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To date, most research on amyloid aggregation has focused on describing the structure of amyloids and the kinetics of their formation, while the conformational stability of fibrils remains insufficiently explored. The aim of this work was to investigate the effect of amino acid substitutions on the stability of apomyoglobin (ApoMb) amyloids. A study of the amyloid unfolding of ApoMb and its six mutant variants by urea has been carried out. Changes in the structural features of aggregates during unfolding were recorded by far-UV CD and native electrophoresis. It was shown that during the initial stage of denaturation, amyloids' secondary structure partially unfolds. Then, the fibrils undergo dissociation and form intermediate aggregates weighing approximately 1 MDa, which at the last stage of unfolding decompose into 18 kDa monomeric unfolded molecules. The results of unfolding transitions suggest that the stability of the studied amyloids relative to the intermediate aggregates and of the latter relative to unfolded monomers is higher for ApoMb variants with substitutions that increase the hydrophobicity of the residues. The results presented provide a new insight into the mechanism of stabilization of protein aggregates and can serve as a base for further investigations of the amyloids' stability. [ABSTRACT FROM AUTHOR]

Amyloid formation is linked with serious human diseases that are currently incurable. Usually, in the study of amyloid aggregation, the description of the protein's association is in focus. Whereas the mechanism of the cross-β-structure formation, and the presence of aggregation reversibility, remain insufficiently explored. In this work, the kinetics of amyloid aggregation of apomyoglobin (ApoMb) have been studied using thioflavin fluorescence, electron microscopy, and non-denaturing electrophoresis. An analysis of the concentration dependence of the aggregation rates allows the conclusion that ApoMb amyloid formation includes the stages of conformational rearrangements in the aggregates, followed by their association and the fibril formation. The study of the mutant variants aggregation kinetics showed that the association rate is determined by the amino acids' hydrophobicity, while the rate of conformational rearrangements is affected by the localization of the substitution. An unexpected result was the discovery that ApoMb amyloid formation is reversible, and under native-like conditions, the amyloid can dissociate, producing monomers. A consequence of the reversibility of amyloid aggregation is the presence of the monomer after aggregation completion. Since the aggregation reversibility indicates the possibility of dissociation of already formed fibrils, presented data and approaches can be useful in finding ways for amyloid diseases treatment. [ABSTRACT FROM AUTHOR]

Bacteriophages are widely recognized as alternatives to traditional antibiotics commonly used in the treatment of bacterial infection diseases and in the food industry, as phages offer a potential solution in combating multidrug-resistant bacterial pathogens. In this study, we describe a novel bacteriophage, Kirovirus kirovense Kirov, which infects members of the Bacillus cereus group. Kirovirus kirovense Kirov is a broad-host-range phage belonging to the Caudoviricetes class. Its chromosome is a linear 165,667 bp double-stranded DNA molecule that contains two short, direct terminal repeats, each 284 bp long. According to bioinformatics predictions, the genomic DNA contains 275 protein-coding genes and 5 tRNA genes. A comparative genomic analysis suggests that Kirovirus kirovense Kirov is a novel species within the Kirovirus genus, belonging to the Andregratiavirinae subfamily. Kirovirus kirovense Kirov demonstrates the ability to preserve and decontaminate B. cereus from cow milk when present in milk at a concentration of 104 PFU/mL. After 4 h of incubation with the phage, the bacterial titer drops from 105 to less than 102 CFU/mL. [ABSTRACT FROM AUTHOR]

The number of infections caused by antibiotic-resistant strains of bacteria is growing by the year. The pathogenic bacterial species Enterococcus faecalis and Enterococcus faecium are among the high priority candidate targets for the development of new therapeutic antibacterial agents. One of the most promising antibacterial agents are bacteriophages. According to the WHO, two phage-based therapeutic cocktails and two medical drugs based on phage endolysins are currently undergoing clinical trials. In this paper, we describe the virulent bacteriophage iF6 and the properties of two of its endolysins. The chromosome of the iF6 phage is 156,592 bp long and contains two direct terminal repeats, each 2108 bp long. Phylogenetically, iF6 belongs to the Schiekvirus genus, whose representatives are described as phages with a high therapeutic potential. The phage demonstrated a high adsorption rate; about 90% of iF6 virions attached to the host cells within one minute after the phage was added. Two iF6 endolysins were able to lyse enterococci cultures in both logarithmic and stationary growth phases. Especially promising is the HU-Gp84 endolysin; it was active against 77% of enterococci strains tested and remained active even after 1 h incubation at 60 °C. Thus, iF6-like enterococci phages appear to be a promising platform for the selection and development of new candidates for phage therapy. [ABSTRACT FROM AUTHOR]

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES. [ABSTRACT FROM AUTHOR]

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A). [ABSTRACT FROM AUTHOR]

This study describes two novel bacteriophages infecting members of the Bacillus pumilus group. Even though members of the group are not recognized as pathogenic, several strains belonging to the group have been reported to cause infectious diseases in plants, animals and humans. Bacillus pumilus group species are highly resistant to ultraviolet radiation and capable of forming biofilms, which complicates their eradication. Bacteriophages Novomoskovsk and Bolokhovo were isolated from soil samples. Genome sequencing and phylogenetic analysis revealed that the phages represent two new species of the genus Andromedavirus (class Caudoviricetes). The phages remained stable in a wide range of temperatures and pH values. A host range test showed that the phages specifically infect various strains of B. pumilus. The phages form clear plaques surrounded by halos. Both phages Novomoskovsk and Bolokhovo encode proteins with pectin lyase domains—Putative depolymerases. Obtained in a purified recombinant form, the proteins produced lysis zones on the lawn of a B. pumilus strain. This suggests that Novomoskovsk and Bolokhovo may be effective for the eradication of B. pumilus biofilms. [ABSTRACT FROM AUTHOR]

The tribomechanical properties of the wear-resistant ultrahigh molecular weight polyethylene (UHMWPE)-based composites loaded with wollastonite microfibres silanized with various coupling agents ("KH-550", "Penta-1006", and "OTS") were investigated. It was demonstrated that the mechanical properties of UHMWPE-based composites filled with various amounts of wollastonite (7–23 wt. %) increased by 1.3 times (yield strength) and by 1.8 times (elastic modulus), while the wollastonite silanization further improved yield strength by 9% in some cases. It was demonstrated that the composite loaded with 23 wt. % wollastonite silanized with the "KH-550" coupling agent possessed the maximum wear resistance under "moderate" conditions of tribological loading. Under "severe" conditions, the composites containing 23 wt. % wollastonite silanized with the less efficient "OTS" and "Penta-1006" agents showed the greatest wear resistance during dry sliding friction. Wear resistance significantly depended on filler weight fraction and the load–speed mode of the tribological tests. Based on the obtained experimental data on the mechanical (including impact toughness) and tribological properties of the UHMWPE-based composites loaded with wollastonite, the optimal compositions (the filler content and the type of the coupling agent) for two load–speed modes were designed using the developed computer algorithm. The composites provided the predefined high tribomechanical properties for operation in the metal-polymer friction units compared to neat polymer. [ABSTRACT FROM AUTHOR]

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins' aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding β-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions. [ABSTRACT FROM AUTHOR]