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The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.
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Ceftriaxona , Simulación de Dinámica Molecular , Neisseria gonorrhoeae , Proteínas de Unión a las Penicilinas , Ceftriaxona/farmacología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/metabolismo , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/metabolismo , Antibacterianos/farmacología , Mutación , Farmacorresistencia Bacteriana/genética , Acilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , D-Ala-D-Ala Carboxipeptidasa de Tipo SerinaRESUMEN
Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.
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COVID-19 , Ensayos Analíticos de Alto Rendimiento , Biblioteca de Péptidos , SARS-CoV-2 , Humanos , SARS-CoV-2/inmunología , COVID-19/inmunología , COVID-19/virología , Ensayos Analíticos de Alto Rendimiento/métodos , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Inmunoglobulinas/inmunología , Inmunoglobulinas/genética , Anticuerpos Antivirales/inmunología , Mapeo Epitopo/métodosRESUMEN
Manganese complexes [(arene)Mn(CO)3]+ were prepared in one step from arenes and Mn(CO)5Br. They were found to be efficient catalysts in the carbonyl cyanation with TMSCN, CO2 fixation by epoxides, and direct reductive amination in the presence of syngas. The amination reaction tolerated various reducible functional groups. The synergy of carbon monoxide and hydrogen in syngas provides high efficiency of the catalytic system. The developed protocols do not require an inert atmosphere, and the catalysts can be handled in air.
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Heterosynaptic plasticity, along with Hebbian homosynaptic plasticity, is an important mechanism ensuring the stable operation of learning neuronal networks. However, whether heterosynaptic plasticity occurs in the whole brain in vivo, and what role(s) in brain function in vivo it could play, remains unclear. Here, we used an optogenetics approach to apply a model of intracellular tetanization, which was established and employed to study heterosynaptic plasticity in brain slices, to study the plasticity of response properties of neurons in the mouse visual cortex in vivo. We show that optogenetically evoked high-frequency bursts of action potentials (optogenetic tetanization) in the principal neurons of the visual cortex induce long-term changes in the responses to visual stimuli. Optogenetic tetanization had distinct effects on responses to different stimuli, as follows: responses to optimal and orthogonal orientations decreased, responses to null direction did not change, and responses to oblique orientations increased. As a result, direction selectivity of the neurons decreased and orientation tuning became broader. Since optogenetic tetanization was a postsynaptic protocol, applied in the absence of sensory stimulation, and, thus, without association of presynaptic activity with bursts of action potentials, the observed changes were mediated by mechanisms of heterosynaptic plasticity. We conclude that heterosynaptic plasticity can be induced in vivo and propose that it may play important homeostatic roles in operation of neural networks by helping to prevent runaway dynamics of responses to visual stimuli and to keep the tuning of neuronal responses within the range optimized for the encoding of multiple features in population activity.
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We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.
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Antibacterianos , Calcio , Bacterias Grampositivas , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Calcio/metabolismo , Estructura Molecular , Bacterias Grampositivas/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Daptomicina/farmacología , Daptomicina/química , Membrana Dobles de Lípidos/química , MicelasRESUMEN
Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.
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Neoplasias Encefálicas , Glioblastoma , Modelos Biológicos , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Epigenómica , Genómica , Glioblastoma/genética , Glioblastoma/patología , Análisis de la Célula Individual , Microambiente Tumoral , Heterogeneidad GenéticaRESUMEN
BACKGROUND: The TERT promoter mutation (TPM) is acquired in most IDH-wildtype glioblastomas (GBM) and IDH-mutant oligodendrogliomas (OD) enabling tumor cell immortality. Previous studies on TPM clonality show conflicting results. This study was performed to determine whether TPM is clonal on a tumor-wide scale. METHODS: We investigated TPM clonality in relation to presumed early events in 19 IDH-wildtype GBM and 10 IDH-mutant OD using 3-dimensional comprehensive tumor sampling. We performed Sanger sequencing on 264 tumor samples and deep amplicon sequencing on 187 tumor samples. We obtained tumor purity and copy number estimates from whole exome sequencing. TERT expression was assessed by RNA-seq and RNAscope. RESULTS: We detected TPM in 100% of tumor samples with quantifiable tumor purity (219 samples). Variant allele frequencies (VAF) of TPM correlate positively with chromosome 10 loss in GBM (Râ =â 0.85), IDH1 mutation in OD (Râ =â 0.87), and with tumor purity (Râ =â 0.91 for GBM; Râ =â 0.90 for OD). In comparison, oncogene amplification was tumor-wide for MDM4- and most EGFR-amplified cases but heterogeneous for MYCN and PDGFRA, and strikingly high in low-purity samples. TPM VAF was moderately correlated with TERT expression (Râ =â 0.52 for GBM; Râ =â 0.65 for OD). TERT expression was detected in a subset of cells, solely in TPM-positive samples, including samples equivocal for tumor. CONCLUSIONS: On a tumor-wide scale, TPM is among the earliest events in glioma evolution. Intercellular heterogeneity of TERT expression, however, suggests dynamic regulation during tumor growth. TERT expression may be a tumor cell-specific biomarker.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Oligodendroglioma , Telomerasa , Humanos , Neoplasias Encefálicas/patología , Glioma/patología , Glioblastoma/genética , Glioblastoma/patología , Oligodendroglioma/genética , Mutación , Biomarcadores de Tumor/genética , Isocitrato Deshidrogenasa/genética , Telomerasa/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Ciclo Celular/genéticaRESUMEN
The global spread of antibiotic resistance marks the end of the era of conventional antibiotics. Mankind desires new molecular tools to fight pathogenic bacteria. In this regard, the development of new antimicrobials based on antimicrobial peptides (AMPs) is again of particular interest. AMPs have various mechanisms of action on bacterial cells. Moreover, AMPs have been reported to be efficient in preclinical studies, demonstrating a low level of resistance formation. Thanatin is a small, beta-hairpin antimicrobial peptide with a bacterial-specific mode of action, predetermining its low cytotoxicity toward eukaryotic cells. This makes thanatin an exceptional candidate for new antibiotic development. Here, a microorganism was bioengineered to produce an antimicrobial agent, providing novel opportunities in antibiotic research through the directed creation of biocontrol agents. The constitutive heterologous production of recombinant thanatin (rThan) in the yeast Pichia pastoris endows the latter with antibacterial properties. Optimized expression and purification conditions enable a high production level, yielding up to 20 mg/L of rThan from the culture medium. rThan shows a wide spectrum of activity against pathogenic bacteria, similarly to its chemically synthesized analogue. The designed approach provides new avenues for AMP engineering and creating live biocontrol agents to fight antibiotic resistance.
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The commensal microflora provides a repertoire of antigens that illicit mucosal antibodies. In some cases, these antibodies can cross-react with host proteins, inducing autoimmunity, or with other microbial antigens. We demonstrate that the oral microbiota can induce salivary anti-SARS-CoV-2 Spike IgG antibodies via molecular mimicry. Anti-Spike IgG antibodies in the saliva correlated with enhanced abundance of Streptococcus salivarius 1 month after anti-SARS-CoV-2 vaccination. Several human commensal bacteria, including S. salivarius, were recognized by SARS-CoV-2-neutralizing monoclonal antibodies and induced cross-reactive anti-Spike antibodies in mice, facilitating SARS-CoV-2 clearance. A specific S. salivarius protein, RSSL-01370, contains regions with homology to the Spike receptor-binding domain, and immunization of mice with RSSL-01370 elicited anti-Spike IgG antibodies in the serum. Additionally, oral S. salivarius supplementation enhanced salivary anti-Spike antibodies in vaccinated individuals. Altogether, these data show that distinct species of the human microbiota can express molecular mimics of SARS-CoV-2 Spike protein, potentially enhancing protective immunity.
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COVID-19 , Microbiota , Humanos , Animales , Ratones , Glicoproteína de la Espiga del Coronavirus , Formación de Anticuerpos , Imitación Molecular , SARS-CoV-2 , Anticuerpos Monoclonales , Anticuerpos Antivirales , Inmunoglobulina A Secretora , Inmunoglobulina G , Anticuerpos NeutralizantesRESUMEN
Using the methods of scanning and transmission electron microscopy, the features of the structural-phase state of a vanadium alloy of the V-Cr-Ta-Zr system after a combined treatment, which consisted in cyclic alternation of thermomechanical and chemical-heat treatments, were studied. The values of yield strength and ductility of the V-Cr-Ta-Zr alloy were determined, depending on the stabilization and test temperatures. It was established that, after the combined treatment, the structural-phase state of the V-Cr-Ta-Zr alloy was composite, in which the joint implementation of dispersion and substructural strengthening ensured the formation of a gradient grain structure with a polygonal state, the elements of which were fixed by nanosized ZrO2 particles characterized by a high thermal stability. Such modification of the microstructure was accompanied by an increase in the high-temperature strength and a shift in the upper limit of the temperature stability interval towards high temperatures, of up to 900 °C. It was assumed that the polygonal state inside the grains contributed to the implementation of cooperative mechanisms of the dislocation-disclination type, which ensured the accommodation of the material in the "high-strength state" under loading.
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Synaptic plasticity is currently considered the main mechanism underlying the plastic modification of neural networks. The vast majority of studies of synaptic plasticity are carried out on reduced preparations, but the situation in vivo is fundamentally different from that in vitro. In this work, we used the Hebbian paradigm, which is known to induce long-term changes in synaptic strength in vitro, to manipulate the properties of a single pyramidal neuron in the mouse visual cortex. We have shown that optogenetic stimulation of a ChR2-expressing pyramidal neuron in the primary visual cortex of Thy-ChR2 mice paired with the presentation of a visual stimulus of non-optimal orientation induces long-term changes in the properties of the receptive field, manifested in alteration of the orientation selectivity of the cell. Non-paired stimulation did not lead to changes in the properties of the receptive field of the neuron during the experiment. Thus, we have demonstrated the role of associative plasticity in the dynamic organization of the receptive fields of neurons in the visual cortex.
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Optogenética , Corteza Visual , Ratones , Animales , Estimulación Luminosa , Plasticidad Neuronal/fisiología , Neuronas/fisiologíaRESUMEN
Natural compounds continue to serve as the most fruitful source of new antimicrobials. Analysis of bacterial genomes have revealed that the biosynthetic potential of antibiotic producers by far exceeds the number of already discovered structures. However, due to the repeated discovery of known substances, it has become necessary to change both approaches to the search for antibiotics and the sources of producer strains. The pressure of natural selection and the diversity of interactions in symbiotic communities make animal microbiomes promising sources of novel substances. Here, microorganisms associated with various animals were examined in terms of their antimicrobial agents. The application of alternative cultivation techniques, ultrahigh-throughput screening, and genomic analysis facilitated the investigation of compounds produced by unique representatives of the animal microbiota. We believe that new strategies of antipathogen defense will be discovered by precisely studying cell-cell and host-microbe interactions in microbiomes in the wild.
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Antibacterianos , Microbiota , Animales , Antibacterianos/farmacología , Frutas , Genoma Bacteriano , GenómicaRESUMEN
Variants of SARS-CoV-2 keep emerging and causing new waves of COVID-19 around the world. Effective new approaches in drug development are based on the binding of agents, such as neutralizing monoclonal antibodies to a receptor-binding domain (RBD) of SARS-CoV-2 spike protein. However, mutations in RBD may lower the affinity of previously developed antibodies. Therefore, rapid analysis of new variants and selection of a binding partner with high affinity is of great therapeutic importance. Here, we explore a computational approach based on molecular dynamics simulations and conformational clusterization techniques for the wild-type and omicron variants of RBD. Biochemical experiments support the hypothesis of the presence of several conformational states within the RBD assembly. The development of such an approach will facilitate the selection of neutralization drugs with higher affinity based on the primary structure of the target antigen.
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ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.
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Proteasa La , Proteasas ATP-Dependientes/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Péptido Hidrolasas/metabolismo , Proteasa La/genética , Proteasa La/metabolismo , Proteoma/metabolismoRESUMEN
The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.
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Animales Salvajes/microbiología , Antibacterianos/administración & dosificación , Bacillus pumilus/química , Escherichia coli/crecimiento & desarrollo , Microbiota , Probióticos/administración & dosificación , Staphylococcus aureus/crecimiento & desarrollo , Animales , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/efectos de los fármacos , Genoma Bacteriano , Metaboloma , Familia de Multigenes , Probióticos/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacosRESUMEN
The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre-steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre-steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.
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Gram-negative pathogens represent an urgent threat due to their intrinsic and acquired antibiotic resistance. Many recent drug candidates display prominent antimicrobial activity against Gram-positive bacteria being inefficient against Gram-negative pathogens. Ultrahigh-throughput, microfluidics-based screening techniques represent a new paradigm for deep profiling of antibacterial activity and antibiotic discovery. A key stage of this technology is based on single-cell cocultivation of microbiome biodiversity together with reporter fluorescent pathogen in emulsion, followed by the selection of reporter-free droplets using fluorescence-activated cell sorting. Here, a panel of reporter strains of Gram-negative bacteria Escherichia coli was developed to provide live biosensors for precise monitoring of antimicrobial activity. We optimized cell morphology, fluorescent protein, and selected the most efficient promoters for stable, homogeneous, high-level production of green fluorescent protein (GFP) in E. coli. Two alternative strategies based on highly efficient constitutive promoter pJ23119 or T7 promoter leakage enabled sensitive fluorescent detection of bacterial growth and killing. The developed live biosensors were applied for isolating potent E. coli-killing Paenibacillus polymyxa P4 strain by the ultrahigh-throughput screening of soil microbiome. The multi-omics approach revealed antibiotic colistin (polymyxin E) and its biosynthetic gene cluster, mediating antibiotic activity. Live biosensors may be efficiently implemented for antibiotic/probiotic discovery, environmental monitoring, and synthetic biology.
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The breastfeeding of infants by mothers who are infected with SARS-CoV-2 has become a dramatic healthcare problem. The WHO recommends that infected women should not abandon breastfeeding; however, there is still the risk of contact transmission. Convalescent donor milk may provide a defense against the aforementioned issue and can eliminate the consequences of artificial feeding. Therefore, it is vital to characterize the epitope-specific immunological landscape of human milk from women who recovered from COVID-19. We carried out a comprehensive ELISA-based analysis of blood serum and human milk from maternity patients who had recovered from COVID-19 at different trimesters of pregnancy. It was found that patients predominantly contained SARS-CoV-2 N-protein-specific immunoglobulins and had manifested the antibodies for all the antigens tested in a protein-specific and time-dependent manner. Women who recovered from COVID-19 at trimester I-II showed a noticeable decrease in the number of milk samples with sIgA specific to the N-protein, linear NTD, and RBD-SD1 epitopes, and showed an increase in samples with RBD conformation-dependent sIgA. S-antigens were found to solely induce a sIgA1 response, whereas N-protein sIgA1 and sIgA2 subclasses were involved in 100% and 33% of cases. Overall, the antibody immunological landscape of convalescent donor milk suggests that it may be a potential defense agent against COVID-19 for infants, conferring them with a passive immunity.
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Since periodontitis and type 2 diabetes mellitus are complex diseases, a thorough understanding of their pathogenesis requires knowing the relationship of these pathologies with other disorders and environmental factors. In this study, the representability of the subgingival periodontal microbiome of 46 subjects was studied by 16S rRNA gene sequencing and shotgun sequencing of pooled samples. We examined 15 patients with chronic periodontitis (CP), 15 patients with chronic periodontitis associated with type 2 diabetes mellitus (CPT2DM), and 16 healthy subjects (Control). The severity of generalized chronic periodontitis in both periodontitis groups of patients (CP and CPT2DM) was moderate (stage II). The male to female ratios were approximately equal in each group (22 males and 24 females); the average age of the subjects was 53.9 ± 7.3 and 54.3 ± 7.2 years, respectively. The presence of overweight patients (Body Mass Index (BMI) 30-34.9 kg/m2) and patients with class 1-2 obesity (BMI 35-45.9 kg/m2) was significantly higher in the CPT2DM group than in patients having only chronic periodontitis or in the Control group. However, there was no statistically significant difference in all clinical indices between the CP and CPT2DM groups. An analysis of the metagenomic data revealed that the alpha diversity in the CPT2DM group was increased compared to that in the CP and Control groups. The microbiome biomarkers associated with experimental groups were evaluated. In both groups of patients with periodontitis, the relative abundance of Porphyromonadaceae was increased compared to that in the Control group. The CPT2DM group was characterized by a lower relative abundance of Streptococcaceae/Pasteurellaceae and a higher abundance of Leptotrichiaceae compared to those in the CP and Control groups. Furthermore, the CP and CPT2DM groups differed in terms of the relative abundance of Veillonellaceae (which was decreased in the CPT2DM group compared to CP) and Neisseriaceae (which was increased in the CPT2DM group compared to CP). In addition, differences in bacterial content were identified by a combination of shotgun sequencing of pooled samples and genome-resolved metagenomics. The results indicate that there are subgingival microbiome-specific features in patients with chronic periodontitis associated with type 2 diabetes mellitus.
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In this paper, the structural characteristics of a W-Ta-Mo-Nb-V-Cr-Zr-Ti non-equiatomic refractory metal alloy obtained by spark plasma sintering (SPS) of a high-energy ball-milled powder mixture are reported. High-energy ball milling resulted in the formation of particle agglomerates ranging from several tens to several hundreds of micrometers. These agglomerates were composed of micrometer and submicrometer particles. It was found that, during ball milling, a solid solution of A2 structure formed. The grains of the sintered material ranged from fractions of a micrometer to several micrometers. During SPS, the phase transformations in the alloy led to the formation of a Laves phase of C15 structure and ZrO and ZrO2 nanoparticles. The microhardness of the ball-milled alloy and sintered material was found to be 9.28 GPa ± 1.31 GPa and 8.95 GPa ± 0.42 GPa, respectively. The influence of the processing conditions on the structure, phase composition, and microhardness of the alloy is discussed.