Paramagnetic centers in the blood of the pig used as the model animal were examined with an X-band (9.3 GHz) electron paramagnetic resonance spectroscopy. The EPR spectra were measured with Bruker spectrometer at 230 K with a modulation frequency of 100 kHz. The EPR lines of the high spin Fe3+ in methemoglobin, high spin Fe3+ in transferrin, Cu2+ in ceruloplasmin, and free radicals were observed in the multicomponent spectra of blood. For the application of the apitherapeutic nanofiber dressing, the amplitudes of the EPR signals of Fe3+ in methemoglobin were similar up to 10 days. For the experiment with the apitherapeutic formulation, the heights of EPR signals of Fe3+ in transferrin were lower after 10 days and 21 days of therapy, compared to day 0. For the application of the apitherapeutic formulation the signals of Cu2+ in ceruloplasmin and free radicals, strongly decreased after 10 days of therapy, and after 21 days it increased to the initial values characteristic for day 0. The apitherapeutic formulation caused that after 21 days the EPR spectrum of Cu2+ in ceruloplasmin and free radicals was considerably high. The apitherapeutic formulation interaction after 10 days and after 21 days of therapy resulted in the low EPR lines of Fe3+ in methemoglobin. EPR spectra of blood may be useful for presentation of the changes in its paramagnetic centers during the healing process of the burn wounds.The evolution of the paramagnetic center system in blood during the healing of skin burn wounds dressed with a biodegradable apitherapeutic nanofiber dressing was examined. The aim of this study was to determine the changes in paramagnetic centers in blood during the influence of apitherapeutic nanofiber dressings on the healing process. The blood samples were tested before burn infliction (day 0) and, respectively, on the 10 Bee  pathway . In thisth and 21st. The procedure of collection of blood samples for laboratory analysis was based on a marginal ear vein cannulation. After cleaning the surface of the ear, an injection needle was inserted into the lumen of the vessel for sample collection. This procedure was repeated at each collection point [16-week-old, domestic pig was chosen for the evaluation of wound repair because of many similarities between pig and human skin. Eighteen contact burn wounds were inflicted on the right and left flanks of the pig body, according to the methods of Hoekstra et al.  and Branon point . After cNano-nonwoven wound dressings are made with an electrospinning method using poly(lactide-co-glycolide) containing 85 mole-% of lactidyl and 15 mole-% of glycolidyl comonomer units (PLGA 85 : 15) . 6 wt-% Paramagnetic centers in blood samples were examined by the use of electron paramagnetic resonance spectroscopy (EPR) at low temperature equaled 230 K. This spectroscopic method was chosen, because of the direct experimental procedures providing information about molecules containing unpaired electrons . Energy ν is microwave frequency, μB is Bohr magneton, and Br is resonance magnetic field.The electron paramagnetic resonance spectra were measured as the first-derivative curves by an X-band (9.3 GHz) EPR spectrometer produced by Bruker (USA). Magnetic modulation of 100 kHz was used. The low microwave power of 7.9 mW was used to avoid microwave saturation of EPR lines. The cryogenic system to low-temperature measurements of Bruker (USA) was used. For the studied blood samples, the following parameters of EPR spectra were analyzed: g-factors and amplitudes (A). Amplitudes (A) of the EPR lines increase with increasing of the amount of paramagnetic centers in the samples . g-Factoν” and “gμBBr” mean the energy of microwaves and the distance between the energy levels, respectively [ν) was directly measured by the Bruker recorder. The Br values were determined from the electron paramagnetic resonance lines. In the present paper, we followed the same EPR method of examination of paramagnetic centers in the blood as in the previous our work [The expressions of “hectively . Microwaour work  concernist, 10th, and 21st days of burn treatment with a previously used biodegradable dressings additionally containing pharmacologically active substance of natural origin—propolis.The present study is a continuation of our previous experimental studies concerning the usefulness of a new low-temperature electron paramagnetic resonance technique for monitoring molecular complexes containing iron Fe3+  or copper Cu2+ ions (ceruloplasmin) and free radicals in the blood during healing of burn wounds treated with biodegradable dressings containing poly(lactide-co-glycolide). The obtained results indicated that a more complete assessment of biochemical changes taking place in the process of repairing tissue damage after burn insult is needed, especially in the scope of mechanisms regulating the metabolism of iron and copper ion complexes as well as free radicals in the blood. Followed by EPR technic described in our previous work , in thisThe evolution of the healing process undergoing three overlapping phases, (1) hemostasis and inflammation, (2) proliferation, and (3) remodelling, has been summarized and presented in 3+ in methemoglobin (line I), high spin Fe3+ in transferrin (line II), and Cu2+ in ceruloplasmin and free radicals (line III). The low field signals  of the EPR spectra of blood in the experiment with the dressing containing propolis for day 0 (before burn infliction), 10 days, and 21 days of therapy were presented in Figures For all the tested blood samples, the complex EPR spectra were measured , 29. The3+ in methemoglobin (line I), high spin Fe3+ in transferrin (line II), and Cu2+ in ceruloplasmin and free radicals (line III), in blood for burned skin wounds treated with the mentioned biodegradable dressing, was presented in The concentration of the individual types of paramagnetic centers in blood changed with time of therapy for propolis nanofiber dressing. This effect was observed with changes in the individual components of the multicomponent EPR spectra of blood. Their heights depended on the time of therapy with the apitherapeutic formulation. The influence of time of therapy on the amplitudes (A) of the high spin Fe3+ in methemoglobin (line I) were similar up to 10 days, and the amplitude (A) of the (line I) for the blood after 10 days of therapy was only slightly lower than the amplitude (A) of this line measured before burn infliction (day 0) (3+ in transferrin (line II) were lower after 10 days and 21 days of therapy, compared to day 0  strongly decreased after 10 days of therapy, and after 21 days of therapy it increased slightly over the initial value characteristic for the day before burn infliction  of the EPR signals of Fe (day 0) . Its val (day 0) . For theto day 0 . For thefliction . The comThe first phase of wound healing begins immediately upon injury and is dedicated to hemostasis and the formation of a provisional wound matrix. In response to tissue injury, inflammatory cells, including neutrophils, monocytes, and macrophages, are recruited to wounded tissue. Free radicals released mainly from activated neutrophils contribute to local tissue impairment causing oxidative damage of proteins, lipids, DNA, and RNA . In thisRepair of the skin ends with a remodeling phase, which results in would contraction and scar tissue formation. Collagen is remodeled from type III to type I. Cross-linking of collagen reduces scar thickness and makes the skin area of the wound stronger. Adequate level of oxygen is absolutely essential for the cross-linking process –41.The proposed concept of the role of free radicals in each of the wound healing stage has important implications in burn management, indicating that compounds with antioxidant potential, including propolis, can improve the healing of thermal burns.2+ (electron donor) and Fe3+ (electron acceptor) [th day of the experiment may be caused by the use of iron in the course of the Fenton reaction during the acute phase of wound healing [st day of burns management with the novel apitherapeutic dressing, may be connected with the Tf's influence on growth and the formation of extracellular matrix, necessary for the proper regulation of biosynthesis phase of the repair process, manifested by stimulation of the collagen biosynthesis [3+ iron rather than ferrous Fe2+ one, characterised by the decreased ability to bind oxygen, is reported to be reduced in blood after propolis application [Except for the formation of free radicals, injury elicits the acute phase response, which increases in hepatic synthesis of proteins, including among others, ceruloplasmin and transferrin. Taking part in the oxidative stress transferrin, ceruloplasmin, and methemoglobin are also involved in iron metabolism . Previoucceptor) . In the cceptor) . Moreovecceptor) , 46. The healing . Last buynthesis  and enhaynthesis . Anotherlication . The laslication . Furtherlication , propolilication . The inclication , may parlication . Moreovelication . Ceruloplication . Howeverlication . Therefolication , 57. The3+ in methemoglobin, high spin Fe3+ in transferrin, and Cu2+ in ceruloplasmin and free radicals exist in all the tested blood samples, and they are responsible for their multicomponent EPR spectraThe high spin Fe3+ in methemoglobin, high spin Fe3+ in transferrin, and Cu2+ in ceruloplasmin and free radicals in the blood, depends on the time of therapyThe amplitudes of the EPR lines, which reflected the concentrations of high spin FeParamagnetic centers and free radicals' changes indicate a favorable effect of innovative biodegradable apitherapeutic dressings on burns regeneration, suggesting a pluripotent multifaceted influence of propolis on the prooxidative/antioxidative balance changes whose components serve a fundamental function in the repair of tissue damagesThe preformed electron paramagnetic resonance studies of blood from the organisms in the examples of the skin of burned wounds treated with biodegradable, propolis containing, dressing have pointed out that:


A broad spectrum of biological activity as antimicrobial, antiviral, antitumor, cytotoxic, antioxidant, and hemolytic activity was examined. All iodolactones showed bactericidal activity against Proteus mirabilis, and lactones 1,2 were active against Bacillus cereus. The highest cytotoxic activity towards HeLa and MCF7 cancer cell lines and NHDF normal cell line was found for lactone 1. All assessed lactones significantly disrupted antioxidative/oxidative balance of the NHDF, and the most harmful effect was determined by lactone 1. Contrary to lactone 1, lactones 2 and 3 did not induce the hemolysis of erythrocytes after 48 h of incubation. The differences in activity of iodolactones 1–3 in biological tests may be explained by their different impact on physicochemical properties of membrane as the packing order in the hydrophilic area and fluidity of hydrocarbon chains. This was dependent on the presence and type of alkyl substituent. The highest effect on the membrane organization was observed for lactone 1 due to the presence of bulky isopropyl group on the benzene ring.The aim of this work was the examination of biological activity of three selected racemic cis-β-aryl-δ-iodo-γ-lactones. Tested iodolactones differed in the structure of the aromatic fragment of molecule, bearing isopropyl ( Cancer is one of the most serious causes of human death worldwide. Despite the introduction of many new anticancer drugs on the pharmaceutical market, mortality is very high, reaching about 9.6 million deaths in 2018 according to the World Health Organization Cl3 is listed. Although the lactones tested in this project did not show any virulence against adenovirus 36, the literature referenced showed only a 50% reduction of this virus by [Co(NH3)6]Cl3 Cl3 . This ju1, 2, and 3. The results are presented in Selected enzymatic and non-enzymatic oxidative stress markers were measured in the NHDF cell line after exposition to lactones 1–3 and interaction between the type of compound and lactone concentration  showed the least impact on the generation of OS in comparison to control. There was no significant influence of the concentration of lactone 3 on the MnSOD activity (1–3 at MTC but not at starting concentration (1 and 2 had a similar impact on GST activity at MTC (p = 0.337), whereas lactones 2 and 3 influenced the TOS level at SC. Using lactones 1 and 2 at their MTC led to the comparable reduction of deleterious impact of those compounds on TOS (All assessed lactones led to a significant increase in oxidative stress (OS) in the selected cell lines. The statistical analysis of antioxidative stress markers showed that activities of all enzymes, GST, CAT, GPx, Total SOD, MnSOD, and CuZnSOD, as well as the levels of non-enzymatic markers such as TAC, TOS, and MDA were equally and significantly influenced by the type of β-aryl-δ-iodo-γ-lactones used in the experiment as well as by the concentrations of lactones ntration . The mos control . Among tactivity . GST actntration . Lactones on TOS .1) and the least effect was observed for lactone 3 with an unsubstituted phenyl ring.Our results are in the line with other findings, which report a significant increase in oxidative stress parameters in the cancer type of the cell line after exposition to the several groups of lactones . Thomasz2 and lactone 3 did not induce the hemolysis of RBCs in the tested range of concentrations (from 10 to 100 μM) up to 48 h of incubation  or no substituent (3) on the benzene ring are nontoxic, unlike lactone with isopropyl group (1) which is toxic at the highest concentration after 48 h of incubation [According to the toxicity classification of compounds, lactones with methyl . The value of GP for lactone 3 was the same as for the control. An increase in the value of GP indicates a decrease in water content in the area of glycerol with a corresponding increase in the packing order in hydrophilic regions of the phospholipid bilayers.The fluorimetric method was employed to monitor the effect of iodolactones on physicochemical properties of RBCMs like the hydration, stiffness, and packing of membrane building molecules. The monitoring of different areas in the membrane is possible using fluorescent probes. For this research, were used two fluorescent probes: Laurdan and DPH. Laurdan is incorporated into the apolar interior of the bilayer through hydrocarbon chains but fluorescent group is located at the level of lipid ester groups . This fl1 and 2 caused a decrease of the value of anisotropy compared to the control sample. A decrease in the anisotropy was at the level of 14% and 8% for compounds 1 and 2, respectively, and these differences were statistically significant (p < 0.05). The decrease in the value of anisotropy determines an increase of the membrane fluidity and a decrease of the hydrocarbon chains organization. This suggests that lactones 1 and 2 show an impact on the fluidity in the hydrophobic region of the lipid bilayers. However, the results also indicate that lactone 3 did not change the fluorescence anisotropy of the DPH probe in RBCMs in relation to the control sample . Changes of values of anisotropy for RBCMs (control) and RBCMs with addition of compounds are presented in l sample  and did 1 and 2 significantly interact with the membrane causing an increase of the packing order in the hydrophilic area as well as a slight increase in the fluidity in the area of hydrocarbon chains. Probably, these changes in membrane organizations are caused by the alkyl substituents on the para position of the benzene ring (hydrophobic substituents). Iodolactones 1 and 2 penetrate deeper into the bilayer, causing changes in the packing of the molecules which may weaken van der Waals interactions between hydrocarbon chains. These effects are more pronounced for lactone 1, having a bulkier isopropyl group on the benzene ring than lactone 2, which possesses a methyl group at the same position. Consequently, the lack of changes in biophysical parameters of membrane after the addition of lactone 3 is probably due to the lack of any alkyl substituent on the phenyl ring.In summary, the results obtained from fluorimetric analysis showed that lactones As shown by the above results, within the studied group of lactones, type and position of substituent on the benzene ring plays an important role in the interactions with the membrane. This finding can be also confirmed by our earlier studies of enantiomeric pairs of δ-bromo-γ-lactones and their δ-lactone analogs possessing a 2,5-dimethylphenyl substituent at the β-position of the lactone ring. They concentrated mainly in the hydrophilic part of erythrocyte membrane but had practically no influence on fluidity in the hydrophobic region. In that case, it was also found that enantiomers that showed higher interactions with the membrane exhibited higher antiproliferative activity .The FTIR method was employed to extend the information obtained in the flouorimetric research and to determine the effect of tested lactones on the main functional groups of the biological membrane. The most important bands of lipids considered in the RBCMs spectrum were band of hydrocarbon chains, carbonyl groups, phosphate group, and choline fragment. All bands with appropriate type of vibration are shown in −1 associated with lipids vibrations of CH2 (methylene) and CH3 (methyl) groups of alkyl chains. The maximum of asymmetrical and symmetrical stretching vibrations of the methylene groups are located at about 2920 cm−1 (vasCH2) and 2850 cm−1 (vsCH2), respectively. For the methyl groups, we considered only the maximum of asymmetrical stretching vibrations which is located at about 2956 cm−1 (vasCH3) [1. The maximum was shifted very slightly towards lower values of wave numbers compared to the control. The addition of lactones 2 and 3 did not change the signal frequency in this area. Based on these results, we can conclude that only lactone 1 causes a slight change in the ordering of hydrocarbon chains.In the hydrophobic region of the membrane, we considered bands located in the range of 3100–2800 cmvC=O) are located between the polar and non-polar parts of the membrane in the range of 1750–1700 cm−1. This group provides information on the degree of hydration in this region of the membrane [Another important functional group in the lipid molecules is the carbonyl group. The bands of the stretching vibrations of this group  and 1095–1085 cm−1 (vs(PO2−), respectively [1, slightly shifted the maximum of asymmetric vibrations of the phosphate band towards lower values of wave numbers. In the case of symmetrical vibration, a similar situation was observed after addition of lactone 1 that caused a slight shift of this band in the same direction. Addition of the other two iodolactones caused a slight shift of the band in the opposite direction. The results indicated slight changes in the conformation of the phosphate group, especially after adding lactone 1 [−1 (for RBCMs) shifted toward lower frequencies in the presence of lactones probably indicates an interaction via hydrogen bonds between the PO2- group of the lipid and lactone 1 molecules. This suggests decreasing H-bonding between lipid molecules, which is indicative of the immobilization of this fragment of the lipid molecule, presumably due to a hydrogen bond formation. The obtained results confirm the conclusion stemming from the fluorimetric methods that iodolactones, in particular lactone 1, influence the degree of ordering of the erythrocyte membrane lipid bilayer. Taking these results into account, we can confirm the interactions between the investigated compounds  and the phosphate group of lipids. The most external part of the hydrophilic area is the choline fragment, and its characteristic band is located in three regions of the FTIR spectrum. The asymmetrical vibration (C–N+(CH3)) has the highest intensity, with a maximum at around 970 cm−1 [2 and 3 shift the maximum of this band very slightly towards higher wavenumbers.The hydrophilic area of the membrane of the erythrocyte is represented by the phosphate group and the choline fragment. Asymmetrical and symmetrical vibrations of phosphate group are located in the ranges of 1255–1225 cmectively . The asyectively . The preactone 1 ,61. The actone 1 . Similaractone 1 ,65,66,67970 cm−1 . The stu−1 and plays a key role in determining the type of protein secondary structure. Amide II occurs in the range of 1480–1575 cm−1 and is also used for determining the type of secondary structure [In the RBCMs spectra using FTIR methods, the main bands derived from proteins named as Amide I and II were also analyzed. Amide I is located in the range of 1610–1695 cmtructure ,61,68. Ttructure .1 had the highest effect on the membrane which is probably related to the presence of an isopropyl group on the benzene ring. The tested iodolactones only slightly affect the structure of proteins present in the membrane. The conclusions obtained from the FTIR method are very important due to the fact that the membrane components participate directly as messengers or regulators of signal transduction. In addition, protein–lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains [The results obtained from FTIR suggest that all compounds affect the hydrophilic region of head groups of lipids by changing their packing order. However, lactone odomains . Therefocis-β-aryl-δ-iodo-γ-lactones showed that their activity is dependent on the presence and type of the alkyl substituent at the para position of benzene ring. In particular, the highest cytotoxic activity and the highest influence on the antioxidative stress markers was found for the lactone bearing isopropyl group (1), lower activity for lactone with methyl substituent (2), and the least activity for lactone 3, possessing an unsubstituted phenyl ring.The panel of biological assays carried out for testing 1 and more significant changes in membrane organizations . The lack of significant changes in the biophysical parameters of the membrane after the addition of lactone 3 is probably due to the lack of an alkyl substituent.The different activity of lactones with alkyl groups is probably the consequence of their different interactions with the cell membrane, which were particularly demonstrated in fluorimetric studies. The presence of a more bulky substituent at benzene ring results in deeper penetration of the lipid bilayer by lactone In the light of these findings, further research is needed to understand the impact of the lactones on the membrane organization which is crucial to explain the mechanism of their biological activity.


Objective: The aim of this study was to explore the influence of short-term (2-week) exposure to social (SE) and/or physical enrichment (PE) on the anxiety-like behavior of ovariectomized (OVX) NIH Swiss mice.Method: One week after surgery, each OVX mouse was housed under one of 4 social conditions: (1) isolated, (2) accompanied by an intact female, (3) accompanied by an intact male, or (4) in a community of 10 OVX individuals. The animals in each of these environments were divided into 2 subgroups, consisting of the presence and absence of PE. Following a 2-week exposure to the respective conditions, each OVX mouse was subjected to either the light/dark exploration test (LDT) or the elevated plus maze (EPM) to examine anxiety-like behavior.Results: The LDT and EPM showed very similar patterns. Compared to an impoverished environment, PE elicited a significant anxiolytic effect for OVX mice housed alone or in companion of an intact female . By contrast, mice living in community but without PE displayed anxiogenic-like behavior, perhaps due to crowding, compared to the animals living in isolation . Conclusion: This study emphasized the importance of taking housing conditions into account during the screening of new anxiolytic agents and the critical role of OVX in the regulation of anxiety. An ovariectomy (OVX) has been shown by different tests to produce an increase in anxiety-like behavior in rodents , 2, whicTreatment of ovariectomized rats , 8 and mIn line with the above, other studies on adult female rodents indicated that the administration of estradiol and progesterone is linked to anxiolytic-like effects , 11, 12.On the other hand, anxiety-like behavior can also be regulated by environmental enrichment (EE), consisting of physical enrichment (PE) and/or social enrichment (SE). Enrichment of physical surroundings is generally accomplished by providing the home cage with toys or objects that favor voluntary exercise and exploratory behavior . EnrichmWhile most studies utilizing the OVX mouse model and behavioral tests have evidenced a pro-cognitive effect of EE , few havHence, the aim of this study was to evaluate the effect of a short-term (2-week) exposure to social (SE) and/or physical enrichment (PE) on the anxiety-like behavior of OVX mice. The effect of various combinations of SE and PE was herein analyzed by means of 2 well-validated techniques to assess anxiety, the light/dark exploration test (LDT), and the elevated plus maze (EPM).AnimalsNIH Swiss mice, purchased from the Harlan animal house of the National Autonomous University of Mexico (Mexico City), were used to generate breeding stock and experimental animals in the local vivarium of the Autonomous University of Chiapas. The animals were freed of all viral, bacterial, and parasitic pathogens listed in the recommendations by the Federation of European Laboratory Animal Science Association . The micOvariectomyThe OVX procedure was performed on 200 mice interperitoneally administered 25 mg/kg sodium pentobarbital  to produce general anesthesia. After shaving and disinfecting the surgical site, a 0.5-cm ventral midline incision was made with a scalpel. Subsequently, the connective tissue was separated to cut through the peritoneal wall. The ovaries were located and removed bilaterally; then, the peritoneum and the muscle layers were stitched with an absorbable suture  and the skin with a nonabsorbable nylon suture  that spontaneously detached at 2-3 days post surgery.Experimental DesignFollowing one week of recovery from OVX, all animals were assigned to one of 8 groups, which were designed for the purpose of testing the effect of different forms and combinations of EE. There were 4 social environments, consisting of isolation  and 3 distinct types of SE. In the latter cases, an OVX mouse was housed with an intact male, with an intact female, or in a community of 10 OVX individuals. Each of the 4 social conditions was subdivided into 2 physical environments, with and without PE .However, these 8 groups were not all used in the same experiment. There were 2 experiments presently conducted, each with a distinct set of groups to examine the effect of EE on the anxiety-like behavior of OVX mice. For community housing, each of the mice was marked with a number (using a non-toxic permanent marker) in the dorsal region and then put in a large cage (60 cm x 40 cm x 22 cm). For the community group exposed to PE, 10 objects for exploration/exercise were scattered throughout the cage. The objects, consisting of plastic tubes (15 cm long x 4 cm in diameter), rectangular plastic blocks of diverse colors and textures (5 cm x 3 cm x 1 cm), rattles and glass marbles (2 cm in diameter) were randomly moved every other day. The groups with 1 or 2 individuals were put in a small cage (33 cm x 14 cm x 14 cm), which for the condition of PE contained only 1 object. In all cages, the object or objects were carefully cleaned and randomly moved every other day. The first experiment compared the consequences of living under 2 general conditions: in isolation (one OVX mouse per cage) or in the company of another mouse . Accordingly, there were 3 social environments, each subdivided into 2 groups (with or without PE). Hence, 6 independent groups were formed for each behavioral test (LDT and EPM) for a total of 12 groups and 120 animals (10 per group). The second experiment analyzed the results of living in isolation versus in community (in each case with or without PE). Thus, 4 independent groups existed for each behavioral test (LDT and EPM) for a total of 8 groups and 80 animals (10 per group). Behavioral TestsAfter 2 weeks of the respective housing conditions, the behavioral evaluations were performed in the 2 anxiety tests. Each animal was subjected to only one of these tests . All expThe Light/Dark Exploration TestThe LDT is conducted in an acrylic cage (44 x 21 x 21 cm) divided into a relatively small dark compartment constituting 1/3 of the total cage size, and a larger and highly illuminated compartment (560 lx light intensity) occupying 2/3 of the cage. The dark and light areas are separated by a wall with a small opening (13 x 15 cm). For the test, each mouse is introduced (only once) into the light area and the number of transitions through the opening is registered during 10 minutes. An anxiolytic-like effect is considered with a larger number of transitions. The present procedure, chosen as a result of our previous studies , 32, is The Elevated Plus MazeThe EPM is based on the natural tendency of mice to actively explore a new environment, countered by a fear of being in an open area. The apparatus consists of a central platform (5 x 5 cm) connecting 2 open arms (25 x 5 cm) with 2 equal-sized enclosed arms (25 x 5 x 15 cm) that form a cross. The maze is elevated to a height of 50 cm above the ground and illuminated by a red light. The evaluation lasts for 5 minutes, beginning when a mouse is placed in the center of the apparatus. A longer time spent on the open arms reflects an anxiolytic-like effect and a longer time on the enclosed arms an anxiogenic-like effect. The total number of crossings from the open to closed arms (or vice versa) is taken as an indicator of ambulatory activity .Ambulatory Activity TestTo examine possible alterations in motor activity, a spontaneous ambulatory activity test was performed after the LDT. For this test, the animal is put in an acrylic cage (60 x 40 x 40 cm) with a checkerboard pattern (20 x 20 cm) on the floor, and the total number of squares crossed by the mouse is manually counted for 5 minutes.Data AnalysisAll data were analyzed by 2-way ANOVA on SigmaStat v 3.5 . PE was assigned as factor A and SE as factor B. In both experiments, factor A was defined as the presence or absence of PE and factor B as one of the housing conditions investigated herein . A significant influence on anxiety-like behavior was exerted by PE  and SE , but not by the interaction of both factors . A shorter time was spent on the open arms by the mice housed in community versus in isolation, independently of the factor of PE . Apart from this exception, the multiple comparison analysis between groups tested on the EPM demonstrated anxiolytic-like behavior similar to that observed in the LDT. Regarding ambulatory activity, no statistical difference existed between the ambulatory activity test (number of squares crossed) and the EPM  .For OVX mice living under different housing conditions, spontaneous ambulatory activity was measured by counting the number of squares crossed or total arm entries in the EPM. Two-way analysis of variance revealed nonsignificance of motor activity in all cases. The results of the 2-way ANOVA showed that for number of squares crossed, PE , SE , AxB interaction ; for the EPM, PE , SE , AxB interaction .Both behavioral tests showed a substantially higher level of anxiety for isolated mice in an impoverished versus enriched environment. Reports in the literature on isolated mice with and without PE have described a similar anxiogenic-like effect for the impoverished environment , 36, thoThe removal of ovaries is a known risk factor for the development of anxiety symptoms in rats . The curIn the absence of PE, an anxiogenic-like behavior was exhibited by isolated OVX mice, but not by OVX mice accompanied by a gonadal-intact female partner. Likewise, it has been documented that pair-housed OVX prairie voles maintain a low level of anxiety compared to isolated animals , and thaAccording to the aforementioned reports, SE is significant as long as the companions have similar characteristics . However, anxiogenic-like behavior was herein displayed by OVX mice living in community (10 per cage) in the absence of PE, which may have been related to the smaller space available per mouse under such conditions . Indeed, an increase in housing density of mice is a well-known stress factor  linked tThere have been descriptions in the literature of mice housed under conditions similar to those used herein, but with distinct results , 40. ThiExcept for the OVX mice with a male partner, the beneficial impact of PE was clearly demonstrated in this study, in line with previous reports of a PE-induced reduction in behavioral and physiological responses to anxiogenic stimuli , 44. ForThe evaluation of experiments was herein limited to the observation of anxiolytic- and anxiogenic-like behavior. Since biochemical analysis was not performed, there was no interpretation of possible mechanisms of the findings. In the future, it is necessary to employ a paradigm of OVX mice housed in isolation and in distinct social conditions in the presence and absence of PE, and attention should be paid on measuring the activity of some neurotransmitters  and the expression of certain receptors .The evaluation of experiments was herein limited to the observation of anxiolytic- and anxiogenic-like behavior. Since biochemical analysis was not performed, there was no interpretation of possible mechanisms of the findings.The results of this study emphasized the importance of OVX in the regulation of anxiety and perhaps in responsiveness to stress. Additionally, different social environments affected the anxiety-like behavior of OVX mice in the presence and absence of PE. Thus, it is necessary to conduct studies using a biochemical approach to provide insights into the mechanisms involved in the regulation of anxiety when OVX animals are housed under distinct social and physical contexts.


High-entropy alloy coatings (HEAC) exhibit good frictional wear and corrosion resistances, which are of importance for structure materials. In this study, the microstructure, surface morphology, hardness, frictional wear and corrosion resistance of an AlCoCrFeNi high-entropy alloy coating synthesized by atmospheric plasma spraying (APS) were investigated. The frictional wear and corrosion resistance of the coating are simultaneously improved with an increase of the power of APS. The influence of the APS process on the microstructure and mechanical behavior is elucidated. The mechanisms of frictional wear and corrosion behavior of the AlCoCrFeNi HEAC are discussed in detail. High-entropy alloys (HEAs), comprised of at least five principal elements with the atomic percentage of each element varying from 5 to 35%, have attracted increasing attention because of their appealing properties and potential applications ,2,3. The0.5 HEACs were fabricated by laser  in the passive film. The third peak at about 532.5 eV indicates a bound water (H2O) in the passive film. The bound water is considered as a capable species that can assemble the dissolving metal ions, finally forming a new film to prevent further corrosive attack. In this case, it can be inferred that the surface of the H4 coating mainly contained Al2O3, Co3O4, CoO, Cr(OH)3, Fe2O3, Fe3O4, FeO, NiO and the bound water (H2O).Regarding the best corrosion resistance of the four coatings, the H4 coating is chosen for the XPS analysis.  and CoO a,b. The nd Cr2O3 c. The Fe and FeO d. The pe and FeO e. Howeveuttering ,40. The − tends to adhere to the passive film at the boundary, which weakens and breaks the passive film, and finally results in the pitting initiation.2O) in the passive film. By changing the processing parameters, it is feasible to acquire an AlCoCrFeNi HEAC with both excellent wear resistance and outstanding corrosion resistance.The frictional wear and corrosion resistance of the AlCoCrFeNi HEACs synthesized by atmospheric plasma spraying with four different process parameters were investigated. All the coatings were mainly composed of BCC (body-centered cubic) and FCC (face-centered cubic), containing some oxides and unsolvable metal elements. With the increasing the power of APS, the solid-solution structure was formed in the HEACs. Multi-component metal oxides were generated and randomly distributed on the surface of the coating in the process of the frictional wear process, which can prevent the serious adhesion wear from occurring. The denudation of multi-component metal oxides and the formation of the particle-like debris on the surface of the coatings can adequately grease the friction process between the rubbing-pair and the coating surface. The coating synthesized by the APS with the highest power of APS of the four coatings with different processing parameters demonstrated the most positive corrosion potential, and the lowest corrosion current density, suggesting an improvement in corrosion resistance. The improved corrosion resistance was attributed to the appearance of bound water (H


In the paper, we provide a detailed description of the enzyme catalytic cycle, model calibration based on a series of in vitro kinetic data, and model validation using experimental data on the regulatory properties of PGHS-1. The validated model of PGHS-1 with a unified set of kinetic parameters is applicable for in silico screening and prediction of the inhibition effects of NSAIDs and their combination on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandin biosynthesis in platelets and endothelial cells expressing PGHS-1.The kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was developed to investigate its complex network kinetics and non-steroidal anti-inflammatory drugs (NSAIDs) efficacy in different in vitro and in vivo conditions. To correctly describe the complex mechanism of PGHS-1 catalysis, we developed a microscopic approach to modelling of intricate network dynamics of 35 intraenzyme reactions among 24 intermediate states of the enzyme. The developed model quantitatively describes interconnection between cyclooxygenase and peroxidase enzyme activities; substrate  and reducing cosubstrate competitive consumption; enzyme self-inactivation; autocatalytic role of AA; enzyme activation threshold; and synthesis of intermediate prostaglandin G The enzyme state containing heme with two oxidizing equivalents—one on the iron and another one on the porphyrin radical cation—is referred to as Intermediate 1  state. The E2 state can be converted back to the resting state E1 by 2 consecutive reactions with RC by followed release of oxidized cosubstrate, OC (reactions 12 and 10). Alternatively, the E2 state can undergo one-electron internal reduction via intramolecular electron transfer from Tyr385 residue in the COX site to protoporphyrin (reaction 13) resulting in the formation of the E5 state  containing Tyr385 radical (Tyr385*) and a ferryl heme. This state is referred to as Intermediate II  referred to as Intermediate II  (E9), containing AA in the COX site. Reaction 2 corresponds to the abstraction of a hydrogen atom from AA by Tyr385 radical and formation of the E13 state containing AA radical in the COX site, . Further, AA* reacts with two oxygen molecules, and the intermediate product PGG2 radical (PGG2*) is produced (reaction 3). We assumed that binding of the first and second oxygen molecules is a rapid process, during which the redox state of the heme remains unchanged  with one oxidising equivalent on iron, and therefore, each of the enzyme states E5, E8, E9, and E13 can potentially participate in redox reactions with RC , producing catalytic states E12, E15, E16, and E20. The later states can be considered as intermediates of an additional COX2 cycle. Note that similar COX cycle was included in the model developed by Kulmacz et al.  (state E11). Note, that similar COX cycle was considered in the extended BC mechanism model , which further converts into intermediate,  (E13), containing the arachidonyl radical AA* (reaction 56). Consideration of two alternative mechanisms of AA radical formation in our model allowed us to analyse their possible contributions to overall COX activity.In our model, we also included an alternative route for initiation of COX reactions, which was proposed in an early version of the TC mechanism of PGHS-1 function —a directPGHS-1 activity undergoes irreversible self-inactivation (SI) during catalysis . The exaBased on the experimental data, we considered several possible mechanisms for enzyme SI in our model. These mechanisms included separate inactivation of the COX and POX activities, originating from enzyme intermediates with different redox state of heme group and containing one or two radicals in the catalytic domains . All theFirst, inactivation of POX activity resulting in fully inactivation enzyme  Reactions 38–42, 53, and 67 originate from Intermediate I and lead to POX and further COX inactivation due to radical state of heme prosthetic group () ,22. In o5, E9, E15, and E20) and lead to POX and further COX inactivation due to the presence of oxidising equivalent Fe(IV) in heme.(b) Reactions 43–46 originate from Intermediate II (states E5 cycle (reactions 25–27). A similar POX cycle was considered in the model developed by Kulmacz et al.  (E17) and  (E4). It should be noted that the remaining POX activity can also further be inactivated due to damage caused by the presence of the radical state in heme (reaction 42).Second, we considered the direct inactivation of COX activity with remaining POX activity which can be caused by the presence of radical states in the COX site in the model . In our z et al. . AccordiAs a result of the joint fitting of three series of the experimental data (see Methods), a unified set of kinetic parameters was determined. The results of fitting are shown in 2 and PGH2 production by PGHS-1 treated by AA and phenol as RC , E2) by RC was obtained to be about zero 8k (0.001 μM−1 s−1); that means that the Intermediate I is rather reduced by intramolecular process of electron abstraction from Tyr385 with radical formation than by exogenous RC.A small value of reaction rate 12k of AA binding to the states in the POX2 cycle , while it is sensitive to the dissociation constant d,12K of the same reactions. The model also showed weak sensitivity to parameter in2k, which corresponds to one of the SI reactions of the enzyme, but it is highly sensitive to parameters in1k and ink characterising the other SI reactions considered in the model.To estimate uncertainty of the parameters obtained in the fitting procedure of the model against experimental data, we carried out global sensitivity analysis (GSA) of the model (see Methods). GSA revealed that the calibrated model showed uniform sensitivity to the most kinetic parameters . The mod2O2 and adrenalin as RC  (Ection 18 .5) state much more effectively than to the resting state (E1), Intermediate I (E2), and E3 state. Maximum flux 1(t)V exceeds maximum values of 30(t)V, 31(t)V, and 32(t)V by an order of magnitude (see comparison of 1(t)V and 31(t)V in 31V) has a high peak in an initial stage of the COX reaction, but its contribution to overall catalysis is negligible because of the short duration of the peak (1 ms).The performed flux analysis showed that AA binds to the Intermediate II . Thus, reduction of the heme group due to Tyr385 radical formation in the presence of AA in the COX site is weaker than that in the case of the empty COX site. Therefore, substrate–enzyme complex  (E9) is formed mainly from Intermediate II according to the experimental data , as opposed to the kinetic models of PGHS-1 , being common for the COX2 and POX4 cycles, participates simultaneously in the production of the intermediate product PGG2 (reaction 23) and the final product PGH2 (reaction 8). As discussed above, these reactions make maximum contributions to the synthesis of PGG2 and PGH2 . For this, we first calculated the kinetics of the oxygen consumption rate O2V(t) and compared the results with the experimental data  (E15). This conclusion contradicts the models where only the ferril state  (E5) was considered in the COX reactions  E1. This coeactions . To remo14 state in POX4 cycle) occurs, when AA is in the COX site. The flux analysis allowed us to compare this mechanism of AA radical formation with the alternative mechanism through Tyr385 radical, proposed in framework of the BC mechanism   and ferryl states  and  . Alternatively, AA can bind to the enzyme state containing Tyr385 radical . Therefore, it was assumed that the reactions of a similar type would have similar rate constants.In general, all the reactions in the developed catalytic cycle shown in However, in general case, kinetic constants of the elementary reactions taking place in one of the catalytic sites can depend on the state of all other catalytic components, located in the nearest proximity and involved in the further catalytic steps. For example, there is experimental evidence that AA binding to the COX site may depend on the presence/absence of the radical on Tyr385 . PGHS-1 We tried different approaches in grouping the model parameters and analysed the results of fitting of the corresponding models to the same set of experimental data. Analysis of the fitting results allowed us to hypothesise which reaction rates do not depend much on the processes taking place in the neighbour site and therefore which kinetic constants can be assumed to be equal to each other. This analysis also allowed us to make conclusions on the cooperativity between COX and POX activities. The approach we used allowed us to significantly reduce the number of parameters subject to fitting. The best fitting of the model to experimental data was achieved with the set of 18 parameters given in When identifying the model parameters, we were trying to find a unified set of parameters which would allow us to consistently describe a majority of the in vitro kinetic data known for PGHS-1 catalysis. Identification of the model parameters and their further validation was performed as follows. We chose several experimental data sets obtained in different research laboratories to fit the model parameters against the data and then validated the calibrated model by checking its applicability to the description of experimental data which were not taken in the fitting procedure.2 and PGH2 production .To avoid ambiguity, for the initial stage of the fitting, among all kinetic information available for PGHS-1, we chose three homogeneous datasets which were obtained for the same object—purified PGHS-1 from sheep seminal vesicles—under approximately the same experimental conditions (temperature and pH). The first set included kinetic data on PGGd AA see . Note thμ and σ are two sensitivity measures in the Morris GSA method, i.e., μ assesses the impact of a parameter on the model output, and σ describes the non-linear effects and interactions of the parameters. As the model output in the calculation, we took an area under the curve (AUC) of PGH2 time course.Global sensitivity analysis (GSA) of the model to the kinetic parameters obtained in the fitting procedure was based on the Morris GSA method for the calculation of elementary effects ,33. The The key aim of this work was the development of a reliable and validated kinetic model of PGHS-1 which reproduces the main features of its complex kinetics and can be used as a computational tool for accurate prediction of inhibition effect of NSAIDs and their combinations in a range of cellular conditions. The cycle-based network model integrates both the current models of the molecular mechanism of PGHS-1 catalysis and a large dataset of experimental data on the kinetics and regulatory properties of PGHS-1. This integrative approach helped us to overcome the limitation of the previously developed PGHS-1 models mainly applicable to the description of individual in vitro data in a specific range of substrate and cosubstrate concentrations.The developed model of PGHS-1 catalysis is a platform for the further development of a kinetic model of inducible isoform PGHS-2 (COX-2) which is expressed together with PGHS-1 in vascular endothelia cells and other organs and is a target of COX-2 selective NSAIDs. Development of the kinetics models of two isoforms within a unified integrative approach would allow for the investigation of the different physiological roles of these isoforms in the same cells and analysis of the effect of NSAID inhibition on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandins in platelets and endothelial cells.


In recent years, the existence of methylation of mammalian mitochondrial DNA (mtDNA) has been discussed. The current state of knowledge indicates that mtDNA is poorly methylated; in fact, it only accounts for 2–8% methylated sites and its pattern is unknown. The lack of comprehensive information on the mtDNA methylation pattern prompted us to investigate the distribution of guanine-cytosine-rich sequences (CpG) in different animal species. The aim of the study was to determine the localization of CpG sites and islands in mtDNA of model organisms. The CpG sites and islands found in vertebrates and invertebrates indicate a diversified pattern of CpG distribution. Generally, the number of observed CpG sites of the mitochondrial genome was higher in the analysed vertebrates than in the invertebrates. However, there was no relationship between the frequency of the CpG sites in the mitochondrial genome and the complexity of the analysed organism. The distribution of the CpG sites for transfer RNA (tRNA) coding genes was usually cumulated in a larger CpG region in the vertebrates.CYTB, ND6, ND5, ND4, ND3, ND2, ND1, COX3, COX2, COX1, ATP6, 16s rRNA, and 12s rRNA. The CpG distribution in animals from different species was diversified. Generally, the number of observed CpG sites of the mitochondrial genome was higher in the vertebrates than in the invertebrates. However, there was no relationship between the frequency of the CpG sites in the mitochondrial genome and the complexity of the analysed organisms. Interestingly, the distribution of the CpG sites for tRNA coding genes was usually cumulated in a larger CpG region in vertebrates. This paper may be a starting point for further research, since the collected information indicates possible methylation regions localized in mtDNA among different species including invertebrates and vertebrates.The information about mtDNA methylation is still limited, thus epigenetic modification remains unclear. The lack of comprehensive information on the comparative epigenomics of mtDNA prompts comprehensive investigations of the epigenomic modification of mtDNA in different species. This is the first study in which the theoretical CpG localization in the mtDNA reference sequences from various species (12) was compared. The aim of the study was to determine the localization of CpG sites and islands in mtDNA of model organisms and to compare their distribution. The results are suitable for further investigations of mtDNA methylation. The analysis involved both strands of mtDNA sequences of animal model organisms representing different taxonomic groups of invertebrates and vertebrates. For each sequence, such parameters as the number, length, and localization of CpG islands were determined with the use of EMBOSS (European Molecular Biology Open Software Suite) software. The number of CpG sites for each sequence was indicated using the newcpgseek algorithm. The results showed that methylation of mtDNA in the analysed species involved mitochondrial gene expression. Our analyses showed that the CpG sites were commonly present in genomic regions including the D-loop,  Caenorhabditis elegans, which lacks the ATP8 gene [Nearly all animal mitochondrial genomes are about 16. 5 kbp (kilo base pairs) in length, whereas plant mitochondrial genomes range between 200 and 2000 kbp [TP8 gene  and the TP8 gene . MtDNA i5C) was found in beef heart mitochondria [The activity of methyltransferase (DNMT) was first detected in mitochondria isolated from loach embryos in 1970s . Next, 5chondria . Reis anchondria  conductechondria . Scientichondria ,13. Little is known about mitochondrial epigenetic modifications, as studies on mtDNA methylation are not as common as studies on the methylation of nDNA. So far, it has been observed that hypomethylation may occur in mtDNA methylation ,14,15. DIn recent years, the methylation of mammalian mtDNA has gained interest. There are papers rejecting the existence of mtDNA methylation . For insDespite new evidence, the information about mtDNA methylation is still limited. Therefore, this epigenetic modification is a controversial issue. The lack of comprehensive information on the comparative epigenomics of mtDNA suggests that there is a need to conduct comprehensive investigations of the epigenomic modification of mtDNA in different species. The aim of the study was to determine the localization of CpG sites and islands in mtDNA of model organisms and to compare its distribution. The results are suitable for further investigations of mtDNA methylation.Reference sequences of twelve animal mtDNAs obtained from GenBank were analysed. The study was carried out on sequences of animal model organisms representing different taxonomic groups of invertebrates and vertebrates .In order to analyse both strands of mtDNA, sequences from GenBank were rewritten in the EMBOSS revseq algorithm to obtain complementary sequences representing the H-strand of mtDNA . RegionsCaenorhabditis elegans, Daphnia magna, and Drosophila melanogaster, i.e. all invertebrates analysed in the study. In the analysed animal models, the length of the CpG islands varied from 202 bp to 313 bp in the L-strand and from 200 bp to 632 bp in the H-strand. The results of the L-strand showed that one CpG island was located in the COX2 gene , and two CpG islands were found in the sequence from Danio rerio , Crocodylus porosus (4), and Homo sapiens (4) had the highest numbers of CpG islands. CpG islands were found frequently in genomic regions covering loci of 12s rRNA (71%), CYTB (43%), ND5 (57%), and COX1 (43%). Interestingly, two CpG islands were observed in the 12s rRNA and ND5 genes from the mtDNA genome of Crocodylus porosus and the COX1 gene from Gallus gallus , in contrast to avian and crocodilian species [Crocodylus porosus and Gallus gallus in a location analogous to the region of the replication origin in the other vertebrates . After replication of two-thirds of the H-strand, the replication of the L-strand starts and proceeds in the opposite direction [Canis lupus familiaris, a higher number of CpG sites were found on the H-strand of mtDNA  and are essential for ATP synthesis [Homo sapiens including CYTB, COX1, D-loop, and 12s rRNA has been reported by Liu et al. [It is worth noting that four genes i.e. ynthesis . Additiou et al. .ND6 gene was reported in many studies [ND6, COX1, and the D-loop region with the use of quantitative methylation specific-PCR in the context of non-alcoholic fatty liver disease in humans [ND6 gene, which inversely correlated with ND6 transcription and protein expression in the liver affected by NASH [ND6 gene in all the analysed vertebrates and the number of CpG sites varying from 3 to 22. Noteworthy, all NADH dehydrogenase subunits  were rich in CpG sites in the mtDNA of the different species  have CpG sites in the ND1 gene; however, the CpG distribution in the ND6 gene is mainly limited to the H-strand of chordates. Moreover, the number of the CpG sites in ND1 was higher (from 2 to 58) than in ND6 (from 3 to 22), especially in Latimeria chalumnae, Ambystoma mexicanum, Crocodylus porosus, Gallus gallus, Mus musculus, Canis lupus familiaris, Pan troglodytes ellioti, and Homo sapiens  and primates (Pan troglodytes ellioti and Homo sapiens). This may be caused by the 1-methyladenosine (m1A) modification in 16S rRNA catalysed by tRNA methyltransferase 61B (TRMT61B) [The transcription of the H-strand of mtDNA starts at two initiation sites  within the control region. The produced transcript from H1 terminates at the 3′ end of the 16S rRNA gene and processes the two rRNAs and two tRNAs, whereas H2 terminates at the 5′ end of the 12S rRNA gene and generates a polycistronic molecule contributing to the mRNAs and most of the tRNAs encoded in the H strand . The mitnslation ,38. The TRMT61B) .Arabiopsis thaliana, Oryza sativa), invertebrates , and vertebrates  [In the analysis of mtDNA methylation, several challenges that can affect the correct detection of the levels of mtDNA methylation have to be overcome. The first one is the high mtDNA copy number in cells; it naturally varies from hundreds to thousands of copies depending on the cell type. Application of super-resolution microscopy provides more details. Currently, the number of mtDNA molecules per nucleoid in human cells is estimated at 1.4 . Anothersapiens) . Therefosapiens) . Moreovesapiens) .The theoretical study on the distribution of CpG sites and islands in the mitochondrial genome of twelve model animal species provides interesting information about the localization of CpG-rich regions that can be methylated in specific cells in certain conditions. The CpG methylation in mtDNA exerts an impact on various molecular processes, including replication, translation, and gene expression. Since methylation in mtDNA, in comparison to methylation in nDNA, is still not sufficiently understood, research in this area is advisable.


We found that the rate of Ca2+ uptake by mitochondria of the liver and heart in HR rats is higher than that in LR rats, which is associated with a higher level of the channel-forming subunit MCU in liver mitochondria of HR rats and a lower content of the dominant-negative channel subunit MCUb in heart mitochondria of HR rats. It was shown that the liver mitochondria of HR rats are more resistant to the induction of the MPT pore than those of LR rats . These data correlate with the fact that the level of F0F1-ATP synthase, a possible structural element of the MPT pore, in the liver mitochondria of HR rats is lower than in LR rats. In heart mitochondria of rats of the two phenotypes, no statistically significant difference in the formation of the MPT pore was revealed. The paper discusses how changes in the expression of the MCUC subunits and the putative components of the MPT pore can affect Ca2+ homeostasis of mitochondria in animals with originally different tolerance to hypoxia and in hypoxia-induced tissue injury.The work examines the kinetic parameters of Ca Hypoxia/ischemia is a widespread phenomenon that occurs both in conditions of oxygen deficiency in the environment and in various pathologies as a result of a decrease in oxygen delivery to the cell to a level insufficient to maintain its functions and structure. Hypoxic conditions in the body are observed in ischemic and reperfusion injuries of organs, hemodynamic disorders, coronary insufficiency, blood loss, hemorrhagic shock, arterial hypertension, pulmonary insufficiency, systemic inflammatory response syndrome, traumatic shock, exposure to adverse environmental factors, extreme conditions, and other stressful effects.2+, homeostasis. Excessive accumulation of Ca2+ in cytoplasm and mitochondria in hypoxia and subsequent reoxygenation can finally initiate cell injury and death via the opening of the Ca2+-dependent mitochondrial permeability transition pore (MPT pore) and the subsequent release of proapoptotic proteins from organelles  modulate key cellular processes, ranging from aerobic metabolism  to the bs cycle ,6,7,8, abs cycle ,10.2+ uptake: mitochondrial Ca2+ uniporter complex (MCUC), RaM (the rapid mode of Ca2+ uptake), Letm1 (leucine zipper and EF-hand-containing transmembrane protein 1), mitochondrial ryanodine receptor type 1, and uncoupling proteins  increased, indicating a massive release of the ion from the organelles due to the opening of the MPT pore. The amount of Ca2+ released upon permeability transition (defined as Ca2+ retention capacity) was used as a measure of the MPT pore opening probability.The CaTo prepare samples for quantifying the levels of mitochondrial proteins, aliquots of native mitochondria (2 mg/mL) were solubilized in Laemmli buffer in Eppendorf tubes and heated for 3 min at 95 °C. Sample aliquots normalized by the protein concentration  were applied to the lanes and subjected to electrophoresis followed by Western blot analysis. Mitochondrial samples were separated by 12.5% SDS-PAGE and transferred to a 0.45 µm nitrocellulose membrane . The proteins of PageRuler Prestained Protein Ladder  were used as markers. After overnight blocking, the membrane was incubated with the appropriate primary antibody. The monoclonal rabbit anti-MCU (#14997), anti-CBARA/MICU1 (#12524), and anti-ANT2/SLC25A5 (#14671) antibodies were from Cell Signaling Technology Inc. . The total OXPHOS Rodent WB Antibody Cocktail (#ab110413) containing the α-subunit of complex V (CV-ATP5A-55 kDa), anti-VDAC1 (#ab154856), and the polyclonal rabbit antibodies Anti-CCDC109B (#ab170715), anti-cyclophilin F (CypD) (#ab64935), and anti-ANT1 (#ab102032) were from Abcam . The immunoreactivity was detected using the appropriate secondary antibody conjugated to horseradish peroxidase . Peroxidase activity was detected with ECL chemiluminescence reagents . The relative levels of the detected proteins were visualized using an LI-COR system . Optical density measurements were performed using the LI-COR Image Studio software.T method was used to quantify the results [Total RNA was isolated from 100 mg of deep-frozen tissue samples (the liver or the heart) using an ExtractRNA kit  in accordance with the protocol of the manufacturer. The resulting RNA preparation was treated with RNase-free DNase I . The concentration of total RNA was measured spectrophotometrically using a Nanodrop ND-1000 spectrophotometer . Two micrograms of total RNA was taken for cDNA synthesis; reverse transcription was performed using an oligo(dT)15 primer and MMLV reverse transcriptase  according to the manufacturer’s instructions. Real-time PCR was performed using a DTLite5 amplifier  using the qPCRmix-HS SYBR reaction mixture . The selection and analysis of gene-specific primers were performed using Primer-BLAST  the oli. The rel results .p < 0.05 was considered to be statistically significant.The data were analyzed using the GraphPad Prism 7 and Excel software and presented as mean (median) ± SEM of 3–5 experiments. Statistical differences between the means were determined by Mann–Whitney U test; 2+ transport in the liver and the heart of animals with different tolerance to oxygen shortage, LR and HR rats. 2+ (50 μM) by the liver mitochondria of LR (dotted line) and HR rats (solid line) in the presence of CsA, which was necessary to block the possible opening of the MPT pore. One can see that the rate of Ca2+ influx into the liver mitochondria of HR rats is 1.3 times higher compared with that of LR rats   C.2+ uptake is mediated by an electrogenic uniport, referred to as “Ca2+ uniporter”, a complex of proteins of the inner mitochondrial membrane, including the pore-forming subunit MCU and its dominant-negative form MCUb, and the regulatory subunits MICU1, MICU2, EMRE, and MCUR1. It is considered that the contents of MCU, MCUb, and MICU1 and their stoichiometry can predominantly regulate the mitochondrial Ca2+ transport in accordance with physiological needs [2+ faster as compared to the organelles of LR rats, one can assume that the adaptation to hypoxic stress is associated with changes in the relative content of these subunits in the mitochondrial membrane. Therefore, we quantified the uniporter protein constituents and their mRNA level in the liver and cardiac muscle of rats depending on the baseline resistance of animals to hypoxia.Mitochondrial Caal needs ,6,23. Si2+ transfer from the endoplasmic reticulum to mitochondria [The immunoblotting of the members of the MCUC protein family of the mitochondria isolated from the liver of rats of two phenotypes reveals that in HR rats, the levels of MCU and MICU1 were higher than in LR rats by 1.3 and 1.35 times, respectively A,B, wherchondria . One can−ΔCtMCU = 0.014 ± 0.001 and 0.025 ± 0.001 in the liver tissue of LR and HR rats, respectively; 2−ΔCtMCUb = 0. 003 ± 0.0005 and 0.0035 ± 0.00038 in LR and HR rats, respectively). −ΔCtMCU = 0.024 ± 0.005 and 0.034 ± 0.008 in the heart tissue of LR and HR rats, respectively; 2−ΔCtMCUb = 0.007 ± 0.0007 and 0.0038 ± 0.0005 in LR and HR rats, respectively).The results of the real-time PCR analysis confirm the data obtained and also indicate that the changes occur at the level of transcription. The mRNA content of MCU in the liver of HR rats is significantly increased in comparison with that of LR rats. At the same time, the expression profile of MICU1 and MCUb in the liver of rats of two phenotypes does not differ A. So, th2+ uptake in HR rats relative to that in LR rats.On the basis of the results obtained, one can conclude that an increase in the relative amount of the pore-forming subunit of MCUC, MCU, in liver mitochondria and a decrease in the dominant-negative subunit, MCUb, in heart mitochondria underlie the increased rate of mitochondrial Ca2+-induced MTP pore opening is well known to be central to mitochondrial vital functions and can play a lethal role in many pathophysiological conditions including hypoxia cell injury. So, the next objective of our work was to examine whether the resistance of mitochondria to the opening of the MPT pore changes depending on the baseline tolerance of an animal to hypoxic condition. The mitochondrial Ca2+ retention capacity (CRC) is related to the threshold concentration of Ca2+ necessary for the pore to open. One of the ways to assess this parameter is to introduce Ca2+ into the suspension of mitochondria in small successive doses, and the number of such additions until the MPT pore is triggered will reflect the CRC of the organelles. 2+ additions  in the case of the liver mitochondria of HR rats was greater compared to the organelles of the liver of LR rats. The parameter of CRC of HR rat liver mitochondria increased 1.3-fold , ATP synthase, and cyclophilin D, are considered to be essential components of the pore complex ,24. To eAtp5f1a in the liver of HR rats is significantly decreased in comparison with that of LR rats. At the same time, the expression profile of ANT1 and cyclophilin D in the liver of rats of two phenotypes does not differ , a metabolic shift toward glycolysis, and the development of oxidative stress, but also induce cell death pathways via mitochondrial Ca2+ overload and the formation of Ca2+-dependent MPT pore in the inner mitochondrial membrane, leading to the release of proapoptotic proteins into the cytosol [2+ transport pathways might be targets for protective intervention, as well as involved in the formation of the molecular mechanism of cell resistance to hypoxia and ischemia. The purpose of our work was to study in more detail the features of structure and operation of the Ca2+ transport systems in the mitochondria of the liver and heart of rats with originally different tolerance to acute hypoxia. It is generally accepted that mitochondria are one of the main intracellular targets of hypoxia. Structural, biochemical, and functional abnormalities of mitochondria are widely believed to be important pathogenetic factors that underlie hypoxic or ischemic cell injury ,18. Apar cytosol . Consequ2+ homeostasis. In the case of HR rats, the rearrangements of the Ca2+ uniporter can increase the efficiency of Ca2+ accumulation by liver and heart mitochondria. At the same time, the organelles of the liver of HR rats become more resistant to the opening of the Ca2+-dependent MPT pore in comparison with those of LR rats. Along with the well-characterized distinction in the intensity of OXPHOS in mitochondria of the vital organs of LR and HR rats [The results obtained in this work indicate that mitochondria of HR and LR rats are characterized by fundamentally different genetically programmed rearrangements of the systems regulating Ca HR rats ,25, thesIt is well known that individuals of an animal population differ in their tolerance to oxygen deficiency ,16,17,25It was previously shown that mitochondria of the vital organs of LR and HR rats differ in both structural and basic functional parameters. Thus, the mitochondria of the cerebral cortex of HR rats are characterized by a denser packing of cristae and a more electron-dense matrix, smaller sizes, and higher concentrations of the respiratory chain complexes and the respiration substrate, succinate  ,25. In c2+ ions via MCUC. The liver and heart mitochondria of HR rats are characterized by a significant increase in the efficiency of Ca2+ uniport compared to the organelles of LR rats, with the difference being especially pronounced for the liver tissue. To elucidate the causes of these functional effects, we have examined structural changes in the Ca2+ transport system of mitochondria of LR and HR rats and revealed that these changes do occur in the macromolecular complex of the mitochondrial Ca2+ uniporter in the heart and liver tissues.Here, we have demonstrated that, in addition to the above structural–functional features, the mitochondria of the liver and heart of rats with originally different tolerance to hypoxia are distinguished in respect to the rate of influx of Ca2+ channel, which transfers the ion across the inner mitochondrial membrane. MCUb is a dominant-negative MCU paralogue, and its overexpression impairs the ion-transporting function of the complex [The MCUC is considered to consist of two transmembrane channel subunits: MCU and MCUb. MCU forms a highly selective Ca complex . MCU andAs follows from the data obtained, in HR animals, the contents of the pore-forming subunit MCU and the gate subunit MICU1 are substantially increased in liver mitochondria, whereas the level of the dominant-negative subunit, MCUb, is decreased in heart mitochondria. The data are confirmed by the results of real-time PCR analysis and indicate that these changes occur at the level of transcription. At the same time, there is almost no difference between liver and heart mitochondria of LR and HR animals in the content of the regulatory uniporter subunit EMRE and the expression of its gene.2+-conducting pore subunits MCU and MCUb [2+ influx into the organelles. Based on the data obtained, we can conclude that the structural changes in the pore-forming subunits of MCUC in HR rats increase the efficiency of Ca2+ accumulation in the mitochondrial matrix and, as a result, might underlie the fine control of cytoplasmic free Ca2+ at low levels.It is known that the activity of MCUC is regulated by changes in the ratio of the Caand MCUb ,26,27. T2+ into mitochondria can result in the opening of MPT in the inner mitochondrial membrane. As a result, all transmembrane ionic gradients and the membrane potential collapse, and mitochondria swell, which leads to the rupture of their outer membrane and the release of proapoptotic proteins  from the organelles. This can facilitate the activation of the caspase cascade, DNA damage, and eventually cell death. The opening of the MPT pore is believed to be triggered by hypoxia and subsequent reoxygenation [2+ capacity . Similar results were partially obtained by us earlier [On the other hand, excessive accumulation of Cagenation ,14. The  earlier . In orde earlier .However, the question as to which subunit of this multisubunit complex is the central structural component of the MPT pore, or whether it is formed by MPT-specific conformation of dimers of ATP synthase, remains the province of further investigations.2+ buffering capacity is likely explained by the fact that the content of cyclophilin D, a major regulatory protein of the MPT pore [2+ transfer from the endoplasmic reticulum to mitochondria [2+ handling and the MTP opening.It should be noted that the CRC index of mitochondria isolated from the heart of HR rats was slightly lower compared to that of LR rats, although this difference was not statistically significant. This trend toward a decline in the CaMPT pore ,29,30, ichondria ,14. So, 2+ transport in mitochondria is not accompanied by a higher susceptibility of mitochondria to calcium overload and the induction of the MPT pore. As follows from our data, changes in the levels of the MPT-related proteins can play a crucial role in the regulation of pore formation in mitochondria. At the same time, the high rate of mitochondrial Ca2+ uptake may provide a more effective removal of excess Ca2+ ions from the cytosol under physiological conditions and hypoxia.Based on the results obtained, one can assume that the increase in the rate of Ca2+ ion transport, which may be essential for proper mitochondrial function in hypoxia and contribute to the formation of adaptive signs to provide the development of cellular response to oxygen shortage. So, apart from the increased activity of the ATP-sensitive potassium channel and respiratory chain complexes, mitochondria of HR animals are characterized by the increased rate of uptake of Ca2+ ions. At the same time, compared to liver mitochondria of LR animals, the organelles of HR rats are more resistant to the opening of the Ca2+-dependent MPT pore. Taking into account the fact that the MPT pore opening is one of the key pathogenic events in oxygen deficiency, it can be supposed that these changes contribute to the molecular basis underlying cellular sensitivity to hypoxic injury.The results obtained point out that liver and heart mitochondria of HR animals exhibit functional and structural features in Ca