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Contents UBJ, 2024, Volume 96, Issue 2

Research capacity building, networks, define research strategy, and funding opportunities in RECOOP HST Association

Sandor G. Vari

Director, International Research, and Innovation in Medicine Program
Cedars – Sinai Medical Center, Los Angeles, CA, USA;

I built research capacity and networks for the first ten years in the RECOOP Research Consortium from 2002 to 2012. The RECOOP HST Consortium, with the financial support of Cedars–Sinai Medical Center­ (CSMC), applied for the International Visegrad Fund (IVF) grants. We applied for 20 Standard Grants and won 14 Standard Grants to support forming and managing research networks and multinational–multidisciplinary research projects. The IVF grants won by CSMC – RECOOP helped move toward the 21st Century and broaden the scope of activities in RECOOP. Also, the IVF grants helped to build support networks for biosafety and biosecurity, animal use in research, clinical research management, and research and Innovation management training. RECOOP HST Association implemented the Common Mechanism of Diseases (CMD) research program for innovative life science research in member countries. The objectives were to increase the number of young scientists participating in creative research. Knowledge sharing is the most essential element of collaborative research. Within the context of RECOOP, my final endeavor will focus on investigating the diagnosis and management of Post-Traumatic Stress Disorder (PTSD) in Ukraine. As of January 2024, an estimated 6.3 million people have been forced to flee Ukraine, with 94 percent of them hosted in European countries, representing 5.9 million refugees. In Ukraine, an estimated 7.8 million people need health assistance, and 11.5 million need protection assistance and services. Studies of PTSD report volume abnormalities in multiple regions of the cerebral cortex. However, findings for many regions, particularly regions outside commonly studied emotion-related prefrontal, insular, and limbic regions, are inconsistent and tentative. We, as researchers, must continually share our research findings and diligently replicate established methods and protocols. These tasks can often feel akin to the labor of Sisyphus. Moreover, within the scientific community, integrity is paramount; dishonesty is swiftly met with consequences akin to the justice administered by Zeus. Therefore, we researchers must roll a boulder up a hill again and again, and after we have proved that the published scientific work is sound, the “boulder” and the scientist will stay on top of the hill.

Inhibitory action of methylene bisphosphonic acid on metabolic activity and viability of J774A.1 cells

D. O. Labudzynskyi*, E. P. Pasichna, O. I. Krynina, М. M. Veliky

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;

Received: 01 February 2024; Revised: 22 March 2024;
Accepted: 24 March 2024; Available on-line: 30 April 2024

Bisphosphonates (BPs) are primary agents in the current pharmacological arsenal against osteoclast-related bone loss due to osteoporosis, Paget’s disease and bone tumors. Due to the lack of complete understanding of the molecular mechanism of their action in bone tissue and the overlap of key properties between BPs of different generations, integral studies of BPs inhibitory and antiresorptive properties are relevant today. The present work was carried out to establish a comprehensive study of the inhibitory effects of methylene bisphosphonic acid (MBPA) on the mevalonate pathway, metabolic activity and cell death in vitro compared to zoledronic acid (Zol). Farnesyl pyrophosphate synthase activity of MBPA-treated J774A.1 cells was inhibited by 80%, compared with a 79% reduction in Zol-treated samples. The ability of MBPA to decrease the percentage of viable cells in culture is slightly lower compared with Zol. After 24 h of incubation with lowest concentration, the percentage of inhibition of metabolic activity was 10.6 and 25%, respectively. After 48 h, these values were 34.8 and 55.6%, respectively. The inhibitory effects of MBPA and Zol on the intensity of incorporation of radioactively labeled precursor [14C]-acetate to the cholesterol fraction were 76.2 and 59.1%, respectively. In the case of isoprenoid fraction, the inhibitory effects were 40.9% and 51.2%, respectively. MBPA and Zol differently induced apoptosis in the J774A.1 cells culture, increased count of apoptotic cells in 2.4 and 6.3 times, and also increased the number of propidium iodide-positive cells in 7.4 and 19 times, respectively. MBPA and Zol also increased the number of TUNEL-positive cells in macrophage culture in 2.6 and 5 times, respectively. Zoledronate significantly reduced carbonic anhydrase 2 and nuclear factor of activated T-cells 1 gene expression levels compared to the MBPA action. Thus, the use of MBPA in future research and therapy of both cancer and osteoporosis looks promising due to lower cytotoxicity, high efficiency of mevalonate pathway inhibition and the possibility of dosage variation.

ECG patterns in post-COVID-19 patients

M. V. Hrebenyk1, S. M. Maslii1, O. O. Shevchuk1*,
M. M. Korda1, S. G. Vari2

1Ivan Horbachevsky Ternopil National Medical University
of the Ministry of Health of Ukraine, Ternopil, Ukraine;
2International Research and Innovation in Medicine Program,
Cedars–Sinai Medical Center, Los Angeles, CA, USA;

Received: 06 January 2024; Revised: 17 March 2024;
Accepted: 17 March 2024; Available on-line: 30 April 2024

COVID-19 has been associated with a wide range of cardiac sequelae after the acute phase. The goal of the study is to evaluate the spectrum of arrhythmias in the aspects of age, comorbidity and survival rate using ECG patterns in patients after COVID-19 during 2 months of recovery. ECG data of 758 patients were examined and analyzed, including 256 (33.6%) males and 503 (66.4%) females aged 15 to 90 (52.99 ± 11.68) years. A total of 848 ECGs were performed in acute period and recovery. ECG changes were classified according to the Minnesota code (MC) classes. It was found that age, sex, severity of COVID-19, presence of concomitant hypertension and diabetes mellitus have a significant impact on ECG changes. Age correlated with the severity of COVID-19 (r = 0.485, P < 0.001), concomitant hypertension (r = 0.471, P < 0.001), diabetes (r = 0.346, P < 0.001) and obesity (r = 0.179, P < 0.001). Correlations were established between age and the presence of baseline previous pathological ECGs (r = 0.290, P < 0.0001). We established that heart rhythm disorders related to the severity of the COVID course are significantly influenced by oxygen saturation (r = -0.211, P < 0.001) and, to a lesser extent, the percentage of lung damage according to CT data (r = 0.127, P = 0.060). The results of the arrhythmias screening in patients with COVID-19 demonstrate the association mainly with the severity of the disease, and comorbidity, especially diabetes mellitus. So, we may consider arrhythmogenesis in COVID-19 through the prism of inflammation, intoxication, hypoxia, metabolic disorders, and drug effects.

Diet-induced and age-related changes in rats: the impact of N-stearoylethanolamine intake on plasma lipoproteins, adiponectin, and adipocyte cholesterol-phospholipid composition

O. S. Tkachenko*, H. V. Kosiakova

Palladin Institute of Biochemistry,
National Academy of Sciences of Ukraine, Kyiv, Ukraine;

Received: 26 January 2024; Revised: 19 March 2024;
Accepted: 19 March 2024; Available on-line: 30 April 2024

Adiponectin is secreted by adipose tissue, associated with lipoprotein (LP) metabolism, down-regulated­ in insulin resistance states, and reduced in individuals suffering from obesity and cardiovascular diseases. Phospholipids and cholesterol are the main components of cell membranes and play a critical role in storage and secretory adipocyte functions. N-stearoylethanolamine (NSE) is a minor lipid affecting cell membrane lipids’ composition. Our study aimed to investigate plasma levels of adiponectin and cholesterol of low- and high-density LP (LDL and HDL) and adipocyte cholesterol-phospholipid (Chol-PL) composition of different age rats with high-fat diet (HFD)-induced obesity and insulin resistance and their changes under NSE administration. Our study demonstrated that chronic dietary fat overloading leads to obesity accompanied by impairment of glucose tolerance, a manifestation of dyslipidemia, and changes in plasma adiponectin levels in rats from two age groups (10-month-old and and 24-month-old). Prolonged HFD led to a reduction in plasma adiponectin levels and the growth of adipocyte cholesterol content in rats of different ages. A significant increase in plasma LDL-Chol level and main adipocyte PLs (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and lysophosphatidylcholine) was observed in younger rats, whereas not detected in elder animals after dietary fats overloading. The decrease in the content of anionic phospholipids (phosphatidylinositol + phosphatidylserine) was also detected in 10-month-old HFD rats compared to the control animals. NSE administration positively affected the normalization of adiponectin levels in both age HFD groups. It significantly impacted the reduction of LDL-Chol levels and the growth of HDL-Chol concentration in the blood plasma of 10-month-old rats as well as PL-composition of young HFD rats and anionic PL restoring in 24-month-old rats. The positive effect on investigated parameters makes NSE a prospective agent for treating diet-induced and age-related metabolic disorders threatening cardiovascular diseases.

Effect of metal nanoparticles usage on oxidative stress indicators and endotoxemia parameters under DMH-induced carcinogenesis

S. B. Kramar1*, I. Ya. Andriichuk2, N. V. Ohinska1, Yu. V. Soroka3,
Z. M. Nebesna1, S. M. Dybkova4, L. S. Rieznichenko4, N. Ye. Lisnychuk2

1Department of Histology and Embryology,
I. Horbachevsky Ternopil National Medical University, Ukraine;
2Central Research Laboratory, I. Horbachevsky Ternopil National Medical University, Ukraine;
3Department of Anaestesiology and Intensive Care,
I. Horbachevsky Ternopil National Medical University, Ukraine;
4F.D. Ovcharenko Institute of Biocolloidal Chemistry,
National Academy of Sciences of Ukraine, Kyiv;

Received: 10 February 2024; Revised: 19 March 2024;
Accepted: 19 March 2024; Available on-line: 30 April 2024

One of the properties of nanoparticles is their ability to correct manifestations of oxidative stress and endotoxemia, which are critical factors in cancer development. Therefore, the work aimed to investigate the effect of the usage of Au/Ag/Fe nanoparticles on oxidative stress indicators and endotoxemia parameters in experimental colon carcinogenesis. The study was performed on 90 white male rats kept in standard vivarium conditions. The division into groups: I – intact animals; II – intact animals with 21 days NPs administration; III – animals injected with N,N-dimethylhydrazine dihydrochloride for 30 weeks; ІV – animals to which Au/Ag/Fe nanoparticles were intragastrically administered daily for 21 days after induced adenocarcinoma. According­ to our results, the concentration of oxidative stress indicators significantly increases under DMH-induced carcinogenesis conditions. It was established that the 21-day intragastric administration of NP Au/Ag/Fe composition caused a significant (P < 0.001) decrease in the concentration of TBARS in the blood serum by 1.33 times, in the content of diene and triene conjugates by 1.63 and 1.98 times, respectively compared to the third experimental group. The introduction of NPs in the fourth experimental group reduces the concentration of the Schiff bases by 1.34 times (P < 0.001), decreases the content of POMP370 and POMP430 by 1.25 (P < 0.001) and 1.37 times (P < 0.001), respectively, compared to the third experimental group. We also observed the reduction of endotoxemia levels in a fourth experimental animal group based on a significant decrease in MMM indexis and EII percentage.

Circulating levels of potential markers of ischemic stroke in patients with the different forms of atrial fibrillation and chronic heart failure

A. O. Tykhomyrov1*, O. Yu. Sirenko2, O. V. Kuryata2

1Department of Enzyme Chemistry and Biochemistry,
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine;
2Department of Internal Medicine 2, Phthisiology,
Occupational Diseases and Clinical Immunology, Dnipro State Medical University, Dnipro, Ukraine;

Received: 19 January 2024; Revised: 13 March 2024;
Accepted: 17 March 2024; Available on-line: 30 April 2024

Atrial fibrillation (AF) is the most common abnormal type of heart rhythm (cardiac arrhythmia), which is considered the leading cause of stroke. There have been limited studies on the prognostic markers for atrial disease and AF-associated ischemic stroke, despite the high demand for this procedure in daily clinical practice to monitor disease course and assess risk of stroke in patients with AF and chronic heart failure (CHF). Thus, the aim of the present study was to evaluate the levels of serum biomarkers related to ischemic stroke in CHF patients with the different forms of AF. Forty-six patients with various types of AF (paroxysmal, persistent and permanent) with or without ischemic stroke were enrolled in the study, 36 clinically healthy donors served as a control. The levels of inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF) and angiostatins (AS) were evaluated by western blot analysis in the serum. The levels of active matrix metalloproteinases (MMPs) were analysed by gelatin zymography. Elevated levels of iNOS were shown in patients with all AF forms as compared with control, but iNOS levels in post-ischemic patients were significantly higher than that in paroxysmal AF individuals. However, the levels of VEGF and AS did not differ from the baseline value in patients with paroxysmal AF, while dramatic increase of their contents was shown in post-stroke patients with persistent and permanent types of AF. Elevated active MMP-9 levels were shown to be associated with the diagnosis of all AF forms, regardless of the occurrence of stroke. Taken together, our findings­ demonstrate that tested proteins can be considered as valuable biomarkers of AF forms transformation and potentially useful for ischemic stroke risk stratification in patients with AF and CHF. Observed changes in regulatory protein levels may expand our understanding of pathological roles of endothelial function dysregulation, disrupted angiogenesis balance and abnormal tissue remodeling in AF and associated ischemic events.

Vitamin D(3) auto-/paracrine system in rat brain relating to vitamin D(3) status in experimental type 2 diabetes mellitus

I. Shymanskyi1*, O. Lisakovska1, A. Khomenko1,
L. Yanitska2, M. Veliky1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine;
2Bogomolets National Medical University, Kyiv, Ukraine;

Received: 29 January 2024; Revised: 08 March 2024;
Accepted: 11 March 2024; Available on-line: 30 April 2024

Growing evidence suggests that vitamin D3 (D3, cholecalciferol) deficiency and impaired signaling of the hormonally active form of D3, 1α,25(OH)2D3 (1,25D3), through its cellular receptor (VDR) can be significant risk factors for the development of numerous multifactorial diseases, including diabetes. Our investigation was aimed at researching the D3 status in relation to the state of the D3 auto-/paracrine system in the brain and clarifying the effectiveness of the therapeutic use of D3 as a neuroprotective agent in experimental type 2 diabetes mellitus (T2DM). T2DM was induced in male Wistar rats by a combination of a high fat diet and a low dose of streptozotocin (25 mg/kg BW). Diabetic animals were treated with or without cholecalciferol (1,000 IU/kg BW, 30 days). The content of 25-hydroxyvitamin D3 (25D3) in blood serum and brain tissue was determined by ELISA. Analysis of mRNA expression of CYP24A1 and CYP27B1 genes was performed by RT-PCR. Protein levels of VDR, vitamin D3 binding protein (VDBP), CYP27B1 and CYP24A1 were investigated by Western blotting. A significant T2DM-associated decrease in the content of 25D3 in the blood serum was revealed, which correlated with a reduced content of this metabolite in the brain tissue. Impaired D3 status in animals with T2DM was accompanied by an increase in the levels of mRNA and protein of both 25D3 lα-hydroxylase (CYP27B1) and 1,25-hydroxyvitamin D3-24-hydroxylase (CYP24A1), which, respectively, provide local formation and degradation in the nervous tissue of the hormonally active form of D3 – 1,25D3. At the same time, a significant T2DM-induced down-regulation of the brain content of VDBP was shown. In addition, diabetes caused a slight increase in the protein expression of the VDR, through which the auto-/paracrine effects of 1,25D3 are realized in the brain. We have established a complete or partial corrective effect of cholecalciferol on D3 status, its bioavailability in the CNS and the level of protein expression of CYP27B1 and CYP24A1 in the brain of rats with T2DM. Abnormal D3 status in animals with T2DM was accompanied by compensatory changes in the expression of key components of the auto-/paracrine vitamin D3 system. Cholecalciferol was demonstrated to be partially effective in counteracting the impairments caused by T2DM.

Neutrophil activation at high-fat high-cholesterol and high-fructose diets induces low-grade inflammation in mice

G. Bila1, O. Vishchur2, V. Vovk3, S. Vari4, R. Bilyy1*

1Department of Histology, Cytology and Embryology,
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
2Institute of Animal Biology NAAS, Lviv, Ukraine;
3Department of Pathological Anatomy and Forensic Medicine,
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
4International Research and Innovation in Medicine Program,
Cedars-Sinai Medical Center, Los Angeles, California, USA;

Received: 05 February 2024; Revised: 17 March 2024;
Accepted: 17 March 2024; Available on-line: 30 April 2024

Nonalcoholic fatty liver disease (NAFLD), which can progress to nonalcoholic steatohepatitis (NASH), is a significant health concern affecting a substantial portion of the population. This study investigates the role of neutrophil extracellular traps (NETs) in liver inflammation induced by high-fat high-cholesterol diet (HFHCD) and high-fructose diet (HFD). The chronic nature of NAFLD involves low-grade inflammation with cytokine elevation. The research aims to visualize neutrophil elastase (NE) activity during HFHCD and HFD representing conditions of low-grade activation and assess neutrophil functional status. The study employs a mouse model subjecting animals to HFHCD, HFD or a standard diet (SD) for six weeks. Various analyses were used including histological evaluations, in vivo imaging of NE activity using a fluorescent probe, fluorescent microscopy, flow cytometry and assessment of neutrophil function through reactive oxygen species (ROS) levels. Mice on HFHCD and HFD display liver damage consistent with NASH, which was validated pathohistologically. NE activity in blood significantly increases after six weeks indicating systemic NETs involvement. In vivo imaging confirms NE activity in multiple organs. Cellular localization reveals NETs persistence even after neutrophil destruction in splenocytes indicating systemic involvement. Neutrophils under HFHCD exhibit a functional phenotype associated with low-grade inflammation, higher basal ROS levels and reduced activation potential. This study establishes the systemic impact of NETs in HFHCD- and HFD-induced liver inflammation, providing insights into the functional state of neutrophils. The findings contribute to understanding the mechanisms underlying chronic liver conditions and may inform future therapeutic strategies.