Laboratory for Molecular Genetics and Congenital Immunity

• The Laboratory has produced mouse lines (mutants and hybrids) resistant to septic shock, oncogenesis, obesity. We have conducted a sequencing of the genome and a bioinformatic analysis of pro-inflammatory genes in the mouse lines. We selected and identified candidate genes responsible for resistance to TNF. We have conducted a phenotype analysis of homozygous mutants to determine their resistance to transplanted tumors, sensitivity to induced tumors.
• We have developed the testing systems «Diagnostics of the development of inflammation in vessel walls in lipid exchange disorders», «Early diagnostics of cancer of reproductive organs» and «Pre-clinical diagnostics of diseases based on nonlinear dynamics».
• Our researchers have synthesized a group of new nitrogen-containing compounds with high biological activity. By example of model systems we have demonstrated, both in vivo and in vitro, that the reagents possess an expressed anti-tumor and anti-inflammatory effect. Compounds are viewed as prospective components of pharmacological agents.
• It has been demonstrated that cells  of С57ВL/6 mice (hepatocytes) are highly sensitive to Fas-induced apoptosis due to the production of higher levels of cFLIPR (a short isoform of the protein) compared to hepatocytes of the MSM line (that produce the long isoform cFLIPL). cFLIPL binds caspase 8, preventing cell apoptosis.
• It has been determined that the transmembrane protein of endoplasmic reticulum STING (Stimulator of Interferon Genes), which is known as a molecular sensor of cytoplasmic DNA playing an important role in recognizing intracellular pathogens and activating interferon - (type I) dependent pathway in cells of the myeloid series, has a broader range of functions. STING is not only the convergence point of signals sent by other cytosol sensors/receptors, but is also responsible for their specific switching, simultaneously functioning as a receptor and as an adaptation protein. STING can be a specific signaling «hub» whose work is determined by the current molecular context.
• We have found a natural mutant variant of STING that is not responsible for activation by launching the expression of IFN type 1; the N-terminal domain of STING is mutant and not ligand-binding domain (as it was suggested by many Western colleagues), and is linked to disruptions of the STING traffic from the ER to the Golgi apparatus. Using the IL-6 promoter, we researched the impact 47/48 mutations (and S53L) on IL-6 activation, since it is namely in them that the strongest contribution to the defect of IFN signal is observed. It has been found earlier that multiple polymorphisms in STING MOLF (L47V, A48G, S53L, S103F, I114M, Y115C, Y126S and 6-aa deletions 116-122 in NTD, with one substitution, N210D, in CTD STING) will influence the secondary structure of the protein and disrupt transmembrane binding regions of STING. For instance, we managed to detect a natural mutant variant of STING in which АК substitutions (L47V, A48G, and S53L) are located in the N-terminal domain and are associated with disruptions of the STING traffic from ER to the Golgi apparatus.. Furthermore, we have demonstrated that the high production of IL-6 in MOLF is caused by the retention of STING in ER, while 47/48-polymorphisms correlate with a high level of IL-6. We have introduced mutations into the dimerization domain (DD) of STING, which ensured the constitutive activation and translocation of STING from ER, we cloned 47/48-mutations in DD in constitutively activated mutants of STING, which results in the process of STING translocation from ER becoming impossible, which leads to IL-6 switching to the constitutive mode. At the same time, the high frequency of heterogeneousness in the defect allele of STING in a human population (from 20 per cent to 50 per cent) indicates the fact that pharmacological activators of STING cannot be prescribed to all patients.
• We have researched the role of cGAS, IFI16, DDX41, AIM2 in responses to DNA in mice of the MOLF line. The conducted experiments demonstrated that responses to DNA in mice of the MOLF line mainly depended on cGAS, however, other sensors, such as IFI16 or AIM2, had significance for realizing the effect. cGAS is a  Nucleotidyltransferase, it is related to producing the secondary cGAMP messenger, that activates the STING adapter protein. As a result, the TBK1/IRF3 pathway is launched and Ifn genes are produced. Therefore, during infection DNA transmission from the pathogen to the cytosol of the host cell causes the activation of cGAS and STING, which leads to the induction of IFN and the expression of several interferon-stimulated genes  (ISGs), include IFN type 1, Ccl5, Cxcl10, Mx1, Ifit 1, Ifit2, Ifit3. In mice of the  C57BL/6 line it occurs exactly like this. However, a transcription analysis of ISGs in response to an infection (HSV — Herpes simplex virus and Listeria monocytogenes) by MOLF confirmed a defect in their IFN response and some NF-kB-regulated genes, the genes IL-1a, IL-1b, Tnf, Tlr2 and IL-6 were upregulated. The cause of such an effect is also related to a defect in the N-terminal domain of STING.
• For the first time we have characterized the mechanism of Т cell activation with the use of a specific activator of STING, 5,6 dimethlyxanthenone-4-acetic acid (DMXAA).   DМХАА wild-type Т cell activation leads to the development of  apoptosis, while in knockout cells no such effect is observed. Experimental data indicate that one of the mechanisms of DMXAA STING-induced apoptosis can be the accumulation of proteins with defective structure in ER, as a significant increase in the expression of genes participating in protein folding has been found, in particular Bip/ HSPA5 and GADD34. The obtained results suggest the use of this compound as a prospective activator of innate immunity.
• Using the hybrids of a F2 intercross (MOLFхC57BL/6) we managed to determine that the genetic differences in the responses of the immune system are associated with two chromosome locuses. One of the loci on the chromosome 2 contains a gene of the mannose receptor (MRC1). Low levels of the expression of this gene were found only in macrophages of mice of the MOLF line. Another locus located on chromosome 10 causes the low immune response to stimulation by the majority of agonists of TLR receptors (CpG (TLR9), LTA (TLR2), LPS (TLR4), Imiquimod (TLR7)), shows inhibitive effect with respect to TLR-mediated activation. The contribution of this locus to the phenotype is inherited as an autosomal dominant trait. Using the hybrids we found that more pronounced responses in mice of the MOLF line are caused by a locus in chromosome 13. The locus belonging to chromosome 13 stimulates TLR-mediated signaling. A subsequent analysis of candidate genes allowed to detect several representatives that are differentially expressed in MOLF macrophages and contain multiple polymorphic substitutions. According to the proposed working model, the ITIM domain in the cytoplasmic «tail» of these proteins has a disrupted structure in the MOLF line and therefore is not able to recruit inhibitor phosphatase, which gives the immune response in MOLF mice a more pronounced pro-inflammatory nature. Using the knockout technique in primary macrophages, we have demonstrated that some of these genes are responsible for the increase in the production of IL-6 in mice of the MOLF line.
• We have conducted a pilot RNA sequencing (MiSeq, llumina) samples of normal epithelium and all clinical pathological stages of virus-associated carcinomas (cervical cancer, head and neck cancer). We performed a complete bioinformatic analysis of the collected data. The analysis of the expression of genes in groups: 1 – specific lymphatic endothelial growth factors (VEGF-С and -D, HGF, FGF, PlGF) and their receptors (VEGFR3, c-MЕТ/HGFR); 2 – lymphangiogenesis-specific transcription factors (Prox1, FOX-C2, ETS); 3 – transcription factors of epithelial-mesenchymal transition (Twist1, Snail, Slug); 4 – receptors of adhesion of lymph endothelial cells – regulators of invasion (LYVE-1, PDPN); 5 – components of extracellular matrix – regulators of angiogenesis (thrombospondin -1, 2; endostatins) demonstrated that the formation of invasive carcinoma, regardless of the state of the patient, is accompanied by successive changes of the activity of lymphangiogenesis regulators and epithelial-mesenchymal transition. We have found a defined functional signature of genes associated with the processes of angiogenesis, lymphangiogenesis and epithelial-mesenchymal transition. The most important aspect is that in the composition of the signature we were able to detect new groups of genes that had not been noticed earlier in studies of the mechanisms of the development of virus-associated carcinomas but are currently viewed as promising targets for antimetastatic therapy.  First and foremost, it is the membrane receptors of the SEMA-PLXN-NRP system, HGFR/c-MET, TGFbeta/TGFR. 
• We have developed a model that characterizes the molecular mechanisms of the early invasive growth of carcinomas associated with HPV infection. We described the signaling cascades and the functions of genes that ensure the changes in the cell morphology, the mechanisms of «lymphangiogenesis switching» and the formation of the proangiogenic phenotype. In this process a quite important role is attributed to the molecules of the cell that are based on the intersection of signaling pathways. One of the leading positions in this case is occupied by STING. We confirmed data described earlier only for mouse cells concerning the high expression of STING in human Т lymphocytes. For the first time it has been demonstrated that in various fractions of Т cells the pattern of STING expression can change against the background of the progression of invasive cancer associated with chronic viral infection, which can testify to the participation of Т cell STING in controlling HPV and the developing oncopathology. The results of RNA sequencing of blood samples from patients demonstrated a significant increase in the expression of a range of interferon-stimulated genes (ISGs), including transcription factors that regulate the interferon pathway (IRFs and STATs) and antiviral response genes, in particular, OAS1-3, ADAR1, ISG15, ISG20, PKR. Of particular interest is that at the early stage of the invasive process of induction of the STING/TMEM173 gene and cGAS, a nucleotidyltransferase that is related to the production of the secondary messenger cGAMP that activates STING. The expression of genes coding components of alternative cytosolic sensor systems (PRRs), including AIM2, RIG-I/MDA5, IFI16, also turned out to be significantly higher. Such differences in expression patterns point at the fact that with the development of the invasive phenotype, activation of the STING-associated signaling pathway occurs. The results have high importance not only for fundamental but also for clinical oncology, namely for the development of new methods and approaches to targeted therapy. One of the targets for such therapy can be the  STING protein.

Implemented results of research:

• We have developed test systems for the early diagnostics of reproductive system cancers, chronic diffuse liver diseases and pain syndrome in oncology patients. Patents for inventions have been obtained for all the systems. At the International exhibitions  «Bioindustry-2014» (Saint Petersburg, 2014) and «High technologies. Innovations. Inventions» (Saint Petersburg, 2015) the Laboratory’s teem received three gold and 2 silver medals. The test systems have completed initial testing at clinical departments of the the Institute of Medicine of Petrozavodsk State University and are currently being implemented on the grounds of the Emergency Hospital and the Karelian Republican Oncological Dispensary.
• The Laboratory has developed and implemented the database «Passports of laboratory animals». The electronic resource «Pasports of laboratory animals» is a database that includes all the generations of mouse lines used in experiments of the research project. We put into the database both the mouse lines that we had purchased to implement the project and the lines produced in the course of its implementation. Using the database it is possible to obtain all the information concerning a particular animal. The database is constantly being expanded.
• We have developed and implemented a complex of imitators of biological fluids of the human organism (a blood imitator, a urine imitator, a blood plasma imitator, a blood serum imitator, a cerebrospinal fluid imitator, a gastric juice imitator) that can be used as model systems when training students in medical universities. All the imitators of biological fluids have been patented. The imitators are used in the education process of the Institute of Medicine of the Petrozavodsk State University (the Department of Biomedical Chemistry, Immunology and Laboratory Diagnostics; the Department of Human and Animal Physiology, Pathophysiology, Histology; the Department of General and Faculty Surgery) and Petrozavodsk Basic Medical College.

Education and career development:

• From 2011 to 2022 the Laboratory participated in the development of 30 education programs in the training directions: «Biology», «General Medicine», «Pediatrics», «Pharmacy».
• In 2015 a new master»s degree program, «Medico-biological sciences», was opened at  Petrozavodsk State University.
• Employees of the Laboratory have published 27 textbooks and teaching aids. There is the additional training program for young researchers «Modern methods of research in biomedicine», within which 105 young scientists have completed training.
• 12 Candidate of Sciences and two Doctor of Sciences have been prepared and defended.
• The developed electronic courses for school students and biology teachers or the Republic of Karelia. The laboratory supervises a class at the Gymnasium No. 30 named after Dorofey N. Myzalyov in Petrozavodsk. School students participate in the  All-Russian Competition of School Students Research Works and school academic competitions.
• The leading scientist and the employees of the Laboratory participate in conducting classes at the «Sirius» Education Center in Sochi for prize winners of the All-Russian Competition of School Students Research Works in the domain «Genetics, Personalized and Predictive Medicine». 

Organizational and structural changes:

In 2012 the Institute of High Biomedical Technologies (IHBMT) was created at Petrozavodsk State University on the grounds of the Laboratory.  The main goal of the Institute was to structure fundamental and applied research in the field of medicine and biology in accordance with the priority directions of the Russian Federation in the area of biomedical technologies and world trends of science in this domain, as well as to create conditions for conducting R&D to develop innovative technologies ready for implementation into clinical practice. 

In 2020 the IHBMT was renamed to the Research and Education Center of High Biomedical Technologies (R&EC HBMT). 

R&EC HBMT currently incorporates eight specialized research laboratories (the Laboratory of the Molecular Genetics of Innate Immunity,  the Laboratory of Biologically Active Natural and Synthetic Organic Compounds, the Laboratory of Pre-clinical Research, Cell Pathology and Bioregulation, the Laboratory of New Methods of Physiology Research,  the Laboratory of Clinical Epidemiology, the Laboratory of Medical and Environmental Research, the Laboratory of Telemedicine and Electronic Medical Education, the Laboratory of Artificial Intelligence in Medicine), the Accreditation and Simulation Center for Doctors and Pharmacists as well as the Multi-functional Medical Consulting and Education Center.

R&EC HBMT conducts research and development in molecular genetics, oncoimmunology, neurophysiology, bioregulation, cell biology, pharmacology, epidemiology, bioinformatics.

The practical implementation of the Laboratory»s developments is performed on the grounds of the Small Innovative Enterprise «NanoFarm» LLC and the Small Innovative Enterprise «BioGen» created within the project in 2012 and 2013 respectively. Since 2021 the laboratory participated in the implementation of the large-scale scientific project «Multi-component software and hardware complex for the automated collection, processing, marking up scientific and biomedical data, their unification and analysis in data centers on the basis of algorithms artificial intelligence and predictive analysts with subsequent implementation of innovative technologies into scientific and educational activities, practical medicine and real clinical practice, ensuring the comprehensive development of the infrastructure of research activities, the enhancement of its accessibility and for the enhancement of the efficiency of its use», supported by a grant from the Ministry of Science and Higher Education of Russia. 

• Tufts University (USA), «First Oncological Research and Consulting Center» LLC (Russia): joint research.
• University of Eastern Finland (Finland): joint research, student exchanges.
• Saint Petersburg City Hospital No. 40» (Russia): joint research and academic events, student exchanges.
• Karelian Republican Hospital named after V. A. Baranov (Russia): joint research and academic events.
• Karelian Republican Oncological Dispensary (Russia): joint research and academic events.
• Institute of Biology of the Karelian Research Center of the Russian Academy of Sciences (Russia): joint research and academic events, student exchanges.
• Laboratory of the Physical Chemistry of Solutions of Macrocyclic Compounds of the G. A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences (Russia): joint research and academic events.
• Department of Chemistry and Technology of Biologically Active Substances named after  Nikolay A. Preobrazhenskiy of the Institute of Fine Chemical Technologies named after Mikhail V. Lomonosov of MIREA — Russian Technological University (Russia): joint research and academic events, student exchanges.
• S. M. Kirov Military Medical Academy (Russia): joint research and academic, student exchanges.
• Research Institute of Rheumatology of the Russian Academy of Medical Sciences (Russia): joint research and academic events.
• Institute of Age Physiology of the Russian Academy of Education (Russia): joint research and academic events.
• Institute for Biomedical Problems of the Russian Academy of Sciences (Russia): joint research and academic events.
• Koltsov Institute of Developmental Biology of the Russian Academy of Sconces (Russia): joint research.
• Moscow State University (Russia): joint research, student exchanges.
• Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences (Russia): joint research and academic events.
• Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Russia): joint research.
• Research Institute of Pulmonology of the Pavlov First Saint Petersburg State Medical University of the Ministry of Health of Russia: joint research and academic events.
• Petrov National Medical Research Center of Oncology of the Ministry of Health of Russia: joint research and academic events.
• Research Institute of Physical-Chemical Medicine (Russia): joint research and academic events.
• Institute for Oncology and Radiology of Serbia: joint research.
• University of North Carolina at Chapel Hill (USA): joint research.
• Laboratory of the Chemistry of Physiologically Active Compounds of the AB Bekturov Institute of Chemical Sciences (Kazakhstan): joint research and academic events, student exchanges.
• Finnish Institute of Occupational Health (Finland): joint research and academic events, student exchanges.
  
• Tampere University Hospital (Finland): joint research.