Molecular Genetics, Microbiology and Virology最新文献

The aim of this research is to study the distribution, species diversity, and genetic variability of rickettsiae in Dermacentor spp. ticks from Western Siberia and Northern Kazakhstan. Thus, samples from 571 Dermacentor ticks (406 individuals of Dermacentor reticulatus, 136 Dermacentor nuttalli, 21 Dermacentor marginatus, and 8 Dermacentor silvarum) have been examined for the presence of Rickettsia spp. DNA using nested PCR. The rickettsial species have been determined by species-specific PCR and/or sequencing of gltA gene fragments. For a number of R. raoultii samples, the sequences of the ompA (3266 bp) and ompB (4852 bp) gene fragments have been additionally determined. The examined ticks carry DNA of four Rickettsia species and a new genotype Rickettsia spp. Kos-97-Dr, which cannot be assigned to any known species. All tick species are most commonly infected with R. raoultii; the infection rate varies from 47.0 to 86.8%. Rickettsia sibirica DNA has been found in 16.1–45.7% of D. nuttalli from three sites in the Republic of Altai. DNAs of Rickettsia aeschlimannii, Rickettsia aeschlimannii-like, “Candidatus Rickettsia tarasevichiae,” and a new genotype Rickettsia sp. Kos-97-Dr have been found sporadically in D. reticulatus and D. marginatus. Based on analysis of the gltA gene fragment, seven haplotypes of R. raoultii have been identified; four of them correspond to previously described genotypes. The analysis of long fragments of the ompA and ompB genes of R. raoultii samples revealed the presence of three genetic groups corresponding to different genotypes for the gltA gene. Thus, Dermacentor spp. carry both typical and atypical species of Rickettsia spp. The existence of natural foci of Siberian tick typhus in the Republic of Altai has been demonstrated. Based on the analysis of the variable surface protein genes and conserved gltA gene, the existence of three genetic groups of R. raoultii has been shown.

The COVID-19 pandemic has witnessed the emergence of diverse variants of SARS-CoV-2, with spike proteins playing a pivotal role in mutation due to their extracellular projection and exposure to immune system pressures. Clinical manifestations of COVID-19 have shown significant variation, ranging from severe symptoms requiring ICU admission or resulting in fatality to asymptomatic cases. This study aims to investigate genetic variations in the spike protein among two distinct groups of SARS-CoV-2 sequences: asymptomatic and ICU/deceased patients. The objective is to explore the viral genetic factors associated with these two clinical outcomes. Our analysis reveals that four spike protein mutations (P26S, D253G, K417N, and D614G) may be partially linked to the ICU/deceased outcome. Additionally, the Omicron and Delta variants exhibit the highest proportions of overall asymptomatic and ICU/deceased patients, respectively. Further evaluation of the ratio of asymptomatic cases to ICU/deceased within a singular variant demonstrates that the Beta and Gamma variants elicit the greatest proportion of asymptomatic and ICU/deceased cases, respectively. In conclusion, our findings suggest a possible association between four spike protein mutations and the outcome of ICU admission or death. The Gamma variants demonstrate greater lethality, while the Delta variants are associated with higher mortality rates.

The use of efficient and inexpensive substrates (2D matrices) for cultivation and differentiation of nerve cells in vitro is important for the creation of tissue engineering constructs intended for the treatment of nervous system pathologies. Recombinant analogues of the orb-weaver spider dragline-silk proteins spidroins 1 and 2 appear promising in addressing this task. The aim of the study was to evaluate the effect of cell substrates derived from mixtures of recombinant spidroins (RS) rS1/9 and rS2/12 with hybrid proteins (HP) containing rS1/9 monomer fused with biologically active peptides on gene expression levels of key synapse-specific proteins and viability of the human neuroblastoma SH-SY5Y cell line during directed cholinergic differentiation. A two-stage scheme of directed cholinergic differentiation of SH-SY5Y cells using retinoic acid and brain-derived neurotrophic factor (BDNF) was implemented. Cell viability was assessed via MTT assay and crystal violet staining. The mRNA levels of the studied genes were assessed by real-time PCR. Directed differentiation of the SH-SY5Y cells was marked by a significant increase in the gene expression levels of synaptophysin, synapsins I and II, and the postsynaptic protein PSD-95. The highest cell viability and increased PSD-95 expression levels were observed during differentiation on a matrix consisting of RS rS1/9 and rS2/12 mixed with the RGDS peptide (present in extracellular matrix proteins) and heparin-binding peptide (HBP, laminin fragment) containing HPs. The highest efficiency during the differentiation of the SH-SY5Y cells was demonstrated by a matrix consisting of the mixture of RS rS1/9 and rS2/12 and a HP made up by RS rS1/9 monomer fused with RGDS (the ligand of integrins) and HBP (the ligand of growth factors and syndecans). Matrices consisting of RS rS2/12 alone or the mixture of rS2/12 with HP(RGDS) showed lower efficiency, although the use of the GRGGL peptide (which interacts with the neural cell adhesion molecules and is a component of RS rS1/9) led to an increase in efficiency.

The Annexin 2 protein (ANXA2) encoded by the ANXA2 gene performs a number of biological functions that are primarily related to cellular transport. An impaired functional activity of the ANXA2 gene have been primarily detected in oncological diseases; however, we have previously shown an increased expression of the Anxa2 gene at the mRNA level in the early stages of neurodegeneration. In this regard, it is most interesting to study the effect of suppressed expression of the ANXA2 gene on nervous-system functioning in a Danio rerio model with the use of morpholino oligonucleotides (morpholino). The present study examined the effect of morpholino to the anxa2a gene on the development of Danio rerio embryo. The study was conducted with D. rerio line AB, embryos of which were injected with morpholino oligonucleotides to the anxa2a gene. The estimate of the effect of injected morpholino oligonucleotides on embryo development was examined by the changes in the phenotype on the second and fourth day post fertilization (dpf). An injection of experimental morpholino targeting the start-codon and 5'UTR region results in an increased percentage of deformations compared with the Co–In and Co–Mo–D groups at the fourth dpf. The deformations observed at the fourth dpf were evident in both control and experimental groups. In addition, we analyzed an earlier time point such as the second dpf. Phenotype changes at this time point were detected only in the experimental groups. A comparative analysis of data obtained from injected morpholino targeting start-codon and 5'UTR of the anxa2a gene evidence that both variants may be used for further research. In addition, we have shown that it is preferable to carry out analysis of expression at the second dpf. Apparently, the effects detected at the second dpf are specific, while the presence of a hindbrain ventricular deformity suggests that altered expression of the anxa2a gene may result in dysfunction of the nervous system.

Bacterial flagellin (FliC) can be used as a TLR5 ligand-like adjuvant. However, the sequences of hypervariable regions(HVR) of FliC from different bacteria vary, and their effects on adjuvants remain unclear. In this study, FliC_Δ274–406 (deleting of D3 domain) and FliC_Δ174–506 (deleting of D2-D3 domain) from Escherichia coli Nissle 1917 FliC (FliC_EcN) were constructed and expressed in host bacteria, BL21. Purification was conducted using affinity chromatography on a Ni-NTA column, validation was done using SDS-PAGE and western blotting, the antigenicity and immunogenicity were detected using ELISA, and adjuvant effects were evaluated in Caco-2 cells and mice. The results showed that FliC_EcN was mainly expressed in the bacterial supernatant, and the two truncated flagellins were expressed as inclusion bodies. Compared with FliC_EcN, both FliC_Δ274–406 and FliC_Δ174–506 had considerable decreased antigenicity and immunogenicity. In Caco-2 cells, FliC_Δ174–506 had a higher ability to promote the secretion of IL-8 than FliC_EcN and FliC_Δ274–406. In mice, FliC_Δ174–506 showed a comparable adjuvant level to FliC_EcN, while FliC_Δ274–406 was less effective. Our data shows that adjuvant effects of FliC_EcN with deletion of different regions of its HVR are inconsistent, and deleting its entire D2–D3 domain is better.

Hypertrophy of the pharyngeal tonsil (HPT) is considered one of the most common diseases of the ENT organs. The aim of the present study was to investigate the role of interleukin 10 (IL-10) gene polymorphism and infections caused by human herpesviruses 6 (HHV6), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) in children with HPT. The study included 106 children with HPT and 38 healthy children at the age from 2 to 11 years. All children with HPT were divided into three subgroups depending on the pharyngeal tonsil size. Viruses were determined by real-time quantitative polymerase chain reaction using commercially available kits (QIAGEN, Germany). In patients with HPT, HHV6 was more frequently detected as compared with CMV and EBV. Among the three subgroups of children with HPT, infections with HHV6 and EBV viruses predominated in children with a maximal degree of hypertrophy of the pharyngeal tonsil. The frequency of the IL-10 gene rs1800896 GG genotype was higher in the control group of children. Significantly higher frequencies of the G allele and GG and GA genotypes for this gene were detected in the subgroup of children with the lowest size of the pharyngeal tonsil as compared with other subgroups. We hypothesized that infections with HHV6 and EBV can contribute to an increase in the pharyngeal tonsil size. The IL-10 rs1800896 GG genotype can contribute to resistance to hypertrophy of the pharyngeal tonsil.

Antimicrobial drug resistance has made the treatment of microbial infections quite challenging. A Gram-positive, anaerobic, spore-forming, and toxin-producing bacillus, Clostridioides difficile infection causes diarrhea-related deaths globally. The available drugs like vancomycin and metronidazole are becoming less effective against this infection. We have designed this study to identify genes responsible for antimicrobial resistance and have a better understanding of the mutations and their impact on the antimicrobial resistance activity. The Whole Genome Sequencing data of 11 C. difficile clinical isolates was analyzed to determine novel genes playing a significant role in antimicrobial resistance mechanisms. Comparative structure analysis of wild and mutant structures of proteins and their functions provided insight into the impact of the identified mutations on antimicrobial resistance. We identified 8 genes common in all the isolates that play a vital role in drug resistance through antibiotic efflux, ribosomal protection, and antibiotic inactivation. Variations in the functional domains of tetA(P), tetM, and ermB genes were found to be the most promising novel drug targets. Our findings suggest that these novel gene mutations would be beneficial in designing new drugs to combat C. difficile infection.

Abstract

The spread of the disease caused by monkeypox virus (MPox) since 2022 has shown the urgency of developing countermeasures. The development of modern methods of clinical laboratory diagnostics of MPox contributes to this. Enzyme-linked immunosorbent assay (ELISA) is an accessible and sensitive platform for developing diagnostic tools. Detection of MPox antigens using ELISA kits based on monoclonal antibodies (MAbs) is promising due to the quick time of analysis and minimal requirements for sample preparation. We have developed and deposited two strains of Escherichia coli that produce recombinant proteins. Mice were immunized with the AgPOX protein, which contains unique antigenic sequences of MPox. The Trx + A29 protein for selecting MAb producers includes the original amino acid sequence A29L. The absence of antibody crossover to Trx protein and native preparations of variola virus and vaccinia virus tested by ELISA. As a result of hybridization of splenocytes from immunized mice, MAb producers were obtained. Fifteen MAb-producing hybridomas were selected based on ELISA results with three specific MPox antigens and three nonspecific ones. Three hybridomas were selected for deposit according to the productivity criteria. The possibility of detection by means of its MAbs of the native MPox antigen at various concentrations was tested and method sensitivity was determined. The MAbs a-A29L_MPoxV of three hybridomas detected the native antigen MPox at a concentration of 10² PFU/mL. It is likely that the method is even more sensitive when selecting analysis conditions. Based on labeled MAbs a-A29L_MPoxV, it is possible to develop a sensitive and specific indirect two-step ELISA kit for immunodiagnostics of MPox.

Abstract

Introduction: Cancer is one of the leading causes of death worldwide and oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity in which there is a poor prognosis. Viruses, including Human papillomavirus (HPV), play an important role in the etiology of this cancer. The aim of this study was to evaluate the prevalence of HPV in patients with oral cancer in Shiraz. Method: In this case-control study, 100 patients with oral cancer as the case group and 100 healthy individuals as the control group were included in the study after applying the inclusion and exclusion criteria, and after receiving demographic information, Nested-PCR for HPV detection was performed on their tissue samples. Finally, after entering the data into SPSS software, the data were statistically analyzed. Results: A total of 200 patients including 100 patients and 100 healthy individuals were examined. Samples of patients from different parts of the mouth but samples of healthy individuals were taken from the tonsils. The mean age of the subjects was 53/66 ± 1.38 years. The minimum and maximum ages of the subjects were 28 and 90 years, respectively. In this study, 70 (34%) of the subjects were female and 130 (65%) were male. 2 out of 100 patients in the control group and 14 out of 100 patients in the case group were infected with HPV virus in oral samples. This difference was statistically significant (p value = 0.008). In HPV-positive people, most cases of HPV were related to the tongue and then the larynx. The relationship between sampling site and HPV infection was statistically significant (p = 0.004). The association between sampling site and HPV infection in people with oral cancer was also significant. Conclusion: The results of this study showed that HPV plays a direct role in development of oral cancer in individual however, factors such as age and gender also involved. The study also found that people with oral HPV were at higher risk for developing tongue cancer.

Abstract

The effect was analyzed of the flhB2 gene, which is located on the AZOBR_p4 (AZOBR_p410073 gene) plasmid in Azospirillum baldaniorum Sp245 and on the ABSP7_p3 (AMK58_26270 gene) plasmid in A. brasilense Sp7 and codes for the FlhB protein, a flagellar export component that ensures flagellin assembly, flagellation, and motility in these bacteria. We used A. baldaniorum strain Sp245, its Fla^– Laf^– mutant Sp245.1063 (Sp245-flhB1::Omegon-Km), and A. brasilense Sp7. Mutants defective in the flhB2 gene were generated by site-directed mutagenesis. Bacterial morphology and motility were characterized by electron and phase-contrast microscopy. An A. baldaniorum Sp245 mutant, Sp245-flhB2::Km, was generated that had a cloned kanamycin resistance gene in the coding sequence (CDS) AZOBR_p410073. In contrast to the Fla^– Laf^– mutant Sp245-flhB1::Omegon-Km, the flhB1 chromosomal gene of which is inactivated (AZOBR_150177 gene), strain Sp245-flhB2::Km retained the synthesis of a functioning polar flagellum (Fla), but the synthesis and functioning of lateral flagella (Laf) was impaired and the movement and spreading rates of swarming cells in semiliquid agarized media were decreased. Inactivation of the AMK58_26270 plasmid gene in Sp7, which is homologous to the AZOBR_p410073 gene (97% identity), resulted in a similar Laf^– phenotype in the corresponding mutant. Two putative flhB genes are present in the genome of strains Sp245 and Sp7. These genes are located in the chromosome (flhB1) and on the AZOBR_p4 or ABSP7_p3 plasmid (flhB2), respectively. Expression of the flhB2 gene is required for Laf assembly. Transcription of flhB2 is regulated by a mechanosignal, the perception and generation of which is ensured by the functioning Fla, apparently with the involvement of the FlhB protein encoded by the flhB1 chromosomal gene.