Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs

Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study

Розробка методології раціонів харчування для спортсменів в умовах пандемії

The object of research is the diet, physical activity, individual and anthropogenic data of athletes-athletes. A systematic study of scientific literature and a systemic analysis of actual nutrition and sports load were carried out. On its basis, the problems of nutrition of athletes in a pandemic are analyzed and the ways of their solution are determined. During the research, the methods of theoretical analysis of literature, synthesis, formalization, abstraction and experimental studies of the actual nutrition and sports load of athletes-athletes were used. As a result of the research carried out, a methodology of diets for athletes in a pandemic was developed, with its subsequent computerization on the basis of the 1C «Enterprise» platform. This methodology is based on the creation of a database about a specific athlete. This allows in the future to calculate the individual requirements for energy and all nutrients, taking into account the medico-biological requirements for macro- and micronutrients, as well as biologically active substances in consumed food. The program selects the necessary traditional dishes and products, as well as specialized products necessary to fill all the needs of the body of a particular athlete. All the necessary components for a full-fledged diet of an athlete have been analyzed and described. The necessity of eating behavior and the combination of a correct diet with an individual amount of loads have been determined. The methodology of the diet development process is presented. In comparison with analogues, the developed program takes into account the type and time of physical activity, controls the amount of water consumed, calculating an individual food ration, taking into account food preferences and allergic reactions. The introduction of the developed methodology and the use of its computerized program is a rational approach to the diet of athletes in a pandemic, as evidenced by the accumulated experience and achievements of modern nutritional science. Combining a properly balanced diet with a training program will allow an athlete to improve their athletic performance in a pandemic.Объектом исследования является рацион питания, физические нагрузки, индивидуальные и антропогенные данные спортсменов-легкоатлетов. Проведено системное исследование научной литературы и системный анализ фактического питания и спортивной нагрузки. На его основе проанализированы проблемы питания спортсменов в условиях пандемии и определены пути их решения. При проведении исследований использованы методы теоретического анализа литературы, синтез, формализация, абстрагирование и экспериментальные исследования фактического питания и спортивной нагрузки спортсменов-легкоатлетов. В результате проведенных исследований разработана методология рационов питания для спортсменов в условиях пандемии, с последующей ее компьютеризацией на базе платформы 1С «Предприятие». В основу данной методологии положено создание базы данных о конкретном спортсмене. Это позволяет в дальнейшем рассчитывать индивидуальные потребности в энергии и всех пищевых веществах с учетом медико-биологических требований к макро- и микронутриентам, а также биологически активных веществ в употребляемых пищевых продуктах. Программой проводится подбор необходимых традиционных блюд и продуктов, а также специализированных продуктов, необходимых для заполнения всех потребностей организма конкретного спортсмена. Проанализированы и описаны все необходимые составляющие для полноценного рациона спортсмена. Определена необходимость пищевого поведения и сочетание правильного рациона с индивидуальным количеством нагрузок. Приведена методология процесса разработки рациона. По сравнению с аналогами разработанная программа учитывает вид и время физических нагрузок, контролирует количество потребленной воды, рассчитывая индивидуальный пищевой рацион с учетом пищевых предпочтений и аллергических реакций. Внедрение разработанной методологии и использования ее компьютеризированной программы является рациональным подходом к разработке пищевых рационов спортсменов в условиях пандемии, о чем свидетельствует накопленный опыт и достижения современной нутрициологии. Сочетание правильно сбалансированного рациона питания с программой тренировок позволит спортсмену улучшить свои спортивные результаты в условиях пандемии.Об’єктом дослідження є раціон харчування, фізичні навантаження, індивідуальні та антропогенні дані спортсменів-легкоатлетів. Проведено системне дослідження наукової літератури та системний аналіз фактичного харчування та спортивного навантаження. На його основі проаналізовано проблеми харчування спортсменів в умовах пандемії та визначено шляхи їх вирішення. Під час проведення досліджень використано методи теоретичного аналізу літератури, синтез, формалізація, абстрагування та експериментальні дослідження фактичного харчування та спортивного навантаження спортсменів-легкоатлетів. У результаті проведених досліджень розроблено методологію раціонів харчування для спортсменів в умовах пандемії, із подальшою її комп’ютеризацією на базі платформи 1С «Підприємство». В основу цієї методології покладено створення бази даних про конкретного спортсмена. Це дозволяє в подальшому розраховувати індивідуальні потреби в енергії та всіх харчових речовинах із врахуванням медико-біологічних вимог до макро- та мікронутрієнтів, а також біологічно активних речовин у спожитих харчових продуктах. Програмою проводиться підбір необхідних традиційних страв і продуктів, а також спеціалізованих продуктів, необхідних для заповнення всіх потреб організму конкретного спортсмена. Проаналізовані та описані усі необхідні складові для повноцінного раціону спортсмена. Визначена необхідність харчової поведінки та поєднання правильного раціону з індивідуальною кількістю навантажень. Наведено методологію процесу розробки раціону. У порівнянні з аналогами розроблена програма враховує вид та час фізичних навантажень, контролює кількість спожитої води, розраховуючи індивідуальний харчовий раціон із врахуванням харчових переваг та алергічних реакцій. Впровадження розробленої методології та використання її комп’ютеризованої програми є раціональним підходом до харчових раціонів спортсменів в умовах пандемії, про що свідчить накопичений досвід і досягнення сучасної нутриціології. Поєднання правильно збалансованого раціону харчування з програмою тренувань дозволить спортсмену покращити свої спортивні результати в умовах пандемії

Evaluation of the Anticancer and Probiotic Potential of Autochthonous (Wild) <i>Lacticaseibacillus paracasei</i> Strains from New Ecological Niches as a Possible Additive for Functional Dairy Foods

Probiotics such as Lactobacillus spp. could modulate the intestinal microbiota composition, supporting gastrointestinal tract barrier function and benefiting human health. To evaluate the anticancer and probiotic properties of potentially active autochthonous Lacticaseibacillus paracasei strains on proliferating and differentiated enterocytes, human colon adenocarcinoma cell line HT29 was used as a model. The lactic acid bacteria (LAB) were isolated from new ecological niches—mountain anthills populated by redwood ants (Formica rufa L.). Human colorectal adenocarcinoma cells (HT29, ATCC, HTB-38™) were treated for twenty-four hours with supernatants (SNs) derived from four strains of Lacticaseibacillus paracasei: P4, C8, C15 and M2.1. An MTT assay, alkaline phosphatase activity, IAP, Bax and Bcl-2 gene expression analysis (RT-qPCR) and the Bax/Bcl-2 ratio were evaluated. The MTT assay revealed that the observed effects varied among groups. However, 10% neutralized supernatants from P4, C8, C15 and M2.1 strains did not show cytotoxic effects. In contrast to non-differentiated cells, a significant (p p p p p p L. paracasei strains possess anticancer and probiotic properties and could be used as additives for functional dairy foods and thus benefit human health

n-3 polyunsaturated fatty acids provoke a specific transcriptional profile in rabbit adipose-derived stem cells in vitro

Adipose-derived stem cells (ADSCs) possess multipotent properties, and their proper functionality is essential for further development of metabolic disorders. In the current study, we explored the impact of two n-3 LC-PUFAs (long-chain polyunsaturated fatty acids, DHA-docosahexaenoic; C22:6, and EPA-eicosapentaenoic; C20:5) on a specific profile of lipolytic-related gene expressions in the in vitro-differentiated subcutaneous and visceral ADSCs from rabbits. The subcutaneous and visceral ADSCs were obtained from 28-day-old New Zealand rabbits. The primary cells were cultured up to passage 4 and were induced for adipogenic differentiation. Thereafter, the differentiated cells were treated with 100 µg EPA or DHA for 48 hr. The total mRNA was isolated and target genes expression evaluated by real-time RCR. The results demonstrated that treatment of rabbit ADSCs with n-3 PUFAs significantly enhanced mRNA expression of Perilipin A, while the upregulation of leptin and Rab18 genes was seen mainly in ADSCs from visceral adipose tissue. Moreover, the EPA significantly enhanced PEDF (Pigment Derived Epithelium Factor) mRNA expression only in visceral cells. Collectively, the results suggest activation of an additional lipolysis pathway most evident in visceral cells. The data obtained in our study indicate that in vitro EPA up-regulates the mRNA expression of the studied lipolysis-associated genes stronger than DHA mainly in visceral rabbit ADSCs. Eкaterina Vackova1| Darko Bosnakovski2| Bodil Bjørndal3| Penka Yonkova4|Natalia Grigorova1| Zhenya Ivanova1| Georgi Penchev4| Galina Simeonova5|Lyuba Miteva6| Anelya Milanova1| Tatyana Vachkova7| Spaska Stanilova6|Ivan Penchev Georgie

Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia

Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. Objectives and Methods: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 &plusmn; 4 mmHg; age 29 &plusmn; 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 &plusmn; 2 mmHg; controls) were enrolled in the clinical study. Results: PE was associated with a higher placental (0.04 &plusmn; 0.01 vs. 0.49 &plusmn; 0.11 pmol/g; p &lt; 0.01) and plasma MBG (0.5 &plusmn; 0.1 vs. 1.6 &plusmn; 0.5 nmol/L; p &lt; 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 &plusmn; 0.2 vs. 1.5 &plusmn; 0.2 &micro;mol Pi/mL/hr; p &lt; 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p &lt; 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p &lt; 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. Conclusion: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE

Dietary Sodium Restriction Reduces Arterial Stiffness, Vascular TGF-β-Dependent Fibrosis and Marinobufagenin in Young Normotensive Rats

High salt (HS) intake stimulates the production of marinobufagenin (MBG), an endogenous steroidal Na/K-ATPase ligand, which activates profibrotic signaling. HS is accompanied by a blood pressure (BP) increase in salt-sensitive hypertension, but not in normotensive animals. Here, we investigated whether HS stimulates MBG production and activates transforming growth factor-beta (TGF-&beta;) profibrotic signaling in young normotensive rats, and whether these changes can be reversed by reducing salt to a normal salt (NS) level. Three-month old male Sprague&ndash;Dawley rats received NS for 4 and 8 weeks (0.5% NaCl; NS4 and NS8), or HS for 4 and 8 weeks (4% NaCl; HS4 and HS8), or HS for 4 weeks followed by NS for 4 weeks (HS4/NS4), n = 8/group. Systolic BP (SBP), pulse wave velocity (PWV), MBG excretion, aortic collagen 1&alpha;2, collagen 4&alpha;1 and TGF-&beta;, Smad2, Smad3, Fli-1 mRNA, and total collagen abundance were measured at baseline (BL), and on weeks 4 and 8. Statistical analysis was performed using one-way ANOVA. SBP was not affected by HS (125 &plusmn; 5 and 126 &plusmn; 6 vs. 128 &plusmn; 7 mmHg, HS4 and HS8 vs. BL, p &gt; 0.05). HS increased MBG (164 &plusmn; 19 vs. 103 &plusmn; 19 pmol/24 h/kg, HS4 vs. BL, p &lt; 0.05) and PWV (3.7 &plusmn; 0.2 vs. 2.7 &plusmn; 0.2 m/s, HS4 vs. NS4, p &lt; 0.05). HS8 was associated with a further increase in MBG and PWV, with an increase in aortic Col1a2 80%), Col4a1 (50%), Tgfb1 (30%), Smad2 (30%) and Smad3 (45%) mRNAs, and aortic wall collagen (180%) vs. NS8 (all p &lt; 0.05). NS following HS downregulated HS-induced factors: in HS4/NS4, the MBG level was 91 &plusmn; 12 pmol/24 h/kg (twofold lower than HS8, p &lt; 0.01), PWV was 3.7 &plusmn; 0.3 vs. 4.7 &plusmn; 0.2 m/s (HS4/NS4 vs. HS8, p &lt; 0.05), aortic wall Tgfb1, Col1a2, Col4a1, Smad2, Smad3 mRNAs, and collagen abundance were reversed by salt reduction to the BL levels (p &lt; 0.05). HS was associated with an activation of TGF-&beta; signaling, aortic fibrosis and aortic stiffness accompanied by an MBG increase in the absence of SBP changes in young normotensive rats. The reduction of dietary salt following HS decreased MBG, PWV, aortic wall collagen and TGF-&beta;. Thus, HS-induced aortic stiffness in normotensive animals occurred in the context of elevated MBG, which may activate SMAD-dependent TGF-&beta; pro-fibrotic signaling. This data suggests that a decrease in salt consumption could help to restore aortic elasticity and diminish the risk of cardiovascular disease by reducing the production of the pro-fibrotic factor MBG

Canrenone Restores Vasorelaxation Impaired by Marinobufagenin in Human Preeclampsia

Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Recently, we demonstrated that (i) MBG induces fibrosis in rat tissues via a mechanism involving Fli1, a negative regulator of collagen-1 synthesis, and (ii) MBG sensitive Na/K-ATPase inhibition is reversed by mineralocorticoid antagonists. We hypothesized that in human PE elevated MBG level is associated with the development of fibrosis of the umbilical arteries and that this fibrosis can be attenuated by canrenone. Fifteen patients with PE (mean BP = 118 ± 4 mmHg; 34 ± 2 years; 38 ± 0.3 weeks gest. age) and twelve gestational age-matched normal pregnant subjects (mean BP = 92 ± 2 mmHg; 34 ± 1 years; 39 ± 0.2 weeks gest. age) were enrolled in the study. PE was associated with a higher plasma MBG level, with a four-fold decrease in Fli1 level and a three-fold increase in collagen-1 level in the PE umbilical arteries vs. those from the normal subjects (p 50 = 141 nmol/L vs. EC50 = 0.9 nmol/L; p < 0.001). The effects of PE on Fli1 and collagen-1 were blocked by the in vitro treatment of umbilical arteries by 10 μmol/L canrenone. Similar results were obtained for umbilical arteries pretreated with MBG. These data demonstrate that elevated MBG level is implicated in the development of the fibrosis of umbilical arteries in PE, and that this could be blocked by mineralocorticoid antagonists