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Thromboelastographic study of fibrin clot and molecular basis of maximum clot firmness
D. S. Korolova1*, Y. M. Stohnii1, V. I. Gryshchuk1, S. I. Zhuk2,
I. V. Us2, T. M. Chernyshenko1, O. P. Kostiuchenko1, K. P. Klymenko1,
O. M. Platonov1,3, O. I. Ivashchenko3, V. O. Chernyshenko1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Shupyk National Medical Academy of Postgraduate Education, Kyiv, Ukraine;
3ESC “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: d.korolova@gmail.com
Received: 29 January 2021; Accepted: 23 April 2021
Maximum clot firmness (MCF) is the main parameter of thromboelastography (TEG) reflecting the stability of a clot. In this work, we looked for markers that can influence the enhancement of MCF and detected molecular markers and blood clotting parameters that can be involved in such mechanisms. Blood samples of pregnant women with placental disorders were collected in the Kyiv Perinatal Center. TEG was performed on whole blood in EXTEM and INTEM tests. APTT, INR, fibrinogen concentration and platelet aggregation were measured using traditional laboratory approaches. D-dimer was detected in sandwich ELISA using monoclonal antibodies III-3B and II-4D. The relative cross-linking activity of factor XIIIa was measured by the direct quantification of the cross-linked γ-chain of fibrin using Western-Blotting with monoclonal antibody II-4D. D-dimer and fibrinogen concentrations, clotting time in the APTT test, INR and rate of platelet aggregation did not correlate with the MCF. However, we found positive correlations of MCF with factor XIIIa activity: 0.51 and 0.87 for EXTEM and INTEM, respectively. These data indicate that for normal and slightly increased fibrinogen concentrations, fibrin clot firmness will depend mostly on the activity of factor XIIIa. Thus the direct determination of factor XIIIa activity in blood plasma of patients can be relevant for predicting the risk of intravascular coagulation. Evaluation of the content and activity of individual clotting factors or other components of the coagulation system can be useful additions to the TEG diagnostics and should not be neglected.
Oxidative stress suppression contributes to antiseizure action of axitinib and rapamycin in pentylenetetrazol-induced kindling
O. B. Poshyvak1*, O. R. Pinyazhko1,2, L. S. Godlevsky3
1Pharmacology Department, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
2Department of Civilization Diseases and Regenerative Medicine, WSIiZ, Rzeszow, Poland;
3Department of Biophysics, Informatics and Medical Devices, Odesa National Medical University, Odesa, Ukraine;
*e-mail: olesya.poshyvak@gmail.com
Received: 29 January 2021; Accepted: 23 April 2021
Rapamycin and axitinib block different kinases in signaling pathways such as PI3K-Akt-mTOR and BDNF-TrkB, respectively. Both have antiseizure and antioxidative actions, which justify studying the combined effects of these drugs upon seizures and oxidative stress in the chronic model of epilepsy. The investigation aimed to look for the combined effect of rapamycin and axitinib upon pentylenetetrazol (PTZ)-kindled seizures and oxidative stress. Experiments were performed on 300 two- to four-month-old Wistar male rats, which had been kindled daily with PTZ (35.0 mg/kg, i.p.). Malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, and glutathione (GSH) level were determined in brain tissues of kindled rats before and after the treatment. The analysis of antiseizure and antioxidative actions was performed using ED50 of rapamycin and axitinib, with their combined administration using graded dosages of ED50 of each drug. The median effective dose (ED50) for rapamycin and axitinib was 0.93 and 4.97 mg/kg, respectively. ED50 of rapamycin when combined with axitinib (2.0 mg/kg) was 0.60 mg/kg, which was reduced by 35.6% when compared with the ED50 administered alone (P < 0.05). The MDA level increased from 152.9±24.8 to 388.3±49.2 nmol/mg of protein (P < 0.05), while SOD activity reduced from 11.14±2.33 to 3.54±1.08 IU/mg of protein (P < 0.05) in brain tissues of the kindled rats. Combined treatment with rapamycin (0.56 mg/kg, i.p.) and axitinib (2.0 mg/kg, i.p.) resulted in a significant rise in SOD activity (11.09±1.86 IU/mg) and GSH level (7.32±1.34 µg/mg) when compared with the kindled rats (P < 0.05). Combined axitinib and rapamycin therapy have an antiepileptic and antioxidative effect on PTZ-kindled seizures.
Effect of a novel thiazole derivative and its complex with a polymeric carrier on stability of DNA in human breast cancer cells
N. S. Finiuk1,2, O. Yu. Klyuchivska1, I. I. Ivasechko1,
N. E. Mitina3, Yu. V. Ostapiuk2, M. D. Obushak2,
O. S. Zaichenko3, A. M. Babsky2, R. S. Stoika1,2*
1Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine;
2Ivan Franko National University of Lviv, Lviv, Ukraine;
3Lviv Polytechnic National University, Lviv, Ukraine;
*e-mail: stoika.rostyslav@gmail.com
Received: 26 January 2021; Accepted: 2021
Thiazole derivatives are perspective antitumor compounds characterized by a broad range of bioactivity, while polymeric carriers are widely used to enhance the efficiency of biological action of drugs, improve their biocompatibility and water solubility. Previously, we identified that the thiazole-based derivative BF1 (N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide) possessed differential toxicity towards targeted tumor cell lines. The aim of the present work was to investigate the action in vitro of BF1 and its complex with the polymeric carrier (PC) poly(PEGMA-co-DMM) (BF1-РС complex) towards human breast adenocarcinoma cells of the MDA-MB-231 and MCF-7 lines. DNA comet analysis, diphenylamine DNA fragmentation assay, gel retardation assay of plasmid DNA, DNA intercalation assay using methyl green dye and fluorescent microscopy were used to study the effects of BF1 on DNA stability in breast cancer cells. The ІС50 of cytotoxic action towards MDA-MB-231 cells was 26.5 ± 2.9 µМ for BF1, while the ІС50 for the BF1-PC complex was 6.9 ± 0.4 µМ, and the PC demonstrated low toxicity (ІС50 ˃ 50 µМ). The BF1-PC complex possessed higher toxicity towards MCF-7 cells than free BF1, with ІС50 of 9.6 ± 0.8 µМ and 15.8 ± 0.9 µМ, respectively. BF1 and BF1-PC induced an increase in the number of damaged cells of the MDA-MB-231 line with blebbing of plasma membrane, condensed chromatin and/or fragmented nucleus and micronuclei formation. Both BF1 and the BF1-PC complex induced single-strand breaks in DNA and its fragmentation in treated MDA-MB-231 cells. The studied compounds were not bound to plasmid DNA and did not intercalate into DNA molecules.
Locally delivered lovastatin-containing chitosan nanoparticles promote bone regeneration in rats
O. O. Shevchuk1*, Ya. V. Panasiuk2, M. M. Korda3
1Department of Pharmacology and Clinical Pharmacology, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine;
2Department of Functional and Laboratory Diagnostics, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine;
3Department of Medical Biochemistry, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine;
*e-mail: shevchukoo@tdmu.edu.ua
Received: 24 February 2021; Accepted: 23 April 2021
Hypolipidemic statins can stimulate osteoregeneration. However, such effects are observed only after administration methods that are unacceptable for patients (prolonged infusions or huge oral doses). The aim of our research was to compare the osteoregeneration effects of lovastatin administered alone as a common pharmaceutical formulation and as lovastatin-containing chitosan nanoparticles (LCCN) in a drill-hole model of bone damage in rats. White inbred rats were randomly divided into four groups: group 1 – intact rats; group 2 – rats with bone defect without treatment (control group); group 3 – rats with bone defect, which received common pharmaceutical formulation of lovastatin at doses of 0.1, 1.0 and 5.0 mg/kg; group 4 – rats, which received 0.1 mg/kg lovastatin in the form of lovastatin-containing chitosan nanoparticles (LCCN). A dental drill of 2.0 mm in diameter was used to form the tibial bone defect. Rats were sacrificed 3, 7, 14 and 28 days after bone defect formation. Calcium (Ca), phosphorus (P) and sialic acid concentrations, alkaline and acidic phosphatase activities, mineralization index, and collagenolytic activity were measured in blood serum. Computed tomography (CT) and histological study were used to estimate the regenerative processes in the bone. It was found that therapeutic doses of lovastatin (0.1 and 1.0 mg/kg) are ineffective for bone defect healing. Only high doses of lovastatin (5.0 mg/kg) promote osteoregeneration. LCCN were more efficient compared to lovastatin alone, as confirmed by CT examination of bone defects and significant changes of Ca, P, and sialic acid concentrations, alkaline and acidic phosphatase activities, mineralization index, and collagenolytic activity. Lovastatin-containing chitosan nanoparticles effectively enhance fracture healing in used preclinical model. This finding suggests the possibility that a similar approach may be effective in hastening fracture repair in humans.
In silico identification and biochemical validation of plausible molecular targets of 4-thiazolidinone derivative Les-3833 as a potential anticancer agent
L. Kоbylinska1*, D. Khylyuk2, I. Subtelna2,
M. Kitsera3, R. Lesyk2
1Department of Biochemistry, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
2Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
3Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine;
*e-mail: Kobylinska_Lesya@meduniv.lviv.ua; lesya8@gmail.com
Received: 16 January 2021; Accepted: 23 April 2021
Synthetic 4-thiazolidinone derivatives have a broad range of pharmacologic activities. Thus, 4-thiazolidinones are being investigated to create new molecules and develop active pharmaceutical substances for anticancer treatment. In our previous study, we investigated the pyrazoline-thiazolidinone-isatin conjugates, and determined that Les-3833 was the most active compound and might act through inhibition of PARP-, MAPK-, JNK-, Bcl-2-, CDK1/cyclin B, and/or the caspase family. The aim of this research was to perform molecular docking studies to enable the construction of a pharmacophore model for the Les-3833 compound and investigate probable biological targets. Pharmacophore modeling software packages performed molecular docking studies of probable biological targets and enabled the construction of a pharmacophore model. Docking models of Les-3833 with 11 enzymes involved in apoptotic mechanisms were studied. Based on the pharmacophore modeling results for all 11 enzymes, Les-3833 is predicted to be most active in Chk‑1, caspase-6, and caspase-8. Immunoblot analysis proved that the application of Les-3833 led to inhibition of Ser345 phosphorylation, which is induced by etoposide, the most important modification responsible for Chk‑1 activity. Taken together with the results of the docking studies, several mechanisms for the expression of antitumor activity by 4-thiazolidinones are suggested, and such multi-affinity is a characteristic feature of all these derivatives. The docking analysis confirmed the affinity of test compound Les-3833 for a topoisomerase II inhibitor and a high possibility of inhibitory interaction with Chk-1, caspase-6, and caspase-8.
Inflammation is the common mechanism of diseases (CMD) in COVID-19 disease during pregnancy and in gestational diabetes mellitus
Sandor G. Vari
Cedars-Sinai Medical Center, International Research and Innovation in Medicine Program, Los Angeles, California, United States
The Regional Cooperation for Health, Science and Technology (RECOOP HST) Consortium, led by Cedars-Sinai Medical Center was formed in 2006, was transformed into an Association in 2012 and includes 17 universities and academic organizations from eight countries: seven in Central and Eastern Europe (Croatia, Czech Republic, Hungary, Poland, Romania, Slovakia, Ukraine) and the United States. RECOOP builds multinational, multidisciplinary collaborations, and assists as well as coordinates the research activities of the sixteen research groups that are the Cedars-Sinai Medical Center – RECOOP Research Centers (CRRCs). https://www.cedars-sinai.org/research/administration/recoop.html.
Implementations of RECOOP’s strategic goals enable diverse talents geared towards integration of new knowledge derived from multiple specialties to investigate Common Mechanism of Diseases (CMD). While some may consider RECOOP’s CMD research strategy unorthodox, recent and timely scientific evidence shows that inflammation is the triggering event in the change of vascularization and it is the common mechanism of these two diseases: COVID-19 Disease during pregnancy and gestational diabetes mellitus (GDM).
Binding of the SARS-CoV-2 virus to the ACE2 receptor and its entrance into endothelial cells plays a role in vascular thrombosis but has a lesser effect placental endothelial dysfunction. The latter is induced by inflammation and exacerbated by proinflammatory cytokines, resulting in ischemic events and creating an upward spiral of an inflammatory reaction in pregnant women, accompanied by similar conditions in the placenta that will ultimately affect fetal development. In mild or moderate COVID-19 disease, changes in placental vascularization and blood flow have similarities to comorbidities in pregnancy such as GDM. However, during severe or critical stages of COVID-19 Disease, the changes could be harsher than those observed in GDM.
In COVID-19 Disease and GDM the immune status of pregnant women and consequently the newborn is altered due to inflammation and characterized by changes in levels of C-reactive protein (CRP), immunoglobulins (IgG, IgM, IgA) and proinflammatory cytokines that are detectable in maternal and umbilical cord blood and in mother milk.
To examine changes and monitor placental angiogenesis it is necessary to measure Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PLGF), and Umbilical Cord Blood Sclerostin (UCBS) in maternal and umbilical cord blood serum. The angiogenic activity of sclerostin must be validated with the well-known marker VEGF, which is a proven indicator for changes in vascularization. The morphology of the vascular tree and blood flow in the placenta could be evaluated with three-dimensional power Doppler. The proinflammatory and ischemic effects in the placenta should be quantified with histopathology and immunohistochemistry. Changes in blood flow in the placenta and the morphology of the vascular tree in COVID-19 Disease during pregnancy may have similarities to those observed in GDM.
In summary, to improve maternal and fetal outcomes it is imperative to formulate better strategies for managing pregnancies during COVID-19 Disease and comorbidities like GDM. VEGF, PLGF and UCBS could be predictors of placental weight, birth weight, and fetal outcomes. In addition, further studies are needed to investigate the effects, if any, of proinflammatory and anti-inflammatory cytokines on postnatal development.
The discovery of the mechanisms of biological synthesis of nucleic acids: 1959 Nobel laureates S. Ochoa and A. Kornberg
O. P. Matyshevska, V. M. Danilova, S. V. Komisarenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: matysh@yahoo.com
Received: 12 September 2020; Accepted: 17 December 2020
Alongside the chemical and physical research of nucleic acids in the 1940s-50s, the mechanisms of their biosynthesis were investigated. Thus, in 1959, Severo Ochoa and Arthur Kornberg were awarded the Nobel Prize in Physiology or Medicine for the discovery of the mechanisms of biological synthesis of RNA and DNA. The experiments performed by Ochoa and Kornberg are considered today the cornerstone of genetic engineering, as they first demonstrated the possibility of synthesizing RNA and DNA outside the living cell, and also as the enzymes they discovered were among the first tools of this technology.
Prospects of genome editing using CRISPR/CAS or how to master genetic scissors. Nobel Prize in Chemistry 2020
S. V. Komisarenko, S. I. Romaniuk
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: svk@biochem.kiev.ua
The Nobel Prize in Chemistry 2020 was awarded to two researchers in the field of molecular biology – the French Emmanuelle Charpentier, who now heads the Max Planck Unit for the Science of Pathogens in Berlin, and the American Jennifer Doudna from the University of California, Berkeley – for the ‘development of genome editing method’. A press release of the Nobel Committee states that the winners discovered one of the most powerful genetic technology tools – CRISPR/Cas9, or so-called ‘genetic scissors’. This method has contributed to many important results of basic research. In particular, plant researchers have managed to create crops resistant to mold, pests and drought. As for medicine, clinical trials of new cancer treatment techniques are underway, and a dream of curing hereditary diseases is about to become a reality. Genetic scissors have brought the life sciences to a new stage of development and greatly contributed to the benefit of mankind.