno-associated viruses (rAAV). We assessed the tropism of five neurotropic AAV serotypes (1, 2, 6, 9, DJ) for Gαo1-positive neurons from the whole mouse brain. The DJ serotype showed the highest potential as a reporter delivery vehicle, infecting up to 66% of Gαo1-expressing cells without overt cytotoxicity. We demonstrated that AAV-DJ also provides efficient delivery and expression of genetic constructs encoding normal and mutant Gαo1, as well as short hairpin RNA (shRNA) to suppress endogenous Gnao1 in murine neurons. Our results will further simplify the study of the pathological mechanism for clinical variants of GNAO1, as well as optimize the testing of gene therapy approaches for GNAO1 encephalopathy in cell models.

Zhao Y., Xue Y., Wang C., Zhao Z., Cui R., Zhu B.

Shomer I., Mor N., Raviv S., Budick-Harmelin N., Matchevich T., Avkin-Nachum S., Rais Y., Haffner-Krausz R., Haimovich A., Ziv A., Fluss R., Ben-Ze'ev B., Heimer G., Silachev D.N., Katanaev V.L., et. al.

Linthorst N.A., van Vlijmen B.J., Eikenboom J.C.

Wang M., Li H.

Yadav M., Rawat S., Gupta S.

Nikandrova A.A., Petriakova A.D., Izzi A.R., Petrosyan G.A., Tashlitsky V.N., Alferova V.A., Panova T.V., Khrenova M.G., Biryukov M.V., Zakalyukina Y.V., Zvereva M.I., Lukianov D.A., Sergiev P.V.

Antibiotic resistance has been and remains a major problem in our society. The main solution to this problem is to search and study the mechanisms of antibiotic action. Many groups of secondary metabolites, including antimicrobial ones, are produced by the Actinomycetota phylum. The actinobacterial strains isolated from habitats that have not been well studied are of great interest. Due to high resource competition, antibiotics are now considered a ‘trump card in the game of life’ due to their presence in natural substrates with limited nutrients. Potentially, strains isolated from such habitats can be producers of novel or poorly studied antibiotics. In the current research, we identified the strain Streptomyces sp. AP22 from the soils of the Akhshatyrsky Gorge, which is capable of producing pentalenolactone. This study describes the phenotypic and morphological characteristics of Streptomyces sp. AP22 and its biological activity. Pentalenolactone is a known inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important enzyme involved in glycolysis. We identified a previously unknown mutation in the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase that confers resistance to this antibiotic compound. This antibiotic is not used in clinical practice, so its application as a selectable marker will not lead to the creation of pathogens resistant to clinically relevant antibiotics. In this case, the selectable marker is based on a genetic construct containing the glyceraldehyde-3-phosphate dehydrogenase gene with a resistance mutation. The use of this selectable marker can be applied to various genetic and molecular techniques, such as cloning and transformation. This can help to facilitate genetic and molecular biology studies of strains resistant to standard selectable markers such as kanamycin or ampicillin.

Stączek S., Kunat-Budzyńska M., Cytryńska M., Zdybicka-Barabas A.

Antimicrobial peptides (AMPs) constitute a large and diverse group of molecules with antibacterial, antifungal, antiviral, antiprotozoan, and anticancer activity. In animals, they are key components of innate immunity involved in fighting against various pathogens. Proline-rich (Pr) AMPs are characterized by a high content of proline (and arginine) residues that can be organized into Pro-Arg-Pro motifs. Such peptides have been described in many invertebrates (annelids, crustaceans, insects, mollusks) and some vertebrates (mammals). The main objective of this review is to present the diversity of invertebrate PrAMPs, which are associated with the presence of cysteine-rich domains or whey acidic protein domains in the molecular structure, in addition to the presence of characteristic proline-rich regions. Moreover, PrAMPs can target intracellular structures in bacteria, e.g., 70S ribosomes and/or heat shock protein DnaK, leading to the inhibition of protein synthesis and accumulation of misfolded polypeptides in the cell. This unique mechanism of action makes it difficult for pathogens to acquire resistance to this type of molecule. Invertebrate PrAMPs have become the basis for the development of new synthetic analogues effective in combating pathogens. Due to their great diversity, new highly active molecules are still being searched for among PrAMPs from invertebrates.

Kaale S.E., Machangu R.S., Lyimo T.J.

Nemati F., ata Bahmani Asl A., Salehi P.

Noscapine, a tetrahydroisoquinoline alkaloid, was first isolated from Papaver somniferum and identified by Rabiquet in 1817. It has been used as an anti-tussive agent since the mid-1950 s. After the discovery of its anti-mitotic potential, it was into the limelight once again. Due to its low toxicity, high bioactivity and oral administration, It was regarded as a formidable framework for subsequent modification and advancement in the pursuit of innovative chemotherapeutic agents. Up to now, the rational derivatives of the noscapine have been designed and the biological activities of these analogues have been extensively investigated. This review provides a comprehensive examination of the chemical characteristics of noscapine and its semi-synthetic derivatives up to the present, encompassing a concise investigation into the biological properties of these compounds and additionally a discussion about biosynthesis and total synthesis of noscapine is also provided. In summary, our aim is to contribute to a more thorough comprehension of this structure. It can be asserted that a promising future lies ahead for noscapine and its engineered derivatives as noteworthy candidates for pharmaceutical drugs.

Alferova V.A., Zotova P.A., Baranova A.A., Guglya E.B., Belozerova O.A., Pipiya S.O., Kudzhaev A.M., Logunov S.E., Prokopenko Y.A., Marenkova E.A., Marina V.I., Novikova E.A., Komarova E.S., Starodumova I.P., Bueva O.V., et. al.

Puromycin (Puro) is a natural aminonucleoside antibiotic that inhibits protein synthesis by its incorporation into elongating peptide chains. The unique mechanism of Puro finds diverse applications in molecular biology, including the selection of genetically engineered cell lines, in situ protein synthesis monitoring, and studying ribosome functions. However, the key step of Puro biosynthesis remains enigmatic. In this work, pur6-guided genome mining is carried out to explore the natural diversity of Puro-like antibiotics. The diversity of biosynthetic gene cluster (BGC) architectures suggests the existence of distinct structural analogs of puromycin encoded by pur-like clusters. Moreover, the presence of tRNACys in some BGCs, i.e., cst-like clusters, leads us to the hypothesis that Pur6 utilizes aminoacylated tRNA as an activated peptidyl precursor, resulting in cysteine-based analogs. Detailed metabolomic analysis of Streptomyces sp. VKM Ac-502 containing cst-like BGC revealed the production of a cysteinyl-based analog of Puro—cystocin (Cst). Similar to puromycin, cystocin inhibits both prokaryotic and eukaryotic translation by the same mechanism. Aminonucleoside N-acetyltransferase CstC inactivated Cst, mediating antibiotic resistance in genetically modified bacteria and human cells. The substrate specificity of CstC originated from the steric hindrance of its active site. We believe that novel aminonucleosides and their inactivating enzymes can be developed through the directed evolution of the discovered biosynthetic machinery.

Batool Z., Pavlova J.A., Paranjpe M.N., Tereshchenkov A.G., Lukianov D.A., Osterman I.A., Bogdanov A.A., Sumbatyan N.V., Polikanov Y.S.

Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis in vitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.

Babu S.K., Maharana S., Chhatria S., Sahoo D.R., Nanda A., Kanhar S., Behera P.K., Mohanty S., Naik P.K., Sahu P.K.

Objective: To evaluate the antimalarial activity of noscapine against Plasmodium falciparum 3D7 strain (Pf3D7), its clinical isolate (Pf140/SS), and Plasmodium berghei ANKA (PbA). Methods: Using ring-stage survival assay, phenotypic assessments, and SYBR-green-based fluorescence assay, the antimalarial activities of noscapine were assessed compared with dihydroartemisinin (DHA) in in vivo and in vitro studies. In addition, hemolysis and cytotoxicity tests were carried out to evaluate its safety. RT-PCR assay was also conducted to determine the effect of noscapine on papain-like cysteine protease Plasmodium falciparum falcipain-2 (PfFP-2). Results: The antimalarial efficacy of noscapine against Pf3D7 and Pf140/SS was comparable to DHA, with IC50 values of (7.68±0.88) and (5.57±0.74) nM/mL, respectively, and >95% inhibition of PbA infected rats. Noscapine also showed a safe profile, as evidenced by low hemolysis and cytotoxicity even at high concentrations. Moreover, PfFP-2 expression was significantly inhibited in both noscapine-treated Pf3D7 and Pf140/SS (P<0.01). Conclusions: Noscapine has antimalarial properties comparable to standard antimalarial DHA with better safety profiles, which may be further explored as a therapeutic candidate for the treatment of malaria.

Wei X., Zhao L., Wang Y., Wang F., Li C., Liu S., Zhang Q.

In China, substantial amounts of shrimp and crab shells are generated as waste every year. Improper disposal of these shells will damage marine and coastal ecosystems. Due to the low added value of using shrimp and crab shells to produce chitin and chitosan products, a novel approach involves the further degradation of chitin and chitosan. Enhancing the efficiency of enzymatic hydrolysis proves pivotal in advancing the industrialization of enzyme solutions. Trichoderma harzianum stands out because of its enzyme production property. Our lab constructed an induced cDNA library of Trichoderma harzianum and obtained a novel chitinase gene, Chit37. To explore the impact of the 5ˊ- and 3ˊ-untranslated regions (UTRs) on the characterization of the endochitinase Chit37, we conducted heterologous expression experiments of the full-length gene (Chit37-I) and the coding region of the gene (Chit37-II) in Saccharomyces cerevisiae by integrating into plasmid shuttle vector pYES2. Both expressed products were secreted into the medium and exhibited measurable activity. Comparatively, Chit37-II demonstrated higher activity levels than Chit37-I throughout. The optimal reaction temperature, pH, and substrate concentration of Chit37-I and Chit37-II were 40 ℃, 5.0, and 5%, respectively. However, the optimal culture time differed, with 108 h for Chit37-I and 96 h for Chit37-II. Our findings suggest that the UTRs affect the Chit37 transcript level, while its regulation mechanism remains to be elucidated. The results can provide a valuable reference for the efficient utilization of shrimp and crab shells.

Tolicheva O.A., Bidzhieva M.S., Kasatskiy P.S., Marina V.I., Sergiev P.V., Konevega A.L., Paleskava A.

The development of simple, readily available, and sensitive methods for studying bacterial protein synthesis is an important fundamental and applied task. We recently demonstrated the possibility of visualizing short BODIPY-labeled phenylalanine- and leucine-containing peptides using urea-polyacrylamide gel electrophoresis under denaturing conditions. In this work, we expand the range of test sequences and included peptides with valine, lysine, and the modified amino acid εNH2-DOTA-lysine. The described method can also be used to study the mechanism of translation inhibition, as shown by elaboration of the specific action of the antibiotics etamycin A and viomycin.

Belik A.R., Zakalyukina Y.V., Alferova V.A., Buyuklyan Y.A., Osterman I.A., Biryukov M.V.

In the search for new antibiotics, it is a common occurrence that already known molecules are “rediscovered” while new promising ones remain unnoticed. A possible solution to this problem may be the so-called “target-oriented” search, using special reporter microorganisms that combine increased antibiotic sensitivity with the ability to identify a molecule’s damaging effect. The use of such test organisms makes it possible to discover new promising properties even in known metabolites. In this study, we used a high-throughput screening method based on the pDualrep2 dual reporter system, which combines high sensitivity through the use of modified strains of test organisms and makes it possible to easily and accurately identify the interaction mechanisms of a substance and a bacterial cell at the initial stages of screening. This reporter system is unknown in Russia and is significantly superior to its global analogues. In the system, translation inhibition induces the expression of the fluorescent protein Katushka2s, while DNA damage is induced by TurboRFP. Using pDualrep2, we have isolated and described BV-204, an S. phaeochromogenes strain producing K-1115A, the biologically active substance that we have previously described. In our study, K-1115A for the first time has demonstrated antibiotic activity and an ability to inhibit bacterial translation, which was confirmed in vitro in a cell-free translation system for FLuc mRNA. K-1115A’s antibacterial activity was tested and confirmed for S. aureus (MRSA) and B. subtilis, its cytotoxicity measured against that for the HEK293 cell line. Its therapeutic index amounted to 2 and 8, respectively. The obtained results open up prospects for further study of K-1115A; so, this can be regarded as the basis for the production of semi-synthetic derivatives with improved therapeutic properties to be manufactured in dosage forms.

Abramson J., Adler J., Dunger J., Evans R., Green T., Pritzel A., Ronneberger O., Willmore L., Ballard A.J., Bambrick J., Bodenstein S.W., Evans D.A., Hung C., O’Neill M., Reiman D., et. al.

AbstractThe introduction of AlphaFold 21 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design2–6. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein–ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein–nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody–antigen prediction accuracy compared with AlphaFold-Multimer v.2.37,8. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.

Sergiev P., Kabilov M., Komarova E., Pichkur E., Zotova P., Kasatsky P., Volynkina I., Tupikin A., Pavlova J., Lukianov D., Osterman I., Pyshniy D., Paleskava A., Bogdanov A., Dontsova O., et. al.

Abstract Translation is a critical step in gene expression and a target for multiple antibiotics. Inhibition of protein synthesis by many antibiotics depends on mRNA context, making them selective towards particular mRNAs. Toe-printing is a standard low-throughput method to determine ribosome position along mRNA. Hereby we report the development and application of the Toe-seq method, combining toe-printing with next-generation sequencing. Toe-seq proved to be efficient to monitor translation and its inhibition by antibiotics for the library of over 37 000 mRNAs. Context-specificity of antibiotic action could be determined for different antibiotics via Toe-seq. Previously known sequence dependencies of translation inhibition were reproduced by Toe-seq. Data obtained by Toe-seq demonstrated context-specificity of etamycin A (EtaA) action whose binding site is located in the nascent peptide tunnel. Comparison of EtaA specificity with that of erythromycin (Ery) and tetracenomycin X (TcmX) revealed important differences and commonalities. Structural basis for the context-specificity of translation inhibition by EtaA has been deciphered by cryo-electron microscopy at 2.2 Å resolution. Toe-seq method allows one to monitor ribosome progression and stalling in vitro at a codon resolution, making it possible to assess antibiotics context specificity in a multiple parallel assay.

Marina V.I., Bidzhieva M., Tereshchenkov A.G., Orekhov D., Sagitova V.E., Sumbatyan N.V., Tashlitsky V.N., Ferberg A.S., Maviza T.P., Kasatsky P., Tolicheva O., Paleskava A., Polshakov V.I., Osterman I.A., Dontsova O.A., et. al.

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1 to 7 amino acids, BODIPY-labelled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labelled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

Akintola J., Chen Y., Digby Z.A., Schlenoff J.B.

Dhir S., Dutt R., Singh R.P., Chauhan M., Virmani T., Kumar G., Alhalmi A., Aleissa M.S., Rudayni H.A., Al-Zahrani M.

This research presents a straightforward, effective, and eco-friendly method for the production of silver nanoparticles (AgNPs) and copper oxide nanoparticles (CuONPs) using the dried fruit of Amomum subulatum as a reducing, stabilizing, and capping agent. The formation of AgNPs and CuONPs is supported by the presence of a surface plasmon resonance band (SPR) at 440 nm for AgNPs and 245 nm for CuONPs. Additionally, the identification of specific biomolecules responsible for the synthesis of AgNPs and CuONPs was confirmed through FTIR spectra analysis. The Transmission electron microscope (TEM) images demonstrated that AgNPs and CuONPs had spherical shapes, with mean particle diameters of 20.6 nm and 24.7 nm, respectively. X-ray diffraction and selected area electron diffraction (SAED) analyses provided evidence of the crystalline nature of the synthesized AgNPs and CuONPs. Additionally, the presence of silver and copper elements was observed through energy-dispersive X-ray spectroscopy (EDS) analysis. Furthermore, the antibacterial activity of AgNPs was found to be superior to that of CuONPs against human pathogens such as Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The cytotoxic activity of the biosynthesized nanoparticles (NPs) was evaluated in vitro against human cervical cells (HeLa) and human breast cells (MCF-7). In MCF-7 cells, the IC50 value for AgNPs was estimated to be 39.79 µg/mL, while that of CuONPs was 83.89 µg/mL. In HeLa cells, the IC50 value for AgNPs was 45.5 µg/mL, and for CuONPs, it was 97.07 µg/mL. For the first time, an eco-friendly method for the synthesis of AgNPs and CuONPs from fruit extract of Amomum subulatum has been discussed along with their comparative evaluation study. These results highlight the promising applications of the eco-friendly synthesized AgNPs and CuONPs as effective agents against microbial infections and potential candidates for cancer therapy.

Kaczor P., Bazan P., Kuciel S.

The aim of this study is to analyze the strength and antibacterial properties of composites based on structural polyoxymethylene. The base material was modified with the most used antibacterial additives, such as silver nanoparticles, copper oxide, zinc oxide, and titanium oxide. Basic strength and low-cycle fatigue tests were conducted to determine the dissipation energy of the material. The composites were also tested for antibacterial properties against two strains of bacteria: Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538. Strength properties showed no significant changes in the mechanical behavior of the tested composites against the matrix material. The best antibacterial additive was the addition of titanium oxide nanoparticles, providing 100% efficacy against Escherichia coli and almost 100% biocidal efficacy against Staphylococcus aureus. The other antibacterial additives showed biocidal efficacy of about 30–40% against the unmodified material. The added value of the work is the consistency in the methodology of testing materials modified with antibacterial additives, as well as the same compactness of the introduced additives. This study makes it clear which of the introduced additives has the highest biocidal activity.

Tiedje J.M., Fu Y., Mei Z., Schäffer A., Dou Q., Amelung W., Elsner M., Adu-Gyamfi J., Heng L., Virta M., Jiang X., Smidt H., Topp E., Wang F.

Imprudent use of antibiotics in the production of plants and animals raised for food contributes to the spread and evolution of antibiotic resistance. Additionally, the expansion of resistant bacteria and resistance genes among human-associated, animal-associated, and environmental microbiomes in food production systems break boundary barriers, further exacerbating the problem. Therefore, based on the understanding of the fate and dynamics of antibiotic resistance in food production systems, this paper adheres to the One Health framework to support collaborative efforts across multiple sectors to apply preventive measures (e.g., heightening awareness of antibiotic resistance and its confounding factors, strengthening environmental governance and regulatory frameworks, and constructing integrated food production surveillance systems) for the mitigation of antibiotic resistance. This review provides updated information on the sources and transmission of antibiotic resistance in agri-food production and proposes some strategies to alleviate the human burden of antibiotic resistance.

Yılmaz G.E., Göktürk I., Ovezova M., Yılmaz F., Kılıç S., Denizli A.

Microbial colonization on various surfaces is a serious problem. Biofilms from these microbes pose serious health and economic threats. In addition, the recent global pandemic has also attracted great interest in the latest techniques and technology for antimicrobial surface coatings. Incorporating antimicrobial nanocompounds into materials to prevent microbial adhesion or kill microorganisms has become an increasingly challenging strategy. Recently, many studies have been conducted on the preparation of nanomaterials with antimicrobial properties against diseases caused by pathogens. Despite tremendous efforts to produce antibacterial materials, there is little systematic research on antimicrobial coatings. In this article, we set out to provide a comprehensive overview of nanomaterials-based antimicrobial coatings that can be used to stop the spread of contamination to surfaces. Typically, surfaces can be simple deposits of nanomaterials, embedded nanomaterials, as well as nanotubes, nanowires, nanocolumns, nanofibers, nanoneedles, and bio-inspired structures.

de Carvalho G.R., Kudaka A.M., Netto R.A., Delarmelina C., Duarte M.C., Lona L.M.

Pathogen agents, such as bacteria and virus, can contaminate plastic surfaces, particularly those used in food packaging. This study proposed to prepare a polyelectrolyte film with antiviral and antibacterial activity based on sodium alginate (SA) and poly(diallyldimethylammonium chloride) (PDADMAC), a cationic polymer with sanitizing properties. In addition, the physicochemical properties of the polyelectrolyte films were also evaluated. The polyelectrolyte films showed continuous, compact, and crack-free structures. The FTIR analysis confirmed the ionic interaction between SA and PDADMAC. Adding PDADMAC significantly affected the mechanical properties of the films (p < 0.05), increasing the maximum tensile strength (from 8.66 ± 1.55 MPa to 18.1 ± 1.77 MPa). However, polyelectrolyte films showed higher water vapor permeability values due to the strong hydrophilicity of PDADMAC, representing a 43 % average increase compared with the control film. Also, thermal stability improved with the incorporation of PDADMAC. The selected polyelectrolyte film inactivated 99.8 % of SARS-CoV-2 after 1 min in direct contact with the virus, in addition to having an inhibitory effect against Staphylococcus aureus and Escherichia coli bacteria. Therefore, this study demonstrated the efficacy of using PDADMAC in the preparation of polyelectrolyte sodium alginate-based films with improvements in physicochemical properties and especially with antiviral activity against SARS-CoV-2.

Wang L., Xin M., Li M., Liu W., Mao Y.

N-(4-N′, N′, N′-trimethylphosphonium chloride) benzoyl chitosan (TMPCS), N-(4-N′, N′, N′-triphenylphosphonium chloride) benzoyl chitosan (TPPCS), and N-(4-N′, N′, N′-trimethylmethanaminium chloride) benzoyl chitosan (TMACS) were synthesized. The structures of the products were characterized by Fourier transform infrared spectroscopy, Nuclear magnetic resonance spectroscopy and ultraviolet-visible spectroscopy. Their antibacterial activities against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were investigated in vitro using the antibacterial rate, minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the antibiofilm activity was investigated by crystal violet assay. The antibacterial assessment revealed that the chitosan quaternary phosphonium salts of similar structure had superior antibacterial activity than chitosan quaternary ammonium salt. The antibacterial rate of CS, TMPCS, TPPCS and TMACS against E. coli at 0.5 mg/mL was 10.4 %, 42.0 %, 58.5 % and 21.6 % respectively. At the same concentration, the antibacterial rate of TMPCS, TPPCS and TMACS against S.aureus was all up to 100 %. The biofilm inhibition rate of CS, TMPCS, TPPCS and TMACS at a half of MIC against E.coli was 28.4 %, 33.9 %, 56.6 % and 57.6 % respectively, and against S.aureus was 30.8 %, 53.8 %, 62.2 % and 58.5 % respectively. The biofilm removal rate of CS, TMPCS, TPPCS, TMACS against E.coli at 2.5 mg/mL was 20.6 %, 46.4 %, 48.9 % and 41.6 % respectively, and against S.aureus at 2.5 mg/mL was 41.5 %, 60.4 %, 69.9 % and 59.01 % respectively.

Hidayat M.I., Adlim M., Suhartono S., Hayati Z., Bakar N.H.

Most preparation of both silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) was relatively complicated. We proved a more straightforward method not only in the nanoparticle synthesis but also in their immobilization on the solid support. Since both the metal nanoparticles have antibacterial activities, comparing the efficacy and the preparation cost is still interesting to explore. TEM images show both colloidal chitosan stabilized-Ag NPs and ZnO NPs have dispersed and nano-size particles. The immobilized Ag NPs and ZnO NPs on white silica beads (SiG chi-Ag NPs and SiG chi-ZnO NPs) were successfully fabricated with appropriate techniques and suitable characteristics for antibacterial air filter materials. The colloidal and the immobilized metal NPs have high antibacterial properties against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. The inhibition zone diameter of SiG chi-ZnO NPs and SiG chi-Ag NPs against S. aureus and E. coli were relatively similar but needed about ten times more weight zinc than silver to kill the bacteria at a similar inhibition zone size. Silver is ten times more expensive than zinc, but it is relatively simpler to prepare SiG chi-Ag NPs than to formulate SiG chi-ZnO NPs. The cytotoxicity of silver is more than ten times higher compared to zinc cytotoxicity. Therefore, it was reasonable to substitute silver with zinc nanoparticles for antibacterial air filter material.

Pigareva V.A., Marina V.I., Bolshakova A.V., Berkovich A.K., Kuznetsova O.A., Semenova A.A., Yushina Y.K., Bataeva D.S., Grudistova M.A., Sybachin A.V.

Positively charged polyelectrolytes hold significant potential as materials for creating antibacterial coatings. We examined the physicochemical and mechanical properties of the macromolecules in water solutions and in coatings for the series of branched polyethyleneimine (PEI) and linear polydiallyldimethylammonium chloride (PDADMAC) with different molecular weights. The microbiological study was conducted to analyze the biocidal activity of the polycation solutions and coatings towards foodborne bacteria. While the moisture saturation of the polycationic coatings and biocidal activity did not significantly depend on the chemical nature of charged groups or the molecular weight or architecture of macromolecules, the lowering of the molecular weight of polymers resulted in the loss of cohesive forces in the coatings and to a dramatic loss of stability when being washed off with water. The diffusion coefficient (D0) of macromolecules was identified as a key parameter for the wash-off mechanism. Films formed by molecules with a D0 below 1 × 10−7 cm2/s demonstrated a high resistance to wash-off procedures. We demonstrated that PEI and PDADMAC samples with high molecular weights showed high antimicrobial activity towards L. monocytogenes. Our results highlight the importance of macromolecule characteristics in the development of new biocidal coatings based on polycations.

Pigareva V.A., Bol’shakova A.V., Marina V.I., Sybachin A.V.

Polycation-based coatings represent a promising class of protective antimicrobial coatings. Water-soluble complexes of poly(diallyldimethylammonium chloride) (PDADMAC) with sodium polyacrylate (PANa) have been studied by turbidimetry. It has been shown that the addition of the polyanion (12 mol %) to the polycation leads to the formation of an interpolyelectrolyte complex (IPEC) stable with respect to phase separation in water-salt media with salt concentrations as high as 0.1–0.2 M. In contrast to the traditional method of obtaining coatings from IPEC by layer-by-layer deposition, we have studied the preparation of the coatings directly from a solution of water-soluble IPEC on a hydrophilic glass surface and a surface of more hydrophobic polycarbonate. It has been found that the formation of IPEC makes it possible to increase the resistance of the coating to wash-off with water compared to the individual PDADMAC coating on both types of substrates.

Novelli M., Galosi S., Zorzi G., Martinelli S., Capuano A., Nardecchia F., Granata T., Pollini L., Di Rocco M., Marras C.E., Nardocci N., Leuzzi V.

To evaluate clinical phenotype and molecular findings of 157 cases with GNAO1 pathogenic or likely pathogenic variants delineating the clinical spectrum, course, and response to treatments.Clinical phenotype, genetic data, and pharmacological and surgical treatment history of 11 novel cases and 146 previously published patients were analyzed.Complex hyperkinetic movement disorder (MD) characterizes 88% of GNAO1 patients. Severe hypotonia and prominent disturbance of postural control seem to be hallmarks in the early stages preceding the hyperkinetic MD. In a subgroup of patients, paroxysmal exacerbations became so severe as to require admission to intensive care units (ICU). Almost all patients had a good response to deep brain stimulation (DBS). Milder phenotypes with late-onset focal/segmental dystonia, mild to moderate intellectual disability, and other minor neurological signs (i.e., parkinsonism and myoclonus) are emerging. MRI, previously considered noncontributory to a diagnosis, can show recurrent findings (i.e., cerebral atrophy, myelination and/or basal ganglia abnormalities). Fifty-eight GNAO1 pathogenic variants, including missense changes and a few recurrent splice site defects, have been reported. Substitutions at residues Gly203, Arg209 and Glu246, together with the intronic c.724-8G > A change, account for more than 50% of cases.Infantile or childhood-onset complex hyperkinetic MD (chorea and/or dystonia) with or without paroxysmal exacerbations, associated hypotonia, and developmental disorders should prompt research for GNAO1 mutations. DBS effectively controls and prevents severe exacerbations and should be considered early in patients with specific GNAO1 variants and refractory MD. Prospective and natural history studies are necessary to define genotype-phenotype correlations further and clarify neurological outcomes.

Sybachin A., Pigareva V.

The increase of payload is one of the key tasks in creation of nanocontainers for the delivery of bioactive substances (BAS). In this work the adsorption of anionic carboxymethyl cyclodextrins (CMCDs) on the surface of cationic liposomes was studied as mechanism of formation of capacious nanocontainers for the encapsulation and delivery of hydrophobic BAS. The formation and physico-chemical characteristics of complexes were studied by means of laser microelectrophoresis, dynamic light-scattering, conductometry and atomic force microscopy (AFM). As a model, bioactive molecule hydrophobic curcumin was chosen for the investigation. The encapsulation of curcumin was controlled by UV-Vis spectrometry. Interaction of CMCDs/liposomes complexes with model cell membranes was visualized by fluorescent microscopy. Finally, cytotoxicity of nanocontainers was studied by MTT-test. It was estimated that colloid stable complexes with net positive charge could contain up to 2.5÷5 CMCD molecules per one cationic lipid. Incorporation of curcumin in CMCDs does not change the character of interaction of oligosaccharides with liposomal membranes of individual liposome. CMCDs/liposomes complexes adsorb on model cell membranes without significant loss of CMCD molecules. This fact in addition to low cytotoxicity of cationic CMCDs/liposomes complexes demonstrates potential of their application as nanovehicles for the delivery of BAS.