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Open Access
ANAEROBIC FAT FILTERS IN REGENERATIVE MEDICINE
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Храмцова, Н.И.; Плаксин, С.А.; Храмцов, А.А.; Соцков, А.Ю.; Пономарев, Д.Н.
Introduction: Adipose tissue is widely used in plastic, aesthetic and regenerative surgery. Autologous fat grafting is one of the most populat procedures in modern surgery. Adipose tissue is usually harvested by lipectomy and consists of two fractions: fat cells, which are used in volumization, and stromal vascular fraction, which is the main source of stem cells. Then it should be specially processed through anaerobic fat transfers to obtain the maximum number of viable adipocytes of stem cells. Methods: The number of non-destroyed adipocytes and fibroblast-like cells was obtained by hematoxylin and eosin dyeing in fat smears in every passage through each anaerobic fat transfer. Results: With each passage through a 1.4 mm fat filter, the number of non-destroyed adipocytes and fibroblast-like cells decreased. When using a 1.2mm filter, 30 passages did not significantly reduce the number of non-destroyed cells. Each passage through a nanofilter with a mesh diameter of 0.5 mm reduced the number of non-destroyed fibroblast-like cells. The number of non-destroyed cells decreased significantly during the transition from filter to filter. When the fat filter was changed, and the cell diameter was reduced, adipocytes were destroyed to a significantly greater extent, and fibroblast-like cells did not suffer. The use of 1-3 passages through a nanofilter kept viable up to 20% fibroblast-like cells, while the use of a nanofilter with a smallest diameter of 0.15 mm allowed fat cells to be completely destroyed already after 3 passages. Conclusion: The choice of fat filter type should be based on the desired result. To obtain the viable adipocytes while maintaining fibroblast-like cells, it is advisable to use a fat filter with a maximum diameter. It is advisable to use a 1.2mm filter to maintain the same number of fibroblast-like cells, but less number of adipocytes. The use of nanofilters is necessary in those cases when it is necessary to disconnect adipocytes as much as possible, or destroy them, preserving only stromal stem cells.
Open Access
CLINICAL EVALUATION OF THE EFFECTIVENESS OF MARE’S MILK IN PSORIASIS
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Algazina, T.; Yermekbayeva, B.; Batpenova, G.; Kozhakhmetov, S.; Kushugulova, A.
According to the world health organization, the prevalence of psoriasis in the world is about 2%. However, according to a number of studies, this figure is significantly higher in developed countries and can range from 4.6 to 7%. According to official statistics in the Republic of Kazakhstan, psoriasis takes the third place in the structure of dermatoses, giving way to eczema, associated with this ailment of allergic skin diseases and mycoses. Moreover, the majority of patients diagnosed for the first time in their life are people aged 16 to 30 years. The aim of the study was to evaluate the clinical effect of Mare’s milk in the complex treatment of psoriasis patients of mild and moderate severity. The study included 20 patients with mild to moderate psoriasis, whose average age was 34.95±4.11years. In addition to standard psoriasis therapy, patients received freeze-dried Mare’s milk at 60 mg / day for 12weeks. Evaluation of the effectiveness of Mare’s milk was carried out using the dynamics of the Psoriasis Area Severity Index (PASI). Evaluation of the effectiveness of including Mare’s milk in standard therapy for psoriasis was carried out taking into account the dynamics of the dermatological status at the 6th and 12th weeks of treatment: a decrease in the PASI index by 68% and 88% (p=0.0003), respectively. In general, the dynamics of the overall PASI index for mild psoriasis decreased PASI from 10±0.62to 3.52±0.7, with an average severity of 29.51±0.7 to 3.05±0.5. The degree of severity of individual components of the psoriatic process – erythema, infiltration and peeling decreased. Complex therapy of psoriasis with the inclusion of Mare’s milk, which has a prebiotic and immunomodulating effect, to the standard therapy of psoriasis, helps to achieve an earlier and more stable resolution of the skin manifestations of the disease.
Open Access
Correlation of Serum Biomarkers With the Risk Factors of Colorectal Cancer
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Akhmaltdinova, L.L.; Avdienko, O.V.; Ibraeva, A.S.; Zhumaliyeva, V.A.; Tauesheva, Z.B.
Introduction: The search of effective serum biomarkers to diagnose, forecast and evaluate the severity of cancer is a hard and so far unsolved problem. Studying their dynamics, limitations as well as their interaction with clinical signs could help understand their points of application and their effective use. Methods: a group of patients first time diagnosed with colorectal cancer took part in the investigation (n=100). The age: (Ме [25%;75%]) 66.6 years old [62;72]; 45% men and 55% women. A standard clinical examination and patient reported outcomes to reveal the risk factors were performed. Biomarker blood test was done prior to surgery. To study serum biomarkers, 24 Milliplex Human Circulation Biomarker Set was used. PD-L1was determined using Human ProcartaPlex™ Kit. Oncomarker CA 72-4 was identified using ELISA kit by Xema. For Statistic processing Gamma Correlition was taken. It was found that BMI correlated with the contents of Afp (gamma correlation) (0.41), CEA (-0.25), He4 (-0.39), SFas (0.32) and PDL-1(-0.46). A regular polyvitamin ingestion correlated with PDL-1(0.40) and IL-6 (0.35). Regular NSAIDs ingestion related to IL-6 level (0.30). CA125 (0.44), Leptin (-0.55), MIF (0.44) and OPN (0.59) were found to correlate with smoking, and CA19-9 (0.66), IL-6(0.52), MIF (0.53), TNF (0,49) and VEGF (0,53) - with the number of cigarettes smoked. As to the severity and the localization, the following correlations were found: AFP was related to the tumour localization C18.4 (0.8) and the accompanying diverticulosis (0.68) and the presence of metastases (0.62). CA19-9 correlated with С18.2localization. CEA and IL-8 were found to correlate with КРР (0.33 and 0.34 correspondingly), and with the size of the tumour (0.29 and 0.31). IL-6, MIF and TGF markers were linked with the presence of metastases (0.84;0.66; 0.57). CA15-3 was found to correlate with diverticulosis (0.75). Speaking about separate markers correlation, the strongest correlations were revealed between sFasL and TNF (0.41); TGF and FGF (0.49). Thus, on the one hand, the diversity of factors influencing the character of serum biomarkers in patients with oncological disease interfere background values, but on the other, can serve a basis for a complex diagnostic panel.
Open Access
CRYOGELS CONTAINING POLYELECTROLYTE COMPLEX FOR TISSUE ENGINEERING
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Berillo, D.; Zheng, Y.
The design of scaffolds for tissue engineering is an important task. The focus of our research is the design of various biocompatible scaffolds based on natural polymers such as gelatin, chitosan and casein using cryogelation technique.1-4 Cryogelation involves a process of the formation of macroporous polymer systems, so called cryogels, with well-developed 3D structure of interconnected pores. Typically, cryogels have porosity of 90% and macrochannels of 30-200 μm in size, depending on the preparation conditions. Cryogels has ability of unrestricted penetration of solutes as well as high surface area for attachment and proliferation of mammalian cells.2,3 Previously, the preparation of gelatin based cryogel was performed in environmentally friendly way using enzymatic reaction under cryoconditions.3 We used dextran dialdehyde as a mild nontoxic cross-linker and additional physical cross-linking via formation of polyelectrolyte complex(PEC) between oppositely charged groups of polymers. The advantage of PEC based scaffold preparation is the simultaneous existence of positive and negative charges on the surface at physiological pH, facilitating attachment of tissue components via electrostatic interactions, that is favourable for a tissue engineering2. Human hepatic epithelial cell line and fibroblasts were used for evaluation of biocompatibility. It is important to mention that the gelatin type A and B significantly different and therefore affecting the migration, proliferation of cells and also microscopic morphology of the material. This phenomenon may be related to different chemical composition effecting isoelectric point of gelatin. In the present study gelatin was utilised from cold skin fish and bovine type A were used. The PEC scaffold containing gelatin from fish exhibited better fibroblast growth compare to cryogels based on only gelatin and aldehyde dextran. The same composition cryogels based on gelatin(bovine A) and dextran dialdehyde revealed proliferation of hepatocytes inside of the material, whereas hepatocytes formed clusters on the surface of the PEC cryogel. References: 1. Progress in the development of chitosan-based biomaterials for tissue engineering and biomedical application. Biomolecules (2019). 2.Oxidized dextran as crosslinker for chitosan cryogel scaffolds and formation of polyelectrolyte complexes between chitosan and gelatin. Macromolecular bioscience(2012). 3.Biocompatible scaffolds for regenerative medicine. International (Int) journal of biological macromolecules( 2018).
Open Access
DELIVERY OF MONOCLONAL ANTIBODIES FROM MICROENCAPSULATED CELLS
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Ashimova, A.; Hortelano, G.
Monospecific immunoglobulins (monoclonal antibodies, MAb) of therapeutic interest have received significant attention since their development by Milstein and Kohler in 1975. The exquisite specificity of MAb, or the ability to bind exclusively to its target makes it a very promising tool in medical diagnosis and therapy. A large number of MAbs have been licensed and are used in the clinic today, particularly as anticancer agents. Nonetheless, the very high cost associated to the production and purification of MAbs is a significant challenge to their successful commercialization, since a treatment can cost upwards of $100,000 per patient. Since MAb purification is the greatest technical and economic challenge this proposal aims at exploring a novel strategy for the sustained and constant delivery of MAbs. The microcapsules are produced with electrostatic bead generation of the hydrogel alginate, which after gelation are cross-linked with cationic poly-L-lysine to produce stable microcapsules that are 200-400 micrometers in diameter. In addition to the polymer, the choice of judicious cells suitable for encapsulation is critical for the success of the proposed strategy. The microcapsules are permeable to IgG and nutrients, but not to immune cells. As a result of the immune isolation the enclosed allogeneic cells are not rejected, making tissue matching unnecessary for the treatment. This proposal is aimed at investigating the potential of encapsulated myoblasts and MSC cells to produce –and secrete- clinically relevant levels of monoclonal antibodies (MAbs) aimed at preventing tumor growth such as epidermal growth factor receptor (EGFR) as well as MAb against CD-20, to deplete tumorigenic B lymphocytes. Both MAbs have been proved efficacious in patients with carcinoma and lymphoma cancers, respectively. If successful, encapsulated cells will deliver constant, sustained clinically relevant amounts of functional MAb, making antibody delivery cost-effective. This novel strategy could have wide applications in the treatment of medical conditions such as various types of cancer and autoimmune diseases, which are already treated with monoclonal antibodies.
Open Access
DEVELOPMENT OF COSMETIC CREAM BASED ON THE HORSE-PLACENTA
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Saliev, T.; Fakhradiyev, I.; Perfilyeva, Y.; Ostapchuk, Y.; Kali, A.; Abdolla, N.; Tleulieva, R.
Modern cosmetology possesses a huge arsenal of tools and methods for the prevention and treatment of various aesthetic problems. There is a range of medical and cosmetic products consisting of biologically active agents and additives. A number of studies have been devoted to the research on the possibilities of using placenta extracts obtained by lysis of human placental tissues, sheep, goats, and horses for the treatment of different pathologies. Taking into account the therapeutic potential of placental extracts, we developed the composition and technology of fabrication of a cosmetic cream based on the horse placenta extract. The extract was obtained by taking the placenta immediately after vaginal delivery from mares (horse farms, Republic of Kazakhstan). The horse placenta was thoroughly washed and separated with ice-cold phosphate-saline buffer by using tissue homogenizer, centrifugation and lyophilization. Sterilization was performed using γ-radiation employing a source of cobalt-60 isotopes (ILU-10 accelerators, Russia). An emulsion was used as the base of the composition, taking into account its incorporating ability and effect on the human skin. To obtain an emulsion cream (based on horse placenta), we selected lipophilic components as structure-forming substances, including cocoa butter, petroleum jelly, fats as emulsifiers. The cream was prepared by mixing the placental extract with lipophilic components of the base and emulsifiers, followed by the addition of an aqueous phase. The developed cream was tested for organoleptic and physical-chemical properties: colloidal stability during centrifugation, and thermal stability. Organoleptic indicators were evaluated for appearance, colour, and smell. The determination of colloidal and thermal stability was carried out by methods based on the separation of the system into fat and water phases during centrifugation and incubation. The determination of the hydrogen index was carried out in a water extract. The study of rheological properties showed a consistency of the solution that was optimal from a practical and clinical point of view. The study demonstrated the feasibility of fabrication of the cream based on the horse placenta, and its excellent physical and chemical properties. Further research is needed to validate the therapeutic activity and safety of the proposed product.
Open Access
DRUG RESISTANCE OF MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES FROM NEW TB CASES IN KAZAKHSTAN
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Kozhamkulov, U.; Akhmetova, A.; Rakhimova, S.; Daniyarov, A.; Molkenov, A.; Bismilda, V.; Chingissova, L.; Kairov, U.; Akilzhanova, A.
Introduction: Nowadays and worldwide, TB is one of the top 10 causes of death and the leading cause from a single infectious agent. The global incidence of drug-susceptible TB, multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in the past decade led to decreased efficiency of chemotherapy. In Kazakhstan, the 2019 WHO report estimates the TB incidence in 2018 to be 68 per 100 000 population and the MDR-TB incidence to be 26/100,000 (WHO, 2019). Therefore, the goal of the study is to analyze clinical isolates from new TB Kazakhstani patients by determining drug resistance, genotyping and whole genome sequencing. Methods: 192M.tuberculosis drug resistant strains from new TB cases were analyzed. Resistance to the first-line anti-TB drugs were determined using absolute concentration method on L-J media and BACTEC MGIT-960. Spoligotyping was performed using commercially available kit “Ocimum Biosolutions Inc”. The whole genome sequencing was carried out on Roche 454 GS FLX+ platform. Results: The structure of 192drug resistant isolates showed that 98 (51.04%) were MDR, 55 (28.65%) – polyresistant, 39 (20.31%) – monoresistant. Genotyping results showed that W-Beijing family identified in 143 (74.48%) cases, T – 23 (11.98%), LAM – 11(5.73%), Haarlem - 10 (5.21%), U – in 4 (2.08%) and MANU-2– 1(0.52%) case only. W-Beijing family M.tuberculosis was a dominant genotype and composed more than 50% of two resistant groups: polyresistant - 70.9% and MDR - 87.75% clinical isolates of M.tuberculosis. Missense mutation of rpoB gene at codon 531with amino acid substitution Ser/Leu was the prevalent among mutations responsible for rifampicin resistance (87.75%). In case of isoniazid resistance, the most prevalent (96.94%) mutation was substitution at codon 315 Ser/Thr including 3 cases (3.06%) with double mutation when replacement simultaneously took place at katG gene and -15 position of promoter mabA (fabG)-inhA operon. Conclusion: MDR strains are predominant among resistant strains and mostly belong to Beijing family in Kazakhstan. W-Beijing is the most common genotype among all categories of drug resistant M.tuberculosis. As such MDR-TB should be a national priority in National TB Program for Kazakhstan. Acknowledgements: grant# АР05134737 MES RK.
Open Access
EFFECT OF MARE’S MILK PREBIOTIC SUPPLEMENTATION ON THE GUT MICROBIOME AND THE IMMUNE SYSTEM DURING AND AFTER ANTIBIOTIC THERAPY
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Nurgaziyev, M.; Aitenov, Y.; Khassenbekova, Zh.; Akpanova, S.; Rysbekov, K.; Kozhakhmetov, S.; Nurgozhina, A.; Sergazy, Sh.; Chulenbayeva, L.; Ospanova, Zh.; Tuyakova, A.; Mukhambetganov, N.; Sattybayeva, R.; Urazova, S.; Galymgozhina, N.; Gulyaev, A.; Kushugulova, A.
Antibiotic treatment can severely affect the gut microbiome with short-term and long-term consequences. Probiotic and prebiotic supplements are widely prescribed to modulate the composition and function of the human gut microbiome. The current study aims to determine the impacts of mare’s milk prebiotic on the diversity of gut bacterial communities and the local immune system when administered during and after a course of antibiotic therapy. Six children aged 4 to 5 years diagnosed with bilateral bronchopneumonia were prescribed cephalosporin (Cefuroxime) antibiotics. During the 60 days of the study, 3 children consumed mare’s milk prebiotics whereas the other 3 did not. Fecal samples were collected daily during antibiotic therapy and every 5 days after antibiotic therapy. Total DNA was isolated and taxonomic composition of the gut microbiome was analyzed by sequencing of the 16S rRNA gene (V1-V3 region). To evaluate the local immune status the MILLIPLEX MAP platform was used. Counts of 11genera were reduced, which did not recover until the last day of the study. The abundance of Bacteroides were not significantly altered in both groups. Christensenella, Rothia, Abiotrophia, Acinetobacter, Anaerotruncus, Holdemania and Turicibacter numbersare significantly increased at day 5 and remained at the same level during the study period. Cephalosporin administration also reduced pro-inflammatory and anti-inflammatory cyto/ chemokines (MIP1α, TNFα, GMCSF, GCSF, sCD40L, FGF2, TGFα, IL1α, IP10).
Open Access
THE EFFECTS OF PRECONDITIONED MESENCHYMAL STEM CELLS ON GASTRIC ULCER REGENERATION IN MICE
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Zhunusova, M.S.
Introduction: During the last decade, preconditioned mesenchymal stem cells (MSCs) have gained much attention in the field of cell therapy due to their capacity to differentiate into different cell types and to promote immunosuppressive effects in multiple diseases. However, there is little information, however, about the therapeutic effects of preconditioned MSCs in gastrointestinal disorders. In the present study, we examined whether transplantation of preconditioned MSCs could improve gastric ulcer healing. Methods: MSCs were isolated from compact bone of C57BL/6 mice. MSCs were cultured in α-MEM containing 10% FBS and 1% antibiotics. Preconditioning of MSCs was performed with IFN-γ and TNF-α for 24 h. Mice were divided into 3 groups: group 1(PBS), group 2(MSCs) and group 3 (preconditioned MSCs). Induction of gastric ulcer was made with alcohol-acetic acid solution. Each stomach was isolated for macroscopical and histological analysis. The levels of vascular endothelial growth factor (VEGF) and prostaglandin E2(PGE2) in stomach ulcer tissue were measured with ELISA-kits. Results: Our results showed that transplantation of preconditioned MSCs markedly improved the histopathology of the gastric tissue in comparison to MSCs and PBS-treated groups. Also the result of ulcer index was significantly decreased in preconditioned MSC-treated group at day 3 and 5 of the study comparing with the PBS-treated group. ELISA data showed that preconditioned MSCs restored the levels of PGE2to the normal levels and significantly increased the VEGF levels. Conclusion: Thus, our results showed that transplantation of preconditioned MSC significantly improved gastric ulcer healing in mice, possibly through the inhibition of inflammation and induction of angiogenesis in the gastric mucosa via the secretion of VEGF and PGE2.
Open Access
ENGINEERING CHEMOKINE-BASED CCR5 BLOCKERS TO TACKLE INFECTIOUS AND INFLAMMATORY DISEASES
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Vangelista, Luca
The C-C chemokine receptor type 5, CCR5, is expressed on various cell types and is involved in a large number of pathophysiological conditions. In recent years, CCR5 gained a huge attention, as it was found to play a central role in several infectious (e.g., HIV-1and Staphylococcus aureus), inflammatory and autoimmune diseases, and in major pathologies such as cancer and atherosclerosis. Most importantly, CCR5, the major HIV-1cellular co-receptor and the exclusive one in primary infections, is the molecular portal for HIV-1entry and transmission. Interestingly, the CCR5 Δ32gene deletion encodes for a truncated nonfunctional protein, providing resistance to HIV-1infection in homozygous individuals. Consequently, CCR5 and its ligands present a great potential for targeted therapies based on the development of high-affinity receptor antagonists and CCR5 gene editing. Gene editing of CCR5 to introduce the naturally occurring Δ32mutation has the aim to provide protection from HIV-1infection and possibly eradication of the virus from infected individuals. However, individuals naturally carrying the Δ32mutation may have adjusted their chemokine system to compensate for CCR5 absence, hence the de novo introduction of this mutation might bring some pathophysiological burden. Besides gene editing, biochemical CCR5 blockade via the development of potent CCR5 antagonists is a seemingly more realistic large scale therapeutic approach. Significant efforts are under way to understand the fine structural details of the interaction between CCR5 and its ligands and maraviroc, a small chemical drug, has been developed and FDA-approved as HIV-1entry inhibitor acting as CCR5 antagonist. CCL5 is a natural chemokine ligand for CCR5, a small globular protein with a very stable fold. Previous research from my group produced the engineered CCL5 mutant CCL5 5p125m, the most potent in vitro CCR5 antagonist HIV-1inhibitor reported to date (1000 fold more potent than maraviroc). My ongoing research at NU is aimed at the 3D structure rational design and production of even more potent CCL5 derivatives and their investigations as inhibitors in the expanding therapeutic perspective of CCR5 blockade for a growing number of pathological conditions.
Open Access
EVALUATION OF CYTOTOXICITY OF ORGANOSILICA NANOPARTICLES
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Zhaisanbayeva, B. A.; Mun, E. A.; Vorobyev, I. A.; Hortelano, G.; Khutoryanskiy, V. V.
Over the past few decades, nanoparticles have been attracting attention of researches in chemical, biomedical, pharmaceutical sciences, due to their unique physicochemical properties. This includes small size, large surface area, good biocompatibility and high reactivity. Among nanomaterials for biomedical application, silica nanoparticles exhibit great potential due to their straightforward synthesis, low-cost production, safety, biocompatibility and possibility to further functionalization. The most widely-used silica source for the synthesis of silica nanoparticles is tetraethoxysilane (TEOS) 1–3⁠. The surface of silica nanoparticles can be functionalized with different molecules (e.g. antibodies, fluorescent tags), which makes these nanoparticles attractive for imaging, labelling, detection and other biological applications. However, there are no functional groups on the surface of the TEOS nanoparticles. To enable further surface functionalization, an additional step of modification is required to bring thiol-, amino- or other reactive groups to the surface. To avoid those steps, direct synthesis of organosilica nanoparticles from (3-mercaptopropyl)trimethoxysilane (MPTS) was proposed4,5. Their mucoadhesive and diffusive properties, their permeation through mucosal tissues have previously been reported6–8⁠. In this work, we evaluated the cytotoxicity of organosilica nanoparticles synthesized from MPTS. To modulate biological properties, nanoparticles were conjugated with polyethylene glycol (PEG) of different molecular masses (750, 2000, 5000 Da). Effective PEGylation strategy for organosilica nanoparticles reported previously4. It is well known that toxicity and biodistribution depend on the nanoparticle size and surface modifications. Herein, the effect of PEGylated organosilica nanoparticles on varying cell lines studied.
Open Access
GENETIC VARIANTS, METABOLOME, AND GUT MICROBIOME BIOMARKERS FOR OBESITY AND AGING IN RANDOMLY SELECTED KAZAKH INDIVIDUALS
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Akilzhanova, Ainur; Rakhimova, Saule; Kozhamkulov, Ulan; Kairov, Ulykbek; Kushugulova, Almagul R.; Terwilliger, Joseph; Lee, Joseph
Objective: Metabolic syndrome (MS) is a cluster of inter-related and heritable metabolic traits, which collectively impart unsurpassed risk for atherosclerotic cardiovascular disease and type 2diabetes. Considerable work has been done to understand the underlying disease mechanisms by elucidating its genetic etiology. Genome, Metabolome variations and gut microbiome can predict disease risk and diagnosis and help to understand molecular pathophysiology. We aimed toassess plasma metabolom differences and gut microbiome as well as genetic variants among Kazakh population to identify and characterize the genetic, metabolic profiles and host-gut microbiota interactions. Methods: Kazakhs were recruited into study after signing of informed consent in Astana, Kazakhstan. Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) (Metabolon, USA) and NGS16S rRNA gene-sequence-based methods were used. Bioinformatic and statistical analyses were performed. Results: Subjects were stratified by age (young <45y, old ≥45y), gender and BMI. 853 different biochemical indicators of the main pathways for the metabolism were identified in plasma. Results demonstrate alterations in various metabolic pathways in older participants compared to younger subjects. Metabolic differences included changes in metabolites associated with the metabolism of fatty acids, steroidogenesis, secondary carnitine metabolism, inflammation and oxidative stress. Microbiomes of older persons are characterized by a high level of microorganisms involved in the processing of plant substrates, butyrate- producing bacteria and also has higher values of opportunistic microorganisms, representatives of the Tenericutes family. The biodiversity index of the microbiome of older persons is reduced in comparison with the biodiversity index in younger participants. This may indicate the influence on the microbiome characteristics of such factors as genotype, nutrition, lifestyle.Genetic risk factors associated with the obesity and hypertension were identified. Conclusions: Understanding plasma metabolome and gut microbiome is essential to the development of future personalized strategies of healthcare. Genome-wide association studies (GWAS) have been widely utilized albeit with modest success in identifying variants that are associated with more than two metabolic traits. Further studies with detailed analysis are needed to clarify host-gut genetic and metbolome interactions. Study was supported by a grant from the Ministry Education and Science, Republic of Kazakhstan (BR05236508).
Open Access
GENOTYPING OF MYCOBACTERIUM TUBERCULOSIS ISOLATES AMONG RECURRENT CASES OF TUBERCULOSIS IN KAZAKHSTAN
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Akhmetova, A.; Akilzhanova, A.; Toksanbaeva, B.; Bismilda, V.; Chingissova, L.; Kozhamkulov, U.
Introduction: Tuberculosis still remains one of the major health problems in Kazakhstan. According to the World Health Organization data (2018), Kazakhstan is on the list of 30 countries with high rates of multidrug resistant tuberculosis in the world. MIRU-VNTR analysis is widely used genotyping method of M. tuberculosis. The aimof this work is to characterize biological diversity of M. tuberculosis clinical isolates among recurrent cases of tuberculosis in Kazakhstan. Materials and methods: 95 clinical isolates of M. tuberculosis were collected from different regions of Kazakhstan among recurrent cases of tuberculosis. Genotyping of all isolates was performed by 15 MIRU-VNTR approach. PCR products were visualized on 2% agarose gel stained with ethidium bromide. Quantity 1(BioRad) program was used to determine the PCR fragment size and calculate the number of tandem repeats in each MIRU-VNTR locus. 15-digit allelic profiles of all clinical isolates were uploaded to MIRU-VNTRplus web application to identify M. tuberculosis families. Phylogenetic tree was built using UPGMA (Unweighted pair group method with arithmetic mean) algorithm. Results: 40 genotypes were detected based on15 MIRU-VNTR genotyping results. 31(32,6%) genotypes were unique and found only in one isolate in the collection. The rest 64 (67,4%) M. tuberculosis clinical isolates were clustered into 9 clusters which included from 2to 34 isolates in one cluster. 87,4% of all clinical isolates were belonged to Beijing family. The second meaningful family that is found among the isolates was LAM family (10,5%). URAL and CAS families were identified in the rest 2,1% cases. Conclusion: Thus, 15 MIRU-VNTR typing of 95 clinical isolates of M. tuberculosisshowed that Beijing family strains that are associated with drug resistance prevail among recurrent cases of tuberculosis in Kazakhstan (87,4%).
Open Access
IDENTIFICATION OF KAZAKH SPECIFIC GENOMIC VARIANTS USING COMPARATIVE GENOMICS ANALYSIS
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Molkenov, A.; Daniyarov, A.; Sharip, A.; Seisenova, A.; Karabayev, D.; Kairov, U.
Introduction: The modern development of high-performance genomic technologies opens up new possibilities for studying the human genome. Large-scale genomic research generates huge amounts of data, the active development of bioinformatics with the availability of modern methods and approaches of analysis makes it possible to create detailed databases and comprehensively study genomic data. One of contemporary task is to study and identify specific genomic variants of population by detailed analysis of complete genome and complete exome data comparison with open large-scale population datasets. Materials and methods: Materials of the study are 14 complete genomes and 125 complete exomes of Kazakhstani individuals. Our dataset was replenished with data from large whole genome population datasets (SGDP, PRJEB26349, HGDP and 1000 Genomes) for comparative population genomics and to search and identify specific genomic variants. The data in the raw format was mapped and aligned on a single reference genome hg19, then genomic variants were searched and an individual map of the found variants was formed for each dataset in the VCF format. For replenished datasets formed a general map of all variants, which were then excluded from the total number variants found for of Kazakh sampling to search for specific genomic variants. Then the filtered variants were annotated and interpreted. Results: For Kazakр whole exomes were found 9 heterozygous or mutant variants unique among formed genomic databases. 7 variants located on the intron region, 1on the upstream and the last variant frameshift deletion on exonic region. For the Kazakh whole genomes were found 4732heterozygous or mutant variants, 517 variants presented among all Kazakh samples and 144 variants were completely mutant. Only 8 SNVs are located at exonic region: 4 synonymous SNV, 3 nonsynonymous SNV, and 1frameshift deletion. Conclusion: We have discovered unique several genomic variants specific for now to the kazakh individuals. These results can serve as a basis for the creation of a Kazakh reference genome, subsequent research and comparative analysis of Kazakh individuals with various populations of the world. Grant references: AP05135430; MES RK.
Open Access
ISOLATION FROM THE KAZAKH TRADITIONAL FOOD PRODUCTS OF A NEW STRAIN OF LACTIC ACID BACTERIA PRODUCING THE HUMAN PLASMINOGEN RECEPTOR
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Shaikhin, S.; Abitayeva, G.; Abilhadirov, A.; Dossova, A.; Pirmanova, A.; Tynybaeva, I.
Lactobacillus bacteria are one of the most important groups of the human intestinal microbiota that promote health. Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. Plasminogen - binding may also contribute to bacterial adhesiveness and invasiveness by nonproteolytic mechanisms. Plasminogen (Plg) binds to bacterial cell surface receptors, as well as to receptors on eukaryotic cells and may thus function as a bridge between the bacteria and the epithelium. The project aims to isolate a new strain of lactobacilli producing a protein- receptor for human plasminogen (Plg-R) from traditional Kazakh food products and to study the mechanism of reception. Lactic acid bacteria (LAB) strains isolated from traditional home-made food products and identified using 16S rRNA nucleotide sequence analysis; samples of extracellular proteins obtained from the cell-free supernatant (CFS) of the 24-hour culture after pH adjustment to 8.0 to dissociate acidic cell surface proteins, followed by concentration. Screening for Plg binding in CFS of LAB isolates carried out by Western Blotting assay. The Plg-R was purified from cell lysates after ultrasonic homogenization by Q-sepharose chromatography and the isoelectric point and subunit structure were determined by Mono P column chromatography and Sephadex G-75 gel filtration respectively. Screening of 35 isolates of LAB for Plg binding revealed a strain L. plantarum 30 isolated from homemade butter with maximal Plg-binding activity. The Plg-R band with comparatively high intensity migrated in the region of 47 K on the PAA gel with SDS and showed pI=4.8. The Plg binding was inhibited by adding an analog of lysine ɛ-aminocaproic acid (EACA). The Plg-R eluted in the exclusion volume in gel-filtration, which indicates the quaternary structure. A new strain from the homemade butter which produces the Plg-R was isolated. The inhibition by EACA of the Plg binding with receptor indicates the specificity of the binding which realized via lysine residues on the molecule of Plg-R. The properties of Plg-R are similar to the glycolytic moonlighting enzyme enolase and identification of the Plg-R in progress.
Open Access
ISOLATION OF NATURAL REPRESENTATIVES OF CARP LACTOFLORA TO CREATE PATHOGEN CONTROL AGENTS THAT CAN BE ALTERNATIVE TO ANTIBIOTICS
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Urazova, M.; Zakarya, K.; Sarmurzina, Z.; Bissenova, G.; Abitayeva, G.; Shevtsov, A.; Tekebayeva, Zh.; Tynybayeva, I.; Abzhalelov, A.
Currently, the microbiome of commercial fish species is being actively studied. In reviews, mainly based on metagenome analysis features and formation of intestinal microflora of various fish species of North America, Asia and Europe have been presented. Results of these researches showed that lactic acid bacteria (LAB) are common microbial group of intestinal microbiota of fish (Wu et al., 2012; Ye et al., 2014; Borsodiet al., 2017; Wang et al., 2018). In our research a characteristic of 22lactic acid bacteria isolated from Common Carp (Cyprinus carpio) intestines were studied. All of them were tested for their ability to inhibit growth of Aeromonas punctate, Shewanella xiamenensis, Pseudomonas aeruginosa,Pseudomonas taiwanensis.In sum, 7 isolates with high antagonistic activity were selected. 16S rDNA gene sequencing identified them as Lactobacillus fermentum(4), Lactobacillus casei/paracasei (2) and Pediococcus pentosaceus (1). All identified isolates can grow in wide temperature range (10° C to 37° C) and in presence of bile. Earlier the species of Lactobacillus, Lactococcus, Streptococcus, Enterococcus,Pediococcus and Carnobacteriumgenera were isolated from carp intestines (Bucio et al., 2006; Wang et al., 2018). We isolated only strains, belonging to 3 species (Lactobacillus fermentum, Lactobacillus casei/paracasei and Pediococcus pentosaceus). It is low rate of species diversity. Perhaps this is due to the conditions, when we made isolationof LAB. It wasmade at the transition period from wintering to active spawning.The climate of Central and Northern Kazakhstan is severe and it is characterized by a cold winter lasting 5,5-6 months. Long wintering is a stress for an animal, and its microflora too. During the wintering period, carps do not feed, the composition of their microflora becomes poorer.The dominance of somespecies among intestinal LAB during cold period should be due to their specific characteristics, such as resistance to low temperatures and unfavorable environmental factors.Selective 7 strains could be alternatives to antibiotics for freshwater aquaculture in Kazakhstan.
Open Access
LAUNCH OF Q-SYMPHONY BIOINFORMATICS COMPUTING SYSTEM: A HIGH-PERFORMANCE CLUSTER FOR ANALYSIS OF LARGE-SCALE GENOMIC DATASETS
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Molkenov, A.; Daniyarov, A.; Sharip, A.; Seisenova, A.; Karabayev, D.; Kairov, U.
Introduction: One whole human genome, provided by next generation sequencing platforms, in raw format takes 20 to 50 GB. In the course of bioinformatics analysis and data analysis, the data volume increases to 300-500 GB per genome. with an increase in the number of samples, the occupied volume increases. Such a large amount of data required for the analysis of whole genomes demands powerful computing power in the form of servers and data warehouses combined into clusters. We at Laboratory of Bioinformatics and Systems Biology have developed and launched Q-Symphony bioinformatics computing system called (“Qazaq Symphony of Bioinformatics”) for bioinformatics analyses of solving large scale genomic datasets. Materials and methods: The Q-Symphony bioinformatics computing system consists 12high-performance HPE servers: 1control node, 8 compute nodes, 1fat-memory compute node, and 2storage nodes. The system runs on Red Hat Enterprise Linux. The management node controls access to user profiles, data warehouse and Moab Workload Manager. The total number of processing cores is 172, the total amount of RAM is 3072GB, and the total storage capacity is 198 TB, a peak performance of the system of 7.3 TFlops. All nodes use high-speed Infiniband network connections, which allow the data exchange between nodes at 100 Gbps speed. The computational capabilities of the Q-symphony system allow us to evenly distribute resources for each task performed, monitor the load on processor and memory resources in real time, and queue and execute sequentially large lists of tasks. Results: Benchmark measurements performed on Q-symphony system showed an increase of subtasks execution from 15 to 54 times compared to standard solutions built on similar computational processors. Conclusion: The presence of Q-Symphony, well-established and proven bioinformatics methods will make it possible to successfully analyze large-scale human genomic data and determine structural genomic variants and carry out complex comparative and population analysis.
Open Access
Measures of Reducing the Burden of Health Care Costs by Rational Usage of Laboratory Tests
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Marchenko, Y.; Akhmetove, R.
Introduction: Every year manufacturers offer new tests, postulating that it will improve the outcomes of treatment. That results that laboratories have to face an increasing workload combined with a reduction of resources. Implementing strategies to reduce the use of unnecessary and redundant tests is an essential tool to manage increasing health care costs. In the study, it was determined to look at the effectiveness of using laboratory tests performed by an express-laboratory, because of the costs of maintenance express-laboratories higher than lab for routine analyses. Methods: The quarterly report of the Center of Laboratory Medicine (CLM - a centralized laboratory) for the first quart of 2019, the Laboratory Information System and Hospital Information System were used to get data. Data were processed by Microsoft Excel. Results: The express-laboratory department performed 21% of the total number of laboratory tests performed by the CLM. The level of the workload of the personnel was examined by assessing TAT. The degree of demand for laboratory data was determined by evaluating the period from the moment of results preparedness to the moment it was viewed by clinicians. TAT was calculated for 4 phases of time and data shows that in 2 phases from 4 am to 16 pm the percentage of samples were processed with an unacceptable range of TAT. There were 81929 analytical requests made with mark “cito”, among them 59070 (72,1%) were visualized by clinicians during 30 minutes after readiness, 21465 (26,2%) were visualized in more than in 4h and 1392(1,7%) were not visualized. Conclusions: As it is seen from the data the staff in the express-laboratory is overloaded in some period and cannot reach the established range of TAT for 31% of tests. However, the findings show that not all results are visualized by clinicians. The not visualized reports mean performing an invasive technique that it will not contribute to benefit the patient. Thus, it is necessary to make more strict regulatory for analytical requests in express-laboratory by updating medical protocols for critical conditions and harmonizing the panel of tests for them as well as by implementing point of care tests.
Open Access
META-ANALYSIS OF CANCER TRANSCRIPTOMES USING INDEPENDENT COMPONENT ANALYSIS
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Seisenova, A.; Sharip, A.; Molkenov, A.; Daniyarov, A.; Karabayev, D.; Kairov, U.
Introduction: Independent Component Analysis (ICA) is a matrix factorization method for data dimension reduction. ICA has been widely applied for the analysis of transcriptomic data for blind separation of biological, environmental and technical factors affecting gene expression. This study aimed to analyze cancer data using the ICA for identification and comprehensive analysis of reproducible signaling pathways and molecular signatures in cancer. Materials and Methods: In this study, four independent cancer transcriptomic datasets GSE26886, GSE69925, GSE32701and GSE21293 (Affymetrix) from GEO databases were used. R Bioconductor and Matlab have been used for normalization. A bioinformatics tool «BiODICA - Independent Component Analysis of Big Omics Data» was applied to compute independent components (ICs). Gene Set Enrichment Analysis (GSEA) and ToppGene uncovered the most significantly enriched pathways. Construction and visualization of gene networks and graphs were performed using the OFTEN method, Cytoscape and HPRD database. Results: The correlation graph between decompositions into 30 ICs was built with absolute correlation values exceeding 0.15. Clusters of components - pseudocliques were observed in the structure of the correlation graph. Top 500 most contributing genes of each ICs in pseudocliques were mapped to the PPI network to construct signaling pathways for gene interaction. Some cliques were composed of densely interconnected nodes and included components common to most cancer types, while others were common to some of them. Conclusion: The results of this investigation may reveal potential biomarkers of carcinogenesis, functional subsystems in the tumor cells, and helpful in predicting the early development of a tumor.
Open Access
Molecular Genetic Approaches in Diagnosis of Duschenne/ Becker Muscular Dystrophy
(International conference "MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES: FROM BENCH TO BEDSIDE”; National Laboratory Astana, 2020) Bayanova, M.; Uahit, R.; Tolegen, N.; Nazarova, L.; Abilkhadirova, A.; Khamzina, A.; Nauryzbaeva, A.; Jaxybayeva, A.
Introduction: Duchenne/Becker muscular dystrophy (DMD/BMD) is inherited X-linked disease with a frequency of 1: 3,500 newborn males. Deletions and duplications in the DMD gene are errors of reading frameshift and premature termination of translation. Structural rearrangements of BMD does not lead to reading frame rule, DNA polymerase can “skip” deleted exons , which leads to the synthesis of truncated protein, which can fulfill its functions some extent. Methods: We studied the DNA of 104 patients with suspected DMD / BMD and heterozygous, as well as chorionic villi samples (CVS) obtained by prenatal diagnostics. Multiplex Ligation - dependent Probe Amplification(MLPA) (MRC Holland) was used to study of copy number variations of 79 exons of DMD gene. Validation MLPA results and whole exome sequencing (WES) of patients with MLPA negative results were performed at Centogene (Germany). Detection of nonsense mutations leading to the appearance in mRNA a premature stop codons of DMD gene are relevant to stratify patients for antisense-targeted therapy of Duchenne muscular dystrophy. Results: Mutations of (36) 37 % of patients were revealed in the DMD gene. Deletions were detected in (27) 75 %, duplications in (9) 25% of cases. WES of 41patients was conducted for validation and MLPA negative results. Point mutations were identified in 30 (73%), frameshift missense mutations - in 7 (23%), nonsense mutations - in 9 (30 %) cases. Validation of MLPA results (deletions) were performed in 14 patients (46 %). Conclusions: Algorithm of molecular diagnostic of DMD /BMD by MLPA is a method for the detection of large deletions and duplications 79 exons of DMD gene and WES should be to determine the missense and nonsense mutations of DMD gene. Our results formed the basis of clinical protocols of diagnostic and treatment of Duchenne/Becker muscular dystrophy in Kazakhstan.