Biotechnologies developed in Institute of Molecular Biology and Genetics of NAS of Ukraine

Identification of tumor-associated antigens for the latest immunochemical methods of cancer diagnosis and treatment (Valeriy V. Filonenko, Dr. Sci., Professor, Corresponding Member of NASU)

Tumor-associated antigens (PAA) are the basis for the creation of modern anti-tumor vaccines and specific anti-tumor antibodies.

Application of the SEREX technology (serological identification of tumor-associated antigens) made it possible to identify more than 60 tumor-associated antigens of melanoma, colon, thyroid, and breast cancer. In the future, these antigens can be used to create human antitumor antibodies with subsequent use in clinical practice for the treatment of oncological diseases.

Currently, a number of monoclonal antibodies have been obtained against PAA and signaling molecules that undergo significant changes in the process of malignant cell transformation, namely antibodies against: S6K1, S6K2, TSC1, TSC2, Ki67, PTEN, mTOR, CK2, FGFR1, FGFR3. These antibodies are suitable for detailed diagnosis and prediction of the course of oncological diseases by methods of immunohistochemical and serological analysis.

Using the CRISPR/Cas9 system, editing of the expression of oncogenic ribosomal protein kinase S6 (S6K) isoforms in MCF7 breast carcinoma cells was carried out, thanks to which model sublines of MCF7 cells with signs of epithelial-mesenchymal transition and inhibition of estrogen receptor (ESRα) expression, which is inherent in the most aggressive and resistant for the treatment of breast tumors of the triple-negative molecular subtype. Thus, these cell lines can be used to study the specificity and effectiveness of antitumor agents for the treatment of these types of tumors.

Development of marker gene panels for diagnosis and prognosis of tumor development (Alla V. Rynditch, Dr. Sci., Professor, Corresponding Member of NASU)

The Department of Functional Genomics conducts large-scale gene expression studies to create test systems with a panel of marker genes that would contribute to more accurate diagnosis and/or prognosis of breast and thyroid cancer. In particular, our research focuses on the analysis of such genes as TKS5, WIP, TTP, CR16, AMPH1, BIN1, WASL, and TKS4 gene isoforms. The proteins encoded by these genes are involved in such an important carcinogenic process as invasion, which plays a key role in the development and spread of cancer in the body. The results obtained so far allow us to deepen our understanding of the mechanisms of tumor development and contribute to the confirmation of TKS4, TTP and WIP as prognostic markers of breast cancer with their further use in clinical practice.

From high-performance analysis - to the creation of a set of tumor markers for the diagnosis of various epithelial tumors (Volodymyr І. Kashuba, Ph.D. Professor, Corresponding Member of NASU)

  • Development of PCR methods for diagnosis of tumor-associated mutations and methylation of promoter regions of oncogenes and tumor suppressor genes in various human malignant neoplasms.
  • Development of methods for early diagnosis of oncological diseases based on detection of methylation and mutations in liquid biopsy by sequencing and PCR methods.
  • Development of approaches to predicting cancer diseases and sensitivity to therapy based on the detection of tumor-associated genes expression.
  • Development of approaches to PCR diagnostics of pathogenic microorganisms.

Development of effective PCR-based multiplex test systems for identification of pathogens of widespread infections and assessment of the state of the immune system of patients with infectious diseases, as well as tests for the detection of infection in biological fluids (Mykhailo A. Tukalo, Doctor of Science, Professor, Full Member of NASU; Zenoviy Yu. Tkachuk, Ph.D., Senior Research Scientist)

The full-scale war with the Russian Federation and especially the undermining of the Kakhovskaya HPP not only led to an ecological and humanitarian disaster, but also acutely raised the issue of biosecurity in Ukraine, since problems with biosecurity will only increase, both for people and animals. The Laboratory of Innovative Biotechnology (LIB) of the Department of Enzymology of Protein Synthesis of the IMBG of the National Academy of Sciences of Ukraine owns a number of patented modern biotechnologies, namely: PCR test system for simultaneous diagnosis of RNA-containing viruses based on ingredients of Ukrainian production; PCR test system for determining dangerous bacteria; combined PCR test systems for diagnosis and analysis of gene expression of innate immunity in dangerous viral infections; diagnosis of dangerous strains of pathogenic viruses by the method of express sequencing. The experience of the laboratory staff allows us to create a PCR test system both with the help of dye intercalates and on the basis of labeled probes. LIB employees possess methods of synthesis of primers for PCR analysis, which makes it possible to create such systems regardless of import purchases. On the basis of this methodology, under the conditions of appropriate funding, PCR test systems will be created for the simultaneous diagnosis of influenza, parainfluenza, measles, coronaviruses, rotaviruses, enteroviruses, hepatitis viruses and other epidemic viruses. It is planned to develop PCR test systems for diagnosing dangerous bacterial infections such as anthrax, cholera, typhoid, paratyphoid, shigellosis and staphylococcal infection. In connection with the appearance of new mutants of dangerous viruses and bacteria in the laboratory, it is planned to conduct their express sequencing and create DNA banks of dangerous infectious agents for their further identification and classification. Since LIB owns "clean" premises of the BSLII type, it is planned to create a pilot production of PCR test systems for the needs of the Ministry of Health of Ukraine.

Development and implementation of a new diagnostic test system for rapid identification of Pneumocystis jirovecii in clinical material (Olena V. Moshytets, Ph.D., Senior Research Scientist)

Pneumocystis pneumonia (PCP) is a severe infectious disease with high mortality, which is often the cause of death in patients with acquired immunodeficiency due to HIV, leukemia, oncological chemotherapy, etc. The cause of PCP is an opportunistic fungal infection Pneumocystis jirovecii. Due to the fact that pneumocystis and bacteria or fungi have fundamentally different spectrums of sensitivity to antibiotics and antimicrobial drugs, PCP can be cured only by specific therapy. Therefore, rapid diagnosis and differentiation of PCP from classical bacterial pneumonia is a mandatory step in the successful treatment of the patient. In Ukraine, the diagnosis of PCP is exclusively nonspecific and is represented by cytological analysis of a sample of sputum, bronchoalveolar fluid, or biopsy material. This approach is insensitive, which is why a quick and highly sensitive test for PCP will lead to a significant increase in the standards of PCP diagnosis and improve the treatment of patients from groups at risk of PCP. According to the experience of many national and international organizations, polymerase chain reaction (PCR) is the optimal approach for rapid, specific and highly sensitive diagnosis of PCP.

A laboratory model of a diagnostic test system for PCP diagnosis has been created in the conditions of Ukrainian diagnostic laboratories, which can be diagnostic laboratories at the AIDS control and prevention centers of the Ministry of Health of Ukraine and laboratories at transplant centers.

Development of biologically active compounds (Sergiy M. Yarmoluk, Doctor of Science, Professor)

Using the modern methods of biotechnology, molecular biology, organic chemistry and computer modeling, we have developed a number of biologically active compounds:

  • Phenylpiperazine derivatives with antifungal activity against Cryptococcus neoformans – Patent № 145095 (2020).
  • Application of the compound 5-(5-chloro-2-hydroxybenzylidene)-4-thioxothiazolidin-2-one as an MGMT inhibitor – Patent № 122373 (2020).
  • Application of the small-molecular organic compound 2-amino-3-(1H-indol-3-yl)-N-phenylpropionamide hydrochloride as a compound with antibacterial activity against Acinetobacter baumannii – Patent № 144010 (2020).
  • Small-molecular compounds with antifungal activity based on 6-chlorochromen-4-one. – Patent № 143105 (2020).
  • The method for ajmaline obtaining. – Patent № 120678 (2020).
  • The method for development of novel inhibitors for the reparative enzyme O6-methylguanine-DNA-methyltransferase for use in cancer therapy. – Patent № 127059 (2018).
  • 2-(7-Furan-21-yl-2-morpholin-41-yl-4-oxo-4H-thiazolo[4,5-d]pyridazin-5-yl)-N-naphthalene-1-ylacetamide with antitumor activity. – Patent № 123593 (2018).
  • Small-molecular organic compounds with antituberculosis activity based on benzaldehyde thiosemicarbazone. – Patent № 116134 (2017).
  • Small-molecular organic compounds with antituberculosis activity based on isonicotinic acid hydrazide. – Patent № 117279 (2017).

Nanocomposite complex of cytokine EMAP II and dextran-70 with antitumor effect (Olexander I. Kornelyuk, Dr. Sci., Professor, Corresponding Member of NASU)

A nanocomposite complex of endothelial and monocyte-activating polypeptide II (EMAP II) with dextran-70 was created. Stabilization of EMAP II cytokine in complex with dextran-70 and increase the temperature of local conformational transition in EMAP II up to 49ºC were shown. Aggregation properties of the EMAP II cytokine were studied by light scattering and it was established that dextran-70 prevents protein aggregation in the temperature range of 20-55 ºC. The antitumor activity of EMAP II and its complex with dextran-70 was investigated on the xenografts of human prostate adenocarcinoma in mice at a dose of 10 μg/kg and inhibition of tumor growth was shown by 71% and 77%, respectively.

Nanocomposite preparations of EMAP II has a number of advantages, such as increased stability of protein as a therapeutic agent, prolonged therapeutic effect and low toxicity. Separate stages of preclinical trials of the drug have been carried out. The lyophilized preparation EMAP II is dissolves well in physiological solution within a few seconds with the formation of a transparent solution.

Commercialization proposals: Funding is needed for preclinical and clinical trials of EMAP II and its nanocomposite complex with dextran-70.

Protection of intellectual properties: Patents of Ukraine for utility models No 64374 dated November 10, 2011 and No 141271 dated March 25, 2020.

C-module of tyrosyl-tRNA synthetase as a new antiangiogenic cytokine (Olexander I. Kornelyuk, Dr. Sci., Professor, Corresponding Member of NASU)

A new biotechnological product sendomap cytokine was created and tested in vitro. Bacterial expression and purification of sendomap cytokine allows you to obtain recombinant protein in preparative quantities about 10 mg from 1 liter of bacterial culture. Sendomap cytokine can be widely used as a mediator of procoagulant and antiangiogenic action, an inducer of apoptosis and possibly in future as a putative antitumor drug in clinical oncology.

System for detection of mutations in patients with myeloid neoplasms "MN-multitest" (Gennadiy D. Telegeev, Dr. Sci., Professor)

A system was created for the differential molecular genetic diagnosis of myeloid neoplasms, which is designed to detect: chimeric BCR/ABL1 gene; mutations in the kinase domain of the BCR/ABL1 chimeric gene; JAK2 gene mutations; CALR gene mutations; MPL gene mutations. The use of the proposed system allows for complex differential molecular genetic diagnosis of myeloid neoplasms no worse than world analogues at a lower cost. The method was tested on more than 150 patients from different regions of Ukraine.

Test system for detection of BTK gene mutations in patients with primary immunodeficiencies "BTK-test" (Gennadiy D. Telegeev, Dr. Sci., Professor)

A system has been created for molecular genetic diagnosis of blood samples from patients with hereditary hypogammaglobulinemia. This system is based on detection of BTK gene mutations. using specific primers using reverse transcriptase polymerase chain reaction with direct sequencing of PCR products. The use of the system makes it possible to carry out molecular genetic diagnostics in patients with hereditary hypogammaglobulinemia at the level of world analogues at a lower cost and to detect possible carriers of mutations in the BTK gene.

Development of methods for complex molecular diagnosis of chronic myeloid leukemia and acute lymphoblastic leukemia based on PCR and specific polyclonal antibodies (Gennadiy D. Telegeev, Dr. Sci., Professor)

The method was tested on more than 200 patients from different regions of Ukraine on the instructions of the Institute of Hematology and Transfusion of the Ministry of Health of Ukraine and regional hematology departments. The method provided full correlation with other diagnostic methods and provided high sensitivity (10–4–10–5 detection of transformed cells). This method allows you to identify the nature of the rearrangement and completely exclude a false negative result, which is extremely important for the prognosis of the course of the disease and the development of therapy protocols.

Biotechnology of obtaining secondary metabolites (Gennadiy D. Telegeev, Dr. Sci., Professor)

A method of increasing the yield of glycyrrhizin (Gl) and flavonoids from cells and roots of Glycyrrhiza licorice transformed with plasmids of agrobacteria was developed. An endophytic mycorrhizal fungus was isolated from the roots of the medicinal plant Potentila alba L., which serves as an elicitor (stimulator) to increase the biosynthesis of secondary metabolites in transformed cells and root segments of licorice by an average of 1.5 times.

Biotechnological production of glycyrrhizin is of great practical importance because this triterpene saponin is a sugar substitute for diabetics, and its glycyrrhizic acid aglycon inhibits II-beta dehydrogenase activity in liver and kidney tissues both in vitro and in vivo. Flavonoids and 18-dehydroglyceric and glycyrrhizic acids have a pronounced antioxidant effect. It is the anti-inflammatory effect that explains their positive therapeutic effect in case of radiation damage to the lungs, liver pathology, as well as anti-sclerotic, anti-inflammatory, anti-cancer and anti-ulcer effects.

Development and implementation of test systems for non-invasive diagnostics of renal cancer based on the determination of extracellular DNA and plasma microRNA (Inessa Y. Skrypkina, Ph. D., Senior Research Scientist)

The aim of the study is to identify genetic and epigenetic changes in kidney neoplasms associated with a clear cell type of cancer, and in the future can be used for more accurate diagnosis and personalized treatment.

Recently, the possibility of using circulating cell-free nucleic acids (ccfNAs) in blood plasma as potential diagnostic markers for various clinical diseases, including malignant neoplasms, has been demonstrated. The significance of extracellular NCs as tumor markers is assessed by comparing their concentrations in the blood plasma of cancer patients and healthy donors and by determining genetic (mutations, deletions) and epigenetic (hypermethylation) changes in tumor-associated genes associated with the development of kidney cancer. The advantages of such systems are high sensitivity and specificity due to the use of several biomarkers, the possibility of using the system for early cancer diagnosis, a non-invasive sample selection method, and affordable equipment cost.

As a result of the research, the proposed diagnostic methods were implemented in clinical practice at the Institute of Urology of the National Academy of Sciences of Ukraine in Kyiv, and a patent was obtained for a useful model:

  • "The method of using molecular markers in the early diagnosis of renal cell cancer" (2017)
  • "The method of using miRNA in the early diagnosis of renal cell cancer" (2020).
  • Utility model patent No. 134876 "Method for early diagnosis of kidney cancer". Grigorenko V.M., Skrypkina I.Ya., Pereta L.V., Onishchenko K.V., Kashparova O.V. Registered in the State Register of Patents of Ukraine for a utility model on June 10, 2019.

Telomerase inhibitors based on specific ligands of quadruplex DNA (Igor Ya. Dubey, Dr.Sci., Senior Research Scientist)

Telomeric DNA and telomerase are currently considered promising targets for cancer therapy. Telomeres are guanine-rich DNA sequences located at the ends of the chromosomes that can adopt a specific quadruplex structures (G4). Tumor cells express high levels of telomerase enzyme responsible for maintaining the telomere length, whereas normal somatic cells are devoid of telomerase activity. Small molecules specifically binding to G4 structures can inhibit telomerase in cancer cells and thus demonstrate antitumor properties.

We have developed new telomerase inhibitors of two structural classes based on porphyrins and acridines.

Inhibitors of the first class are cationic porphyrin derivatives, metal complexes and conjugates that efficiently bind to G-quadruplexes. Some compounds inhibit telomerase in vitro at micro- and submicromolar concentrations (TRAP assay). Metal complexes of porphyrins conjugates with intercalating agents demonstrated antiproliferative activity in tumor cell culture with EC50 in the range 5-11 μM, comparable to that of doxorubicin and vincristine.

New polysubstituted acridines contain strongly basic N,N-dialkylaminoalkyl group, pyridyl fragment and activity-modulating small substituents. They inhibit telomerase in vitro at low micromolar concentrations (IC50 1.6-3.5 μM) and have high affinity to G4-DNA (binding constants (1-5)×106 M-1). Efficient inhibitors demonstrate high antiproliferative activity in vitro in the nanomolar range, with EC50 50-700 nM.

We obtained the patents of Ukraine UA118375 “4,5,9-Substituted acridine derivatives and method of their synthesis” (inventors Kostina V.G., Alexeeva I.V., Lysenko N.A., Kuziv Ia.B., Dubey I.Ya.) and UA133622 “Method of inhibition of human telomerase in vitro by polysubstituted acridine derivatives” (inventors Negrutska V.V., Saraieva I.V., Kostina V.G., Lysenko N.A., Alexeeva I.V., Dubey I.Ya.); patents owner IMBG of NASU.

New inhibitors of topoisomerase I with antitumor activity (Igor Ya. Dubey, Dr.Sci., Senior Research Scientist)

Topoisomerase I (TooI) is a nucleic acid biosynthesis enzyme performing the relaxation of supercoiled DNA and required for the replication, transcription and reparation of DNA. TopoI changes the topology of DNA via the formation of temporary single-strand breaks. The level of expression of this enzyme is significantly increased in quickly dividing cells, in particular, tumor and bacterial cells. Specific inhibition of TopoI allows suppressing the bacterial and tumor growth. So TopoI is an important biological target for the development of new antitumor agents. A number of TopoI inhibitors clinically used in oncology are known (camptothecin, topotecan, etc.).

Topoisomerase inhibitors are typically based on heterocyclic structures efficiently binding to DNA and contain the polycyclic fragment of specific macrostructure. We have designed a series of new TopoI inhibitors based on acridines and cyanines. Their development involved the biophysical analysis of the affinity of compounds to duplex DNA, screening in the system of DNA relaxation by topoisomerase I in vitro, and computer modeling of ligands interaction with a molecular target. The drugs inhibit the enzyme at low micro- and nanomolar concentrations, with IC50 0.4-4.5 μM. Upon the testing of active inhibitors in tumor cell cultures we have identified the compounds with antiproliferative activity in the nanomolar range (EC50 0.4-0.9 μM).

The patent of Ukraine UA117194 “The use of new 4,5,9-functionally substituted acridine derivatives as efficient inhibitors of topoisomerase I enzyme in vitro” was obtained (inventors Negrutska V.V., Kostina V.G., Alexeeva I.V., Lysenko N.A., Dubey I.Ya., patent owner IMBG of NASU).

The system of screening of potential antitumor and antimicrobial agents based on the specific ligands of duplex and quadruplex DNA (Igor Ya. Dubey, Dr.Sci., Senior Research Scientist).

Enzymes of the system of nucleic acid biosynthesis (DNA and RNA polymerases, helicases, topoisomerases, telomerase) are the established molecular targets for novel therapeutic agents. Their inhibitors often demonstrate the biological activity, in particular, antitumor, antimicrobial and antiviral properties. The search for such drugs commonly employs their testing in the in vitro enzymatic systems; however, it is not suitable for the primary screening of large compound libraries. At the same time, molecules of the inhibitors of nucleic acid biosynthesis enzymes are usually able to efficiently bind to NAs that can be used for their identification.

We have elaborated a screening system for low molecular compounds based on the evaluation of their affinity to DNA using a FID (Fluorescent Intercalator Displacement) method followed by biological testing of the affine ligands. FID screening is performed in parallel against duplex and G-quadruplex (G4) DNA. Screening of the series of compounds in microplate format (semi-automated or manual) allows fast identification of of compounds with high affinity to duplex or G4-DNA. Identification of DNA-ligands opens the way to specific inhibitors of nucleic acid biosynthesis enzymes. The testing of compounds selected during the fast FID screening in enzymatic systems based on polymerases or topoisomerases (for dsDNA ligands) and telomerase (for G4-ligands) allows to find highly efficient inhibitors of corresponding enzymes. The subsequent testing in cell cultures results in identification of compounds with specific activity – antitumor, antibacterial, etc.

The long-term routine use of the developed screening system allowed to discover numerous affine ligands of duplex and quadruplex DNA with binding constants (1–6)×106 M-1 and potent inhibitors of TopoI and telomerase with antitumor activity at nanomolar concentrations, as well as antimicrobial agents. The system is sufficiently universal to be used in the search for potential drugs targeting other enzymes of nucleic acid biosynthesis.

Reagents based on 3-hetarylcoumarins for the fluorescent labeling of biomolecules and cell imaging (Igor Ya. Dubey, Dr.Sci., Senior Research Scientist)

Last achievements of life sciences, biotechnology and medicine are to a significant extent due to the progress of fluorescence-based technologies. The most sensitive and informative methods of detection and quantification of biomolecules and investigation of their structures, functions, metabolism and interactions with other biological and synthetic molecules are based on fluorescent techniques. Of particular interest are the conjugates of biomolecules with fluorescent dyes widely used in the studies of biomolecular interactions, PCR, sequencing of nucleic acids, fluorescence microscopy, immunofluorescent analysis, in sensor and microarray technologies, etc.

We have developed a series of new reagents for the covalent fluorescent labeling of biomolecules based on the derivatives of 3-hetarylcoumarins. They contain the reactive carboxyalkyl linkers and after the activation under mild conditions react with amino groups of biomolecules, and at certain conditions with OH-groups. The dyes are excited with long-wavelength UV light and fluoresce in blue spectral region (438-475 nm) where the limited number of known fluorophores emits, with high fluorescence quantum yields (up to 0.85). The range of new reagents includes both pH-sensitive and pH-insensitive labels. Their efficiency was fully confirmed by labeling the biomolecules of various classes (carbohydrates, nucleosides, peptides, oligonucleotides), as well as natural and synthetic polymers.

Based on these fluorophores, new water-soluble reagents for the fluorescent cell imaging were developed – the conjugates of D-glucosamine with 3-hetarylcoumarins. The developed conjugates penetrate the cell via the system of active glucose transport (GLUT) and have demonstrated their efficiency in fluorescence microscopy.

Transplantation of preconditioned human umbilical cord MSCs as a way to overcome acute pancreatitis and its consequences (Svitlana Y. Rymar, Ph. D., Senior Research Scientist)

Pancreatitis is a disease that is defined by an acute or chronic inflammatory process of the pancreas, characterized by premature activation of digestive enzymes of pancreatic acinar cells, which leads to their damage. The acute form of pancreatitis causes necrosis of the pancreatic parenchyma, abscess and systemic complications, these lead to patient mortality, which reaches 30-47%. Currently, there are no effective methods of treatment for both acute and chronic pancreatitis. Mesenchymal stem cells have unique properties to suppress inflammation and restore damaged tissues, which makes it possible to consider MSCs as a new tool for the treatment of acute and chronic pancreatitis.

A method of restoring the pancreas after acute pancreatitis by transplantation of preconditioned human umbilical cord MSCs is proposed. It was shown that the use of such cells significantly accelerates the process of restoring both the structure of the pancreas and its function in comparison with the transplantation of the initial MSCs (7 days versus 10 days). The method may be proposed for clinical trials for the treatment of acute pancreatitis with MSCs.

Development of skin equivalents including human stem cells or their derivatives for treatment of traumatic damages of skin (Lubov L. Lukash, Doctor of Biological Sciences, Professor)

The biotechnology for obtaining skin equivalents (dermal coatings) with the inclusion of stem cells of the original 4BL line and human skin fibroblasts or their derivatives has been developed. This cellular technology is intended for medical use: treatment of traumatic skin lesions, especially massive burn wounds (according to WHO, burns rank third place among injuries of different genesis.) We obtained Ukrainian patents for the manufacture of dermal coatings with cellular components (UA №82583, UA № 112584, UA №1287876). New biotechnological products have improved performance characteristics: they are based on natural inexpensive materials using cellular components or their derivatives, effective in the treatment of burns and are safe for the body. Thus, in their research on models of thermal burns in animals, the effects of earlier healing of wounds and anti-inflammatory action were revealed. In clinical trials in a limited contingent of burn patients, the use of dermal coatings with cells shows stimulation of regeneration of the skin and increased efficiency of autogenic transplants implantation (100%).

At present, separate stages of pre-clinical testing of dermal coatings with cellular components on animal models in vivo are carried out, but they pass very slowly due to lack of necessary means. Financial assistance would accelerate pre-clinical and further clinical trials. Biotechnological products that are being developed in this project will cost about 10 times less than their foreign counterparts. In the future, they can be used for introduction into practical medicine for the purpose of treatment of burn wounds, lesions of the skin of another genesis, as well as cosmetic purposes.

Prototype of dual nanoplatform with therapeutic enzymes for drug delivery based on bacterial outer membrane vesicles (Nataliya O. Kozyrovska, Ph.D., Senior Research Scientist)

Inflammation remains a key event in most diseases. The goal of this project is to develop and demonstrate the feasibility of using a nanoplatform for drug delivery based on bacterial extracellular outer membrane vesicles (OMVs) of bacteria, which are able to penetrate the blood-brain barrier and are associated with therapeutic enzymes. OMVs, independently or in combination with drugs, will provide anti-inflammatory therapy and deliver toxic drugs safely to the addressee without side effects for patients. The nanoplatform will ensure the use of OMVs against many diseases as an adjuvant.

Nanocellulose-based composites (Nataliya O. Kozyrovska, Ph.D., Senior Research Scientist; Leonid A. Zaika, Ph.D., Senior Research Scientist)

Antimicrobial biodegradable composites based on nanocellulose and antimicrobials (nanoisatison; polyhexamethylenguanidine chloride, PHMG-Cl) for use in medical practice have been designed. New composite of nanocellulose with multipurpose drug nanoisatizon might be effective for treatment of wound surfaces, ulcers and burns, melanoma, as well as against herpes and other viral infections of the skin. The PHMG-Cl prevents the biofilm formation by a microorganisms and could be further exploited as a wound dressing for healing and the regeneration of a scarless skin.

Express method of the Klebsiella oxytoca authentication by PCR (Nataliya O. Kozyrovska, Ph.D., Senior Research Scientist)

Unique sequence of the pehX gene we cloned from industrial strain of bacterium K. oxytoca IMBG26 enabled us to develop a method of the bacterium identification by polymerase chain reaction. Rapid, sensitive and specific test is recommended for discrimination of K. oxytoca between similar species of bacteria of the genus Klebsiella, as well as for environmental monitoring of K. oxytoca.

Development of arginine-selective sensors for the analysis of food and dietary supplements used in preventive and rehabilitation medicine (Olexy P. Soldatkin, Dr. Sci., Professor, Full Member of NASU; Sergiy V. Dzyadevych, Dr. Sci., Professor, Corresponding Member of NASU)

Accurate, rapid and selective determination of individual components in the amino acid profile of functional foods, food supplements and foods for a special diet is essential for the development of an easy quality control of such samples. The use of special purpose foods enriched with amino acid L-arginine (Arg) has become widespread over the last 5-10 years due to the proven effectiveness of using increased doses of L-arginine for improvement in health status and treatment of a number of functional disorders in the body.

In particular, L-arginine was found to exhibit positive effects in all manifestations of endothelial dysfunction of atherosclerotic genesis (cardiac, cerebral, peripheral), hypertension (arterial, pulmonary, renal), liver diseases, diabetes mellitus, obesity, immunodeficiency states, osteoarthrosis, etc. Arginine plays an extremely important role in the body because despite it is the protein component, it is also a precursor of such important compounds as urea, proline, glutamate, creatine, agmatine, nitric oxide NO (a signaling compound, which ensures neurotransmission, vasodilation, cytotoxicity and other functions in the body).

Despite the satisfactory criteria of analytical performance, the use of the traditional methods for arginine detection in the complex matrices such as food and pharmaceuticals is frequently associated with the time consuming sample pre-treatment performed by skilled personnel using costly and bulky equipment and reagents. To ensure the timely decision-making at the control point during analysis of functional foods, dietary supplements and foods for a special diet, the analysis would be limited to 1-2 hours relying on the tests away from the centralized laboratories. Due to this, transitioning to the portable, rapid and at the same time reliable detection instruments has been always of a high demand especially among field workers, farmers, and food inspection agencies. Respective to the agriculture and retail control systems with remote, resource-limited settings where trained personnel is not available, user-friendly, rapid, sensitive, specific, low cost assays are of critical importance.

We developed a conductometric biosensor device based on the functional nanomaterials of various classes, in order to perform highly sensitive and selective determination of arginine in varying model and real samples. The functional layers were based on enzymes (arginase and urease) and nanomaterials (clinoptilolite and calixarene) that were immobilized on the sensor surfaces and were proposed as biosensor selective elements. Thin-film planar conductometric transducers of interdigital configuration, made of varying materials with diverse electrode geometry, served as physical transducers.

As a result of investigations performed by the our team, new nanomaterials with pronounced arginine-selectivity were determined. The utilization of these new nanomaterials will allow the creation of highly sensitive and selective biosensor conductometric devices for arginine determination in model and real samples of different sources (Fig. 2).

The developed conductometric biosensor device was used to measure arginine concentration in various drugs. The obtained results correlated well with the data provided by the manufacturers, as well as with data generated by more traditional methods of arginine determination.

A portable device for determination of arginine concentration was developed and created by our team, which is working in an automated measurement setup and can serve as a prototype for industrial fabrication.

Application of novel nanomaterials in conductometric biosensors opens up new possibilities for commercialization of devices, since developed biosensors has higher sensitivity, signal reproducibility and storage stability compared with other types of sensors.

Development and creation of electrochemical and optical biosensors for the needs of medicine (Olexy P. Soldatkin, Dr. Sci., Professor, Full Member of NASU)

  • Development of potentiometric mono- and multi-biosensors with special sensitivity to glucose, urea and creatinine for medical diagnosis and control of hemodialysis processes.
  • Development of amperometric microbiosensors for in vivo determination of the main metabolites (glucose, lactic acid, ATP) and monitoring of some neurotransmitters (acetylcholine, choline, glutamic acid and D-serine) in the brain of mammals.
  • Development of immuno- and DNA sensors based on surface plasmon resonance for detection of some mutations and pathogenic microorganisms.

Development and creation of analytical systems on electrochemical mono- and multi-biosensors for environmental monitoring and food analyses (S. V. Dzyadevich, Dr. Sci, Professor, Corresponding Member of NASU; Oleksandr O. Soldatkin, Dr. Sci., Senior Research Scientist; Tetyana A. Sergeyeva, Dr. Sci, Senior Research Scientist)

  • Development of potentiometric and conductometric enzyme mono- and multi-biosensors for direct and inhibitory analysis of individual toxic substances and their mixtures (herbicides, pesticides, steroid glycoalkaloids, formaldehyde, heavy metal ions, etc.).
  • Development of technology for the synthesis of synthetic analogs of biological receptors (polymers-biomimics) and creation of a number of electrochemical (conductometric, capacitive, and amperometric) sensors based on them for the determination of herbicides of the triazine series.
  • Development and creation of electrochemical biosensors for control biotechnological processes and quality control of food products
  • Development of amperometric biosensors and sensor arrays for the determination of glucose, glycerin, ethyl alcohol and lactic acid in samples of wine and wine products.
  • Development of potentiometric and conductometric biosensors for the determination of penicillin.
  • Development of a potentiometric enzyme biosensor based on inhibitory analysis for the determination of natural neurotoxic substances - glycoalkaloids - in potatoes, tomatoes and other nightshades.
  • Development of conductometric biosensors and sensor arrays for determination of carbohydrates in the food industry.

Development and creation of multi-target sensor platforms for early diagnosis of diseases (Yaroslav I. Korpan, Ph.D., Senior Research Scientist)

Development of an amperometric sensor platform based on graphene and textiles for the analysis of metabolites in sweat.

Development of a sensor array for discrimination of PTSD and depressive states as a result of military operations.

Development of the foundations for biotechnological production of Rauwolfia serpentine alkaloids exhibiting a wide range of therapeutic activities (Viktor A. Kunakh, Dr.Sci., Professor, Corresponding Member of NASU; Iryna I. Konvaliuk, Ph.D., Research Scientist; Igor O. Andreev, Ph.D., Senior Research Scientist; Liudmyla P. Mozhylevska, Research Scientist)

A technology was developed for producing tissue culture of Rauwolfia serpentina, a tropical medicinal plant, which has high content of indole alkaloids, in particular ajmaline used as the main compound of antiarrhythmic and hypotensive medicines. Biomass production technology that is based on the use of highly productive cell strains with a total alkaloid content of about 3% established in the department were tested at industrial scale.

A new highly technological and the world's most productive K-27M R. serpentina cell line was established, which accumulates more than 4% of total alkaloids in dry biomass. Biomass contains about 1% ajmaline, 0.29% vomilenine, 0.02% ajmalicine, 0.006% yohimbine, 0.006% reserpine, and residual amounts of other alkaloids (20 different alkaloids in total). Based on this cell line, the foundations were developed for biotechnological production of R. serpentinaalkaloids exhibiting a wide range of therapeutic activities, including new hypotensive and antiarrhythmic alkaloids.

A number of pharmacological studies of the extract of the cell biomass of the K-27M R. serpentinacell line were carried out. It was found that the established tissue culture can be used as a source of indole alkaloids exhibiting vasodilator, sedative, and antiarrhythmic activities, which has an α-adrenoblocking effect. The cellular biomass of the K-27M R. serpentina cell line can be used for the development of medicines and dietary supplements.

Development of biotechnological production of Ungernia victoris cellular biomass as a source of biologically active compounds exhibiting a wide range of therapeutic activities (Viktor A. Kunakh, Dr.Sci., Professor, Corresponding Member of NASU; Iryna I. Konvaliuk, Ph.D., Research Scientist; Oksana O. Poronnyk, Ph.D., Senior Research Scientist; Liudmyla P. Mozhylevska, Research Scientist)

A technology was developed for cellular biomass production and vegetative propagation of Ungernia victoris, a rare perennial bulbous plant endemic to the Pamir mountains, a source of alkaloids galantamine and lycorine. Medicines based on galantamine are used in the treatment of myasthenia gravis, progressive muscular dystrophy (myopathy), and radiculitis. Lycorine derivatives are used for treatment of chronic and acute respiratory diseases, as well as bronchial asthma.

Extracts from the cell biomass of the established tissue culture strains of U. victoris have antimutagenic, anticarcinogenic, genoprotective, antiviral, and antimicrobial activities. The cell biomass of U. victoris tissue culture can be used for the development of medicines and dietary supplements.

Establishment of cell strains of Echium plantagineum as a source of raw materials for the production of shikonin, a valuable naphthoquinone compound (Viktor A. Kunakh, Dr.Sci., Professor, Corresponding Member of NASU; Oksana O. Poronnyk, Ph.D., Senior Research Scientist; Iryna I. Konvaliuk, Ph.D., Research Scientist)

A technology was developed for production of Echium plantagineum plant tissue culture biomass with a high content of shikonin (up to 5% in dry biomass). Shikonin and its derivatives are used as a dye, as a component of medicines with antitumor, antibacterial, anti-inflammatory, wound-healing, antioxidant, and antiviral activities, and as a component of cosmetic. Cell biomass of E. plantagineum tissue culture can be used as an alternative source of shikonin and its derivatives.