Mechanisms of autoimmune pathology in post-COVID syndrome

One of the delayed consequences of SARS-CoV-2 infection is post-acute COVID-19 – polymorphic disorders of various organ systems that affect COVID-19 convalescents and persist for more than four weeks after an acute infection. Due to the infectious nature of the COVID-19, we would like to pay special attention to complications from the immune system, especially concomitant and new-onset autoimmune pathology. This review analyzes the current state of the issue of post-acute COVID-19 complications, discusses the molecular features of the SARS-CoV-2 virus and the mechanisms underlying the impaired immune response during acute COVID-19 infection and the occurrence of autoimmune and autoinflammatory conditions during convalescence. Particular attention is paid to the molecular mimicry of antigenic determinants of the SARS-CoV-2 virus, which are structurally similar to the epitopes of human autoantigens. The current data on post-acute COVID-19 autoimmune complications from humoral immunity and the endocrine system, as well as reproductive disorders faced by male patients are presented. For the first time, we hypothesize a role of the structural homology of the human SOX13 autoantigen (HMG box factor SOX13) associated with diabetes mellitus and SARS-CoV-2 envelope (E) protein in the development of the post-acute COVID-19 autoimmune pathologies. Due to the structural similarity of the two proteins and the overlap of their immunogenic regions, we suggest that the increased risk of developing diabetes mellitus and reproductive disorders in men after suffering from COVID-19 may be associated with immunological cross-reactivity

Development of DNA aptamer selection approach based on membrane ultrafiltration of aptamer/target complex

Background. Aptamers are small single-stranded DNA or RNA molecules that have an affinity for a specific target molecule. The main method of aptamers construction is the technology of systematic evolution of ligands with exponential enrichment (SELEX). However, the exact approach depends on the nature of target molecules, and is selected and optimized by each researcher independently. The article describes the technique of production of aptamers to the tick-borne encephalitis virus (TBEV) using membrane ultrafiltration with a molecular weight cut-off of 100 kDa. As a result, the pool of aptamers with observable affinity for TBEV is successfully selected and enriched.The aim. To develop the technique suitable for selection of specific DNA aptamers to a live, crude TBEV suspension directly in cell culture supernatant.Materials and methods. The selection of aptamers was carried out using a modified SELEX DNA aptamer technology in combination with semipermeable membrane ultrafiltration using Vivaspin 6 (Sartorius, Germany) concentrators of molecular weight cut-off of 100 kDa. Enrichment of a specific pool of aptamers was performed using real time polymerase chain reaction. Aptamers were sequenced with automated Sanger sequencing method. The direct virucidal effect of the aptamers was determined by the decrease in the titer of the infectious virus after incubation with the aptamer.Results. The pool of aptamers to TBEV was selected and enriched. This aptamer pool expressed affinity both to the infectious TBEV and to the TBEV antigen. Sixteen aptamers were sequenced from this pool and four of them were synthesized and tested for antiviral activity against TBEV. No antiviral activity was observed.Conclusions. The technique developed that can be successfully used to select aptamers to a live virus culture for the viruses comparable in size to TBEV or larger

6-gene promoter methylation assay is potentially applicable for prostate cancer clinical staging based on urine collection following prostatic massage

The detection of prostate cancer (PCa) biomarkers in bodily fluids, a process known as liquid biopsy, is a promising approach and particularly beneficial when performed in urine samples due to their maximal non‑invasiveness requirement of collection. A number of gene panels proposed for this purpose have allowed discrimination between disease‑free prostate and PCa; however, they bear no significant prognostic value. With the purpose to develop a gene panel for PCa diagnosis and prognosis, the methylation status of 17 cancer-associated genes were analyzed in urine cell‑free DNA obtained from 31 patients with PCa and 33 control individuals using methylation‑specific polymerase chain reaction (MSP). Among these, 13 genes indicated the increase in methylation frequency in patients with PCa compared with controls. No prior association has been reported between adenomatosis polyposis coli 2 (APC2), homeobox A9, Wnt family member 7A (WNT7A) and N‑Myc downstream‑regulated gene 4 protein genes with PCa. The 6‑gene panel consisting of APC2, cadherin 1, forkhead box P1, leucine rich repeat containing 3B, WNT7A and zinc family protein of the cerebellum 4 was subsequently developed providing PCa detection with 78% sensitivity and 100% specificity. The number of genes methylated (NGM) value introduced for this panel was indicated to rise monotonically from 0.27 in control individuals to 4.6 and 4.25 in patients with highly developed and metastatic T2/T3 stage cancer, respectively. Therefore, the approach of defining the NGM value may not only allow for the detection of PCa, but also provide a rough evaluation of tumor malignancy and metastatic potential by non‑invasive MSP analysis of urine samples

ASSESSMENT OF NEUTRALIZING PROPERTIES OF DNA-APTAMERS AND EXTRACTS OF MEDICINAL HERBS AGAINST THE TICK-BORNE ENCEPHALITIS VIRUS

Tick borne encephalitis (TBE) is a dangerous neurological disease that is transmitted to humans through the bite of Ixodid ticks. The disease exhibits an estimated 16 000 cases recorded annually over 30 European and Asian countries. The agent of TBE is the tick borne encephalitis virus (TBEV), belonging to the family Flaviviridae, genus Flavivirus. In spite the significant impact of TBE on human health, there is a serious lack of specific treatment against this disease. The only specific drug available is the human anti-TBEV immunoglobulin from vaccinated blood donors. The drug is produced and used in Russia only, both to prevent and to cure the TBE. In this work, we evaluated the ability of TBEV-specific DNA-aptamers and extracts of traditional medicine plants to neutralize the TBEV Selection of aptamers was performed using SELEX approach. Extracts of the seeds of Momordica cochinchinensis and Terminalia chebula were produced by boiling the ground seeds in water, clarified by centrifugation and filtration steps and filter sterilized. The SELEX had produced two aptamers - My13 and My38. Neither of two was capable to neutralize TBEV in vitro. The ability to shield the antibody binding sites on the surface of TBEV virions was also absent. The extract of M. cochinchinensis exhibited no neutralizing activity as well. Surprisingly, the T. chebula extract completely neutralized the TBEV after 30 min of incubation at 37 °C. The possible explanations and further development of the project are discussed

Online service for interpretation of the resistance prediction results to bedaquiline by the molecular data

Background. Bedaquiline is a new and promising anti-tuberculosis drug, but longterm use requires resistance. This is due to mutations in the atpE and mmpR genes in M. tuberculosis (MBT).The aim of the research was to test a system for automated interpretation of results for predicting resistance to bedaquiline by the molecular data.Materials and methods. DNA was isolated from strains of M. tuberculosis in the Irkutsk region and Yakutia. The total quantity of DNA samples was 27 strains from Yakutia and 21 strains from the Irkutsk region. The study of MBT genomes was carried out on the DNA previously obtained by the authors in the territories of the Irkutsk region (n = 5), Yakutia (n = 4), Buryatia (n = 3), Zabaykalskiy kray (n = 4) and the Far East (n = 8). We used the BSATool program to detect bedaquiline resistance based on  Sanger and genomic data. Sanger sequencing analyzed the atpE and  mmpR genes, and whole genome sequencing examined mutations in the same sequences, as well as additionally in mmpL5, mmpS5, Rv0678, Rv1979c, and pepQ.Results. Complete agreement between the phenotypic and genotypic analysis of resistance to bedaquiline was found for three strains from Yakutia. One genome with significant mutations to bedaquiline was identified. A conclusion was made about the importance of molecular analysis of target genes with subsequent detection of resistance to bedaquiline in silico

Isolation and whole genome sequencing of a lipophilic anaerobic bacterium, a representative of the species complex <i>Corynebacterium tuberculostearicum</i>, from a tuberculosis focus

Background. The study of the lower respiratory tract microbiome has been actively developed inrecent years with the help of whole genome sequencing (WGS) methods. Due to this, it became clear that the nature of the lungs microbiota is very different from other microbial communities inhabiting the human body. One of the important directions in the study of pathological lungs biocenosis is the study of the role of the satellite microbiota of the tuberculosis focus. The aim of the work. To isolate and characterize oxygen-tolerant anaerobes from the necrotic contents of tuberculomas. Materials and methods. Biopsy material from 5 patients with pulmonary tuberculosis was obtained during a planned surgical treatment of tuberculoma. A pure culture was isolated from one sample during anaerobic cultivation. Lipase activity of strain was determined by plating on brain heart infusion agar (HIMEDIA, India) supplemented with 0.1 % Tween-80 and 10 mM of CaCl2. Antibiotic susceptibility was determined by RAPMYCO and SLOWMYCO of TREK Diagnostic Systems (Thermo Fisher Scientific, USA). DNA from the sediment of the broth culture was isolated by the CTAB chloroform method. Whole genome sequencing was performed on a DNBSeq-G400 NGS sequencer by Genomed (Russia). Results. Based on WGS results and phylogenetic analysis, the strain was identified as Corynebacterium kefirresidentii. The strain was characterized by high lipase activity and resistance only to Isoniazid, Ethionamide and Trimethoprim/Sulfamethoxazolin. Conclusion. The isolation of a lipophilic anaerobic representative of the Corynebacterium tuberculostearicum species complex from a tuberculous focus indicates a  possible role of the non-tuberculous microbiota in the liquefaction of caseous necrosis. We assumed that in some cases, favorable conditions are created inside the tuberculous focus for the development of satellite anaerobic lipophilic microbiota

Probiotic consortiums: Structure and antagonistic activity against opportunistic bacteria and human normobiota (using the example of <i>Escherichia coli</i>) <i>in vitro</i>

Background. Using probiotic preparations based on consortia of microorganisms not only helps to restore the balance of the intestinal microbiota, but also increases the therapeutic effect of probiotics. Promising sources for obtaining probiotic consortia are milk products that have undergone natural fermentation with the help of spontaneously formed microbial consortia. The aim. To study the structure of five microbial consortia with probiotic properties from naturally fermented milk products and to assess in vitro their antagonistic activity against opportunistic bacteria and a representative of the human normobiota – Escherichia coli. Materials and methods. The structure of bacterial consortia was analyzed by sequencing methods. The antagonistic activity of the consortia was assessed by the disk diffusion method. Results. It has been established that the studied microbial consortiums are represented by Enterococcus spp. and Streptococcus spp. bacteria. In consortiums No. 1, No.  2, and No.  3, Enterococcus bacteria dominated, while in consortiums No.  4 and No. 5, Streptococcus dominated. Antagonistic activity was shown against four isolates of opportunistic bacteria: Klebsiella pneumoniae No.  493, Enterobacter hormaechei No. 372, Staphylococcus aureus No. 4 and Pseudomonas aeruginosa No. 25 IMB, as well as against one representative of the human normobiota – Escherichia coli No. 495. The highest growth delay zone is found in E. coli No. 495 isolate. Three test cultures (K. pneumoniae No. 509, E. coli ATCC25922 and P. aeruginosa No. 3 IMB) exhibited more dense growth around probiotic consortia. Conclusion. The results of the study showed that the effect of probiotic consortia differing in the composition of microorganisms can be neutral and bactericidal. The presence of antagonistic activity in the studied microbial consortia against multiresistant isolates of opportunistic bacteria is a prospect for creating probiotics with antibacterial properties

Potassium channel gene mutations rarely cause atrial fibrillation

BACKGROUND: Mutations in several potassium channel subunits have been associated with rare forms of atrial fibrillation. In order to explore the role of potassium channels in inherited typical forms of the arrhythmia, we have screened a cohort of patients from a referral clinic for mutations in the channel subunit genes implicated in the arrhythmia. We sought to determine if mutations in KCNJ2 and KCNE1-5 are a common cause of atrial fibrillation. METHODS: Serial patients with lone atrial fibrillation or atrial fibrillation with hypertension were enrolled between June 1, 2001 and January 6, 2005. Each patient underwent a standardized interview and physical examination. An electrocardiogram, echocardiogram and blood sample for genetic analysis were also obtained. Patients with a family history of AF were screened for mutations in KCNJ2 and KCNE1-5 using automated sequencing. RESULTS: 96 patients with familial atrial fibrillation were enrolled. Eighty-three patients had lone atrial fibrillation and 13 had atrial fibrillation and hypertension. Patients had a mean age of 56 years at enrollment and 46 years at onset of atrial fibrillation. Eighty-one percent of patients had paroxysmal atrial fibrillation at enrollment. Unlike patients with an activating mutation in KCNQ1, the patients had a normal QT(c )interval with a mean of 412 ± 42 ms. Echocardiography revealed a normal mean ejection fraction of 62.0 ± 7.2 % and mean left atrial dimension of 39.9 ± 7.0 mm. A number of common polymorphisms in KCNJ2 and KCNE1-5 were identified, but no mutations were detected. CONCLUSION: Mutations in KCNJ2 and KCNE1-5 rarely cause typical atrial fibrillation in a referral clinic population

Dynamic circadian protein-protein interaction networks predict temporal organization of cellular functions.

Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner