Background: To study the effect of allogeneic umbilical cord mesenchymal stem cell transplantation on the structure and function of the thymus in aged C57 mice and provide a new method for the treatment of senile thymic atrophy.Results: The changes in the thymus cortex and medulla volume and the lymphocyte ratio were analyzed by immunofluorescence. For thymus tissue sections, immunohistochemical staining was performed to detect p16, p53, SOD, becline1, LC3b, p62, sirt1, and sirt3. Changes in CK5, CK8, CD4 and CD8 expression were observed. Treatment with mUCMSCs could promote hair regeneration in aging mice and regenerate the thymus structure.Conclusions: mUCMSCs inhibited senescence of the thymus and promoted structural and functional thymus regeneration by downregulating the senescence genes p53 and p16 and upregulating the SOD, Sirt1 and Sirt3 genes, but the mechanism requires further research.7 cells/kg twice per week for 3 weeks. Six weeks after the last transplantation, the thymus was weighed, and the thymus-to-body weight ratio was calculated. The thymus tissue was stained with HE.Methods: C57 mice were obtained and met the requirements of thymic aging. mUCMSCs were infused via the tail vein at a dose of 1×10 The immune system consists of immune organs, immune cells and immune molecules. Its functions include identifying and excluding antigenic foreign bodies and coordinating with other systems of the body to maintain the stability and physiological balance of the internal environment. As a central immune organ in the human body, the thymus is important for maintaining the stability of the immune system. However, the thymus is age-sensitive and is one of the earliest organs to age and atrophy in the human body. Therefore, whether it is possible to regenerate the thymus to slow its aging and shrinking is an important question.As a site of T cell development and maturation, the thymus often undergoes atrophy due to aging. The loss of thymocytes and destruction of the thymus structure caused by thymus atrophy reduce mature T cell output and limit T cell receptor (TCR) diversity. Maintaining normal thymic function is critical for reducing the morbidity and mortality of various clinical conditions, including infection and transplantation , 2.The mammalian thymus is generally divided into two leaves, and each leaf is divided into the central medulla and peripheral cortex. The main constituent cells are thymic stromal cells (TSCs), epithelial Mo-MΦ cells, delayed reaction T cells (TDCs), fibroblasts, and thymocytes. The thymus is at its largest and in its most active periods before birth and during puberty. After these periods, it gradually shrinks and is replaced by fat.+ T cell counts and intracellular GSH levels and decreased plasma levels of HIV RNA [The thymus is mainly composed of hematopoietic thymocytes and nonhematopoietic thymic epithelial cells (TECs) . To prov HIV RNA , which d HIV RNA . 2. Cyto HIV RNA , 11. 3.  HIV RNA . HoweverThe thymic microenvironment consists primarily of TECs and cells of mesenchymal origin. TECs are important in almost all stages of thymocyte development. The mesenchyme is involved in the functional development of epithelial cells and the maintenance of epithelial structure and function. The mesenchyme has been shown to be necessary for the successful reconstruction of thymus tissue . MesenchThis study aimed to systematically demonstrate the improvement of senile thymic atrophy induced by administration of MSCs in naturally aging C57 mice to provide a solution for the treatment of clinical thymic atrophy. This study provides additional experimental evidence for the clinical application of MSCs.Gross view with the naked eye: white hair appeared in C57 mice at 18 months of age , and theThe thymus weight, body weight, and thymus/body weight ratio of C57 mice are shown in After HE staining, the tissue was observed by inverted microscopy. (1) HE staining of the thymus in 2-month-old C57 mice: A thin connective tissue envelope was observed on the surface of the thymus tissue, the thymocyte leaflet interval was not obvious, and the cortex and medulla were clearly defined. The thymus cells in the cortex were dense, and its coloration was deeper. The medulla contained more TECs and had lighter coloration. A large thymus were observed in young mice . (2) HE The umbilical cord tissue pieces were inoculated in a 10 cm culture dish based on the characteristics of adherent mUCMSC growth. After 1 day, the cells were observed climbing out of the dish . After 6The cells obtained by adherent culture of umbilical cord tissue were long, spindle-shaped and fibroblast-like . After cCD29, CD90 and CD34 antibodies were used to label C57 mouse umbilical cord tissue adherent culture cells. The positive proportion of CD29 cells was 99.6% , and theGross view with the naked eye: The model control group showed white hair and partial hair loss on the back . The haiGross view with the naked eye: The thymus in the young control group was yellowish in color, soft in texture and normal in size. The thymus color in the treatment group and the model control group was darker, and the volume was reduced. The volume of the treatment group was significantly larger than that of the model control group . AccordiAfter HE staining, an inverted microscope was used to make observations. (1) The thin layer of connective tissue was visible on the surface of the thymus tissue of the young control group. The thymocyte leaflet interval was not obvious, the cortex and medulla were clearly defined, and the thymus cells in the cortex were dense, so the coloration was deep. The medulla contained more TECs, so the coloration was lighter, and the appearance of the thymus was consistent with that observed in young mice , 9B. (2)Cytokeratin 8 (cK8) and cytokeratin 5 (cK5) are markers of the thymic cortex and medulla, respectively, as shown by the results of thymus staining with immunofluorescent antibodies . (1) The+ and CD8+ cells in the thymus of the model control mice was low  , 7. The By comparing the levels of LC3b, becline1, P62 and other proteins that characterize the level of autophagy in the thymus, we found that the expression of LC3b, becline1, and P62 in the thymus tissue of the treatment group was increased to different degrees compared with that in the other groups. The LC3b activity was lowest in the young control group and highest in the treatment group, and the model control group showed intermediate activity  young c young c  The thymus volume of the C57 mice in the treatment group was significantly larger than that in the model control group. The thymus index was calculated and statistically analyzed. The mean value of the thymus index was compared . However, according to HE staining, the thymus tissue structure of the treatment group was significantly restored compared with that of the model control group. The thymus cortex structure of the model control group was unclear with respect to quality, and no glandular structure was observed. (2) The thymic medulla and cortex were labeled with the keratin antibodies ck5 and ck8, respectively. The statistical analysis showed that the cumulative optical density (IOD) of CK5 expression in the thymic medulla of the model control group was higher than that of the young group (p< 0.01). This result indicated that the density of epithelial stromal cells expressing CK5 in the medulla was increased, the proportion of epithelial stromal cells was increased, and the proportion of lymphocytes that did not express CK5 was decreased. This suggested that the size of the thymic medulla in C57 mice increases with age, the proportion of lymphocytes decreases, and the proportion of thymic medulla stromal cells increases. The expression level of CK5 in the thymus of the treatment group was lower than that of the model control group (p<0.01), indicating that the proportion of lymphocytes that do not express CK5 in the medulla of the senile mice increased after mUCMSC treatment, and the total number of cells as well as the thymus volume increased. CD4 and CD8 immunofluorescence staining revealed a thymic cortex with lymphocytosis. This indicates an increase in the number of lymphocytes that accumulated in the medulla. This suggested that mUCMSC treatment can promote lymphocyte homing to the medulla of aged mice or promote their proliferation in the medulla. The expression of CK8 in the thymic cortex of the model control group and the treatment group was higher than that of the young group. This suggested that the proportion of thymic cortex stromal cells expressing CK8 increases with age, and the proportion of lymphocytes in this region decreases. Compared with that in the model control group, the thymus CK8 expression in the treatment group was higher than that in the model control group (p>0.05), suggesting that the proportion of thymic cortical stromal cells after treatment of elderly mice with mUCMSCs was not significantly changed. The ability of mUCMSC treatment to increase the proportion of lymphocytes in the thymic cortex of aged mice was not obvious. In summary, mUCMSCs can improve senile sensitization, mainly by increasing the proportion and number of lymphocytes in the medulla of mice. mUCMSC treatment can promote lymphocyte homing to the medulla of aged mice or promote proliferation in the medulla region.+/CD8+ ratio decreases with age, which is mainly due to the rapid increase in the number of CD8+ cells, and the CD4+/CD8+ ratio in adulthood increases. This shows that young children and seniors show an immune imbalance. In the former, this reflects immune immaturity, and in the latter, this reflects weakened immunosuppressive function.How do mUCMSCs regenerate the thymus structure of aged C57 mice? A literature review showed that the percentages of lymphocyte subpopulations vary with age. The evidence shows that the changes from childhood to adulthood proceed as follows: the CD4+ T and CD8+ T cells in the treated group was significantly higher than that in the model control group, and the increase in CD8+ T cells was the most obvious. The change in the mUCMSC transplantation group compared with the model control group indicated that mUCMSC transplantation can restore the distribution of lymphocyte subsets from the old age state to the youth state. Intact thymus tissue was digested with type II collagenase into a single-cell suspension. The proportions of the T lymphocyte subsets were analyzed by flow cytometry, which showed that the number of CD4+ T and CD8+ T cells was increased significantly in the treated group compared with the model control group. The most obvious increase was observed in CD8+ T cells. The results obtained by fluorescence staining of CD4 and CD8 antibodies were consistent. Therefore, we asked whether these changes suggest that the mouse thymus tissue changes to a young state after mUCMSC transplantation.Therefore, we asked whether mUCMSCs also change the proportions of different subpopulations of lymphocytes in the thymus. In this study, fluorescence staining with CD4 and CD8 antibodies revealed that the number of CD4By immunohistochemical detection of P53, P16, SOD1, Sirt1, Sirt3 and other aging proteins, we found that the expression of p53 and P16 protein in the thymus of the treated group increased (p>0.05). The levels of SOD1, Sirt1, Sirt3 and other proteins with anti-oxidative stress and anti-aging functions increased to different degrees in the treatment group (p>0.05). This suggests that the degree of aging in tissues is reduced, indicating that treatment with mUCMSCs can confer a certain degree of anti-aging activity. p53 can cause endogenous apoptosis by activating proapoptotic genes . mUCMSCsBy comparing LC3b, becline1, P62 and other proteins that characterize the level of autophagy in the thymus, we found that the expression of LC3b, becline1 and P62 in the thymus tissue of the treatment group was increased to different degrees compared with that in the other groups. The LC3b activity was the lowest in the young control group and was the highest in the treatment group, and the model control group showed intermediate activity  young c young c .Pregnant C57 mice, which were raised in the SPF animal laboratory of the 920For the P4 generation mUCMSCs, the growth morphology during culture was observed under an inverted microscope. The positive expression rates of CD29, CD90 and CD34 in mUCMSCs were analyzed by flow cytometry. Using the induced differentiation assay kit, adipogenic, osteogenic and chondrogenic differentiation was performed according to the instructions of the manual, and the differentiation potential was identified.The C57 mice that met the requirements of thymic aging in the previous screening were randomly divided into the treatment group and the model control group. At the same time, 2-month-old C57 mice were used as the youth control group, with 15 mice in each group. They were all kept in the SPF animal laboratory.7 cells/kg twice a week (Monday and Thursday) for 3 weeks; the other 2 groups were injected with an equal volume of normal saline. After routine feeding and daily recording of mouse activity, the mice were sacrificed by cervical vertebra dislocation 6 weeks after the last transplantation, and the efficacy and mechanism were evaluated.For the experimental group, P4 generation mUCMSCs were infused through the tail vein at a dose of 1×10After assigning the mice to different groups, images were obtained under the same lighting conditions, and the average grayscale value of the head, back and hip in the photos was measured and tested by one-way ANOVA. The thymus specimens were arranged in groups and photographed. The body weight of each mouse and the corresponding thymus weight were recorded. The thymus index was calculated, and one-way ANOVA was performed.C57 mice were sacrificed by cervical dislocation. The abdominal cavity was cut open and cut along the rib cartilage on both sides to the upper edge of the thoracic spine. The thymus was exposed. After weighing and comparing the photos separately, the thymus was fixed with 4% paraformaldehyde. The thymus tissue was collected, embedded in paraffin, sectioned, and stained with HE. The changes in the tissue structure were observed under a light microscope.2). The positive cell density (n/mm2) was determined. The expression of CK8, CK5, CD4, and CD8 in the thymus was scored.A portion of the thymus was fixed with 4% paraformaldehyde, embedded in paraffin, sliced, and subjected to immunofluorescence staining. The green/red fluorescent nuclei with the same labeling were selected by ImagePro Plus 6.0 software as the unified standard for judging all photopositive cells, and each photo was analyzed to obtain the number of positive cells in each field of view , and independent samples were analyzed by t tests. Statistical comparisons between groups were tested by one-way ANOVA. P < 0.05 indicated a significant difference.The C57 mouse model with thymic atrophy showed partial whitening, shedding, alopecia areata, slow movement, decreased activity, atrophy of the thymic gland, disappearance of the medullary gland-like structure, and increased expression of the p53 and P16 genes.C57 mouse mUCMSCs with different growth characteristics, immunophenotypes and differentiation potential in accordance with UCMSC standards were prepared.After treatment with mUCMSCs, the thymus volume of the mice increased, the weight increased, parts of the thymic structure regenerated, the number of lymphocytes in the thymic medulla increased, and the functioning of CD8+ T cells improved.mUCMSCs inhibit the expression of the aging-related genes p53 and P16 by promoting the expression of autophagy- and anti-oxidative stress-related genes, such as LC3b, becline1, P62, SOD1, Sirt1 and Sirt3, thereby increasing the level of autophagy in the thymus tissue, inhibiting apoptosis and exerting therapeutic effects on thymus atrophy.th Hospital of the PLA Joint Logistics Support Force.The experimental protocols were approved by the Experimental Animal Ethics Committee of the 920


In order to discuss the load capacity and displacement of masonry constructed with recycled concrete and self-insulation blocks, one type of 10.6 MPa compressive strength block and three kinds of mortar with M15, M10, and M5 compressive strengths are selected. The constitutive model and corresponding parameters selection of different materials in the ABAQUS numerical simulation are analyzed, and the numerical simulation analysis and experimental tests of the load capacity and displacement of masonry constructed with mortars of different strengths are carried out. The results show that masonry compression failure is controlled by the mortar or block that has the lower compressive strength. The displacement of masonry increases with the mortar compressive strength increase, and the higher mortar compressive strength is beneficial for improving the load capacity and displacement of masonry. Reasonable selection of the constitutive model and parameters will help to obtain reasonable results for the ABAQUS numerical simulation. Construction quality and loading method will affect the load capacity and displacement of the masonry. The above conclusion can provide reference for the engineering application of recycled concrete and self-insulation blocks. In recent years, with the rapid development of China’s economy, the improvement of urban and rural living environments has led to a large amount of building demolition and reconstruction. On the one hand, more than 1 billion tons of construction and demolition waste (CDW) are generated every year. About 40% of this CDW is structural concrete, which could generate about 400 million tons of recycled aggregates (RA) . On the Economic development means that people have a higher requirement in terms of the comfort of their living environment. However, according to statistics, the energy consumption of China’s construction industry accounts for about 30% of the total energy consumption of society, and this proportion is increasing with the annual growth of the national construction area. However, more than 95% of the buildings in China are high energy consumption buildings. The increasing energy consumption demand of the construction industry has led to a huge energy crisis in China. Therefore, building energy conservation has become an important part of social energy conservation and has huge potential for energy saving. The method of self-insulation of exterior walls has gained interest recently because of its outstanding advantages, such as the service life of the building, low cost, convenient construction, safety, and reliability. Reducing the heat transfer coefficient of the self-insulation systems of exterior walls is the key to improving the level of building energy conservation. Therefore, it is of great significance in terms of economic and environmental protection value to find a type of energy-saving building wall material that can comprehensively utilize recycled concrete and metal tailings as building materials and improve the technical level of wall energy-saving [Some scholars have studied the compressive properties of new block masonry. Longo, et.al. experimentally determined the mechanical and the thermal capacities of FRCM-matrices by compressive tests, flexural tests and thermal conductivity measurements . Xiao etReferences ,13,14 shThe recycled concrete and self-insulation block size is the same as that shown in the literature ,2,3. The4 MPa, and the Poisson ratio is 0.3. The compressive strength of recycled concrete and self-insulation blocks is 10.6 MPa, shear strength is 1.1 MPa, elasticity modulus is 503 MPa, and the Poisson ratio is 0.28. The parameters of three types of mortar with different strengths, obtained from technical sheets, are as follows: (1) M15 mortar; compressive strength is 15 MPa and elastic modulus is 12 × 103 MPa, (2) M10 mortar; compressive strength is 10 MPa and elastic modulus is 8.5 × 103 MPa., and (3) M5 mortar; compressive strength is 5 MPa and elastic modulus is 6 × 103 MPa. All of the above parameters will be adopted in the ABAQUS numerical simulation.The experimental compressive strength of block concrete material is 25 MPa, elasticity modulus is 2.55 × 10ψ = 30°, the eccentricity ϵ = 0.1, the ultimate compressive strength ratio of biaxial compression and uniaxial compression σb0/σc0 = 1.16, the invariable stress ratio cK = 0.667, and the viscosity parameter μ = 0.0005 [A plastic damage model is adopted to simulate the properties of recycled concrete and self-insulation blocks and mortar. The plasticity model parameters are as follows: The dilation angle = 0.0005 .The strain values of the block concrete and the mortar are obtained from the corresponding stress by the concrete or mortar constitutive, i.e., the adopted plasticity model and the inelastic strain are calculated by Equations (1) and (2).Here, Taking the proportion, The compression recovery factor takes the default, i.e., Similarly, taking the proportion, Tension recovery factor takes the default, i.e., wt=0 .For simulating the actual test conditions, a rigid plate is used as a loading tool to transmit force through the contact surface. The size of the rigid plate exceeds 10 mm for each side of the block. The influence of geometric nonlinearity is considered by statics analysis. The friction coefficient between the rigid plate and the concrete block is 0.1. Loading mode is displacement control. There are 246,966 C3D8R units used in the model, and the unit density is 8 mm for the block and 6 mm for the mortar. The numerical simulation model and grid partition are shown in 7 N at initiation, the type of evolution is energy and the softening mode is linear, the normal, 1st and 2nd, shear fracture energies are both 63.7 J, and the viscosity coefficient of stabilization is 5 × 10−5. Model constraints and interface processing are detailed in The lower part of the wall is supported by a rigid plate, and the upper part is a rigid plate under pressure. The rigid plate is used to transmit force between the walls. Cohesive contact between mortar and block is taken into account in order to consider the bond relationship between the mortar and the block interface. The contact property mutual parameters between block and mortar are set as follows: The elastic parameters are based on the unit stiffness of the contact surface, and the damage parameters are as follows: the maximum normal stress and shear stress are both 10The Mises equivalent stress, displacement, compression failure, plastic strain, and loading-displacement curve of the masonry are selected and analyzed . Figure The displacement diagrams of masonry constructed by mortars of different compressive strengths are shown in The compression failure diagrams of masonry constructed with mortars of different compressive strengths are shown in The loading force vs. displacement of the center point in the upper surface curve of different kinds of masonry is shown in In order to verify the rationality of the ABAQUS numerical simulation results, the compressive properties of masonry constructed with 10.6 MPa compressive strength recycled concrete and self-insulation blocks and M15, M10, and M5 compressive strength mortar are experimentally tested. The measured compressive strength of M15, M10, and M5 mortar are 15.1, 8.7, and 4.6 MPa, respectively. The experimental device consisted of a rigid frame counterforce device, a pressurizing device, and a measuring system device. For details of the experimental device, see One can see from For improving building energy saving level and achieving the utilization of waste concrete and zinc and lead tailings, a self-insulation block with a three-row-hole arrangement was developed using recycled concrete broken aggregate and activated zinc and lead tailings. The rational selection of the block and mortar constitutive model and corresponding parameters in the ABAQUS numerical simulation were theoretically analyzed, and different masonry was constructed using 10.6 MPa compressive strength blocks and M15, M10, and M5 mortar. The compression performance of the masonry was numerically analyzed by ABAQUS and experimentally tested. Through the analysis of numerical simulation and experimental results, the following two points need to be further discussed.(1) The stress-strain curve of masonry constructed with mortars of different compressive strengths. The experimental study of Corradi et al. showed that the relationship of compressive vertical stress and strain is an approximate linear , but theIt is necessary to mention that, in this experiment, the compressive vertical stress was loaded in a few minutes, and the loading was rigid because the servo-loading system was not used. In this case, the plastic strain of masonry constructed by mortars of different compressive strengths could not be fully developed. The law of strain development and the nonlinear plasticity assumed by the numerical simulation are a little different, which caused the load-displacement curves of the numerical simulation and the experimental tests to be different. For the load-displacement curve of the numerical simulation, the strain of the masonry constructed with mortars of each compressive strength is about 0.5 mm when the load reaches the maximum, but, for the measured load displacement curve, due to the appearance and development of the local cracks, the strain value under the ultimate load of the masonry constructed by mortars of higher compressive strengths increases. For instance, for masonry constructed with M10 and M15 mortar, the strain values corresponding to the ultimate load were 1 mm and 1.2 mm, respectively.It can be seen that the numerical simulation results of the ultimate load are consistent with the experimental results, but the numerical simulation results of the deformation value of masonry structures under the ultimate load are quite different from the experimental results. This is mainly because of the selection of the numerical simulation parameters, and the constitutive model of this new type of masonry needs to be further discussed. The damage diagrams are not consistent because the compression cracks are generally parallel to the load direction, which might be due to the deficiency of the masonry walls because of the insufficient technological level of the laboratory staff. More experimental data need to be accumulated for obtaining reasonable numerical simulation results by adopting parameters that are consistent with the experimental results, which is also the direction of further research.(2) The compressive strength of masonry constructed with different mortars. According to the National Standard of the People’s Republic of China, Appendix B of the Code for the design of masonry structures , the aveHere, Calculated by Equation (5), the average compressive strength of masonry constructed with M15, M10, and M5 compressive strength mortar should be 7.39, 6.09, and 5.0 MPa, respectively. The calculated value is slightly higher than the experimentally measured value for masonry that is constructed with M15 mortar, which may be due to the fact that the masonry quality is not up to the average level or brick-mortar bond strength difference . The det(1)When the mortar compressive strength is higher than the compressive strength of the blocks, because it can provide reliable bond strength, masonry compression is determined by the block compressive strength, the masonry can bear larger displacement, the overall compressive performance is better, and the higher mortar compressive strength is beneficial to improve the overall compressive and deformability of the masonry.(2)When the compressive strength of mortar and block is equal, the masonry can bear a certain deformation and the mortar and block have good synergy. The compression failure of the masonry is controlled by the lower compressive strength of the local block in the middle of the masonry and by the mortar.(3)When the mortar compressive strength is lower than the compressive strength of the blocks, the compression failure of the masonry is mainly caused by the interface bond loss, the deformation performance of the masonry is poor, and the compression failure of the masonry is mainly controlled by the mortar compressive strength.(4)Under the reasonable constitutive model and parameter selection, the ABAQUS numerical simulation can obtain satisfactory results. The masonry quality has a certain influence on the measured results. The effect of the masonry quality and the loading mode on the compressive strength of the masonry should be considered when the results of the numerical simulation are applied to the engineering application.By using recycled concrete aggregate and activated zinc and lead tailing powder, the authors developed a new type of three-row-hole block, which had a better energy saving effect. Based on previous research, this paper studied the compressive strength and displacement of masonry constructed with this kind of block and mortars of different compressive strengths through ABAQUS numerical simulation analysis, and compared them with the experimental results. The following conclusions could be drawn from the above mentioned research:


The article titled “Design and Workspace Analysis of a Parallel Ankle Rehabilitation Robot (PARR)”  has beenThe authors say that the 2-UPS/RRR configuration was used in the PARR, with joints S and U taken as independent joints and the corresponding centers simplified as single points in Figure 6. The spherical joints S1 and S2 were composed of three rotational joints, and the three centers of the rotational joints did not coincide at one point. Similarly, the universal joints U1 and U2 were composed of two rotational joints distributed vertically, and the distance between the centers of the rotational joints should not have been neglected. These simplifications lead to incorrect results of the theoretical workspace. If the simplification is corrected, this provides more accurate results though the error is less than 10%.In addition, the simplifications were not helpful for precise motion control. During the measurement of the effective workspace (EWS) of PARR, although the thigh and calves were tied and fixed with wooden clamps using elastic straps, the thigh and calves still moved slightly and influenced the results. It will be necessary to attach an inertial attitude sensor on the knee to eliminate the influence of the movements and give the confidence interval of the EWS. Hence, the workspace analysis of PARR was insufficient, and a more precise model should be developed.For the first issue, the corresponding details were stated in the second and fourth paragraphs of Section 3, “Kinematic Analysis of PARR.” For the second issue, the corresponding details were stated in the second paragraph of Section 4.1, “PMS of Ankle Joint.” The two issues affected the results depicted in Figures 8, 9, 11, and 13.The editor noted that the assumptions made by the authors in the published article were plausible, though the validity of the simplified model was not thoroughly verified.


The emergence of coronary heart disease is increased with menopause, physical inactivity and with dyslipidemia. Physical training is known to promote the improvement of cardiovascular functions.To investigate the effects of aerobic physical training on the left ventricle in ovariectomized LDL knockout mice.Thirty animals were divided into 6 groups (n = 5): Sedentary non-ovariectomized control; Sedentary ovariectomized control; Trained ovariectomized control; Sedentary non-ovariectomized LDL-knockout, sedentary ovariectomized LDL-knockout and trained ovariectomized LDL-knockout. We analyzed the average parameters of apparent density of collagen fibers types I and III, and metalloproteinase type 2 and type 9, were considered significant p < 0.05.The results showed that the proposed exercise protocol altered the volume of type I collagen fibers, altered collagen remodeling parameters (MMP-2), and also reduced the 8-hydroxy-2’-deoxyguanosine (8OHdG) oxidative stress parameter.Moderate intensity aerobic training acts on collagen fiber volume, on collagen remodeling with the reduction of oxidative stress in the left ventricles of ovariectomized LDL-knockout mice. The animals were kept in a USJT vivarium at a controlled temperature (22-24°C) and lighting (12 hours of light cycle and 12 hours of dark) receiving commercial feed  and water "ad libitum". The animals were divided into 6 groups (n = 5): sedentary non-ovariectomized control (CS), sedentary ovariectomized control (COS); trained ovariectomized control (COT); non-sedentary ovariectomized LDL knockout (LDL-S), sedentary ovariectomized LDL knockout (LDL-OS) and trained ovariectomized LDL knockout (LDL-OT). The division of the animals in the groups was performed by convenicence.The experimental protocol was approved by the Research Ethics Committee of Universidade São Judas Tadeu (CEP-Protocol: 058/2007) and the research was conducted according to the Principles of Laboratory Animal Care formulated by the National Institutes of Health.16At nine months of age animals underwent the ovariectomy procedure. The animals were anesthetized with a ketamine and xylazine solution  and placed in supine position and a small incision in the lower third of the abdominal region, parallel to the line of the body, was made. The ovaries, the uterine horns, and the blood vessels were located, sectioned, and removed. After that, the musculature and the skin were sutured. Confirmation of the efficacy of the ovariectomy was determined through colpocytology of the vaginal secretion performed over four consecutive days. On the last day of analysis, euthanasia was performed on these animals.A maximal training test was performed on all the groups at the beginning and at the end of the exercise training program. The test consists of placing the animal to run on an ergometric treadmill at 0.3 km/h for 3 minutes, and this workload was increased by 0.3 km/h every 3 minutes until the animal reached exhaustion. The time of the test and the speed of the last workload were noted and served to determine the mean value of aerobic capacity of each group.Exercise training began 7 days after the ovariectomy surgery; the trained groups were subjected to a physical training protocol on an ergometric treadmill at low-moderate intensity  for 1 hour a day, 5 days a week, for 4 weeks, with a gradual increase in speed from 0.3 to 1.2 km/h. The animals were adapted to the treadmill for 10 minutes on three days prior to beginning the training.At the end of the training the animals were sacrificed by decapitation. A thoracotomy was performed in which the heart and atria were removed and the right and left ventricles were sectioned. Left ventricle samples were fixed in 10% buffered formalin for 24 hours. Afterwards, the tissue was transferred to a 70% ethyl alcohol solution, dehydrated in increasing ethanol series, diaphanized in xylol and embedded in paraffin. Non-serial, 5 µm thick cuts were performed in which each section received a total of 6 cuts. The sections were stained with the Picrosirius technique, for the analysis of collagen fibers I and III in the left ventricle and visualized by polarized light microscopy.The volume density of collagen fibers I and III (Vv[fc]) expresses the fraction of the volume occupied by the collagen fibers in relation to the total volume of the analyzed image. For this analysis, the test system was used with a total of 475 points, which corresponds to 100% of the image, using the program Image J. .2O2 for 15 minutes, followed by washing in distilled running water and in PBS pH = 7.4. For each slide, a specific antibody was used, For the immunohistochemical techniques, 3 to 4 micrometer thick cuts were made and mounted on slides previously silanized using 4% silane. After the slides were dewaxed, using a heating oven overnight set at 57ºC, after which they were immersed in xylol baths for 30 minutes. Afterwards, they were hydrated in ethyl alcohol with decreasing concentrations of 100%, 80% and 70%, each with a duration of 5 minutes, then washed in running water. In the next step, the antigenic recovery was performed in a water bath at 90°C, the slides were placed in citrate buffer for 30 minutes, then washed in running water. Endogenous peroxidase blockade was performed in HIn all the techniques, observing the presence of a dark brown precipitate, the sample was visualized under an optical microscope. A quantitative analysis of the images was performed using the program ImageLab 2000, where the brown markers were selected and the program quantified immunoexpression intensity, Stata Statistical Software: Release 11. College Station, TX: StataCorp LP.)Absolute and relative values were used to describe the qualitative variables. For the quantitative variables with normal distribution (Shapiro-Wilk > 0.05), mean, standard deviation, minimum and maximum were used, and for the variables with non-normal distribution (Shapiro-Wilk < 0.05), median and percentiles were used. To study the diferences between the variables with the groups, the Kruskal-Wallis and ANOVA tests were used, complemented by the Dunn test or Bonferroni. For all analyzes, a confidence level of 95% was used. The program utilized for these analyzes was Stata version 11.0. (StataCorp. 2009. Our data show a significant reduction in the volume density of type I collagen fibers in the ovariectomized dyslipidemic group (LDL-OS) (-51%). The LDL-OT group showed a decrease of 100% when compared to the COS group and a significant difference in relation to the COT and LDL-S groups. Regarding metalloproteinase type 2 expression, in the control group (CS) there was a significant increase of 24% in the ovariectomized group (COS), when physical training (COT) was performed there was an increase of 72%, and with dyslipidemia (LDL-S), the increase was 92%. In the dyslipidemic group with ovariectomy (LDL-OS), the values decrease 27% in relation to the LDL-S group and 18% in relation to the COT group, but still above the values of the control groups by 42% in relation to the CS group and 14% in relation to the COS group. When the training was performed in the ovariectomized dyslipidemic group LDL-OT, the results obtained present a 15% increase in relation to the sedentary ovariectomized LDL-OS group and a 15% decrease in relation to the LDL-S group. When compared to the control group, there was an increase of 64% in relation to the CS group, 32% in relation to the COS group and a decrease of 5% in relation to the COT group.When analyzing the oxidative stress data, evaluating 8OHdG expression, there was a decrease in the indexes for the control group (CS), in which there was a decrease of 33% in the group with ovariectomy (COT) and a 39% decrease in the trained group. When dyslipidemia was present (LDL-S), there was a 51% decrease in relation to the CS group, and in the dyslipidemic group with ovariectomy (LDL-OS) there was a decrease of 85% in relation to the CS group, 78% in relation to the COS group, 76% in relation to the COT group and 70% in relation to the LDL-S group. When training was performed in the dyslipidemic ovariectomized group (LDL-OT), the data presented a 152% increase in relation to the LDL-OS group and a decrease in relation to the control groups, with 63% for the CS group, 45% for the COS group and 40% for the COT group. For the parameters related to volume density of type III collagen fibers, metalloproteinase type 9 and inflammation (COX2), the values obtained for the control and dyslipidemic groups do not present significant differences in any parameter, 18Physical activity is recognized as an important non-pharmacological treatment for numerous diseases and conditions, including dyslipidemia and menopause. Studies indicate that collagen fibers are present in the process of myocardial remodeling, which occur due to aging and other factors.19 Interestingly, the findings reported in this article have similar results only with the proposed physical activity. Another study where acute myocardial infarction was induced in rats, the gradual increase of types I and III collagen was observed at 4 weeks. The kinetics of collagen I/III increase, in combination with the decrease of the elastic fibers in the infarcted area after an MI, provided evidence that impaired cardiac function after an MI was due to healing or infarct scar formation, with increased rigidity and less flexibility of the heart.20Our data show that in the myocardium, the expression of type III collagen fibers did not differ between groups and parameters. In relation to type I collagen, ovariectomy and training brought a decrease in the levels presented in relation to the sedentary ovariectomized control group. Results similar to those found in our study verified the same variable in animals after a period of obesity induced by a diet rich in unsaturated fat.22 However, when evaluating the LDL-OT groups, there is a clear reduction in the volume of type I collagen fibers. This verification was obtained using the proposed training protocol (intensity or duration).24Our results suggest that the aging period in this study does not appear to interfere with the increase in the expression of collagen fibers presented in other studies.29 The expression of metalloproteinase type 9 did not differ between groups and parameters. Regarding metalloproteinase type 2, an increase was shown in the control group with ovariectomy and physical training even in the presence of dyslipidemia. This process explains the factor with the collagen indexes when these are not elevated, because the degradation of collagen and the extracellular matrix is peformed by the action of the metalloproteinases.32 The elevated presence of MMPs in patients with dyslipidemias found in some studies suggest their participation in the matrix degradation process in atherosclerotic plaques and in the ruptures of these and with exposure of the nucleus33 and are independent predictors for the progression of kidney disease and are independently associated with an increased risk of mortality.34Metalloproteinase type 2 and type 9 also participate in the remodeling process of cardiac tissue, and are present in several pathologies such as inflammatory and cardiovascular diseases, among other injuries.The expression of COX2 in our study did not present statistical significance, indicating that in all groups and parameters, there were no changes for these data. Thus, in this parameter chosen to verify inflammation, no changes are observed showing that the factors associated with the groups do not interfere in such an evaluation.5 In our research, oxidative stress was verified through the expression of 8OHdG, and ovariectomy decreased the values of this parameter in the control and dyslipidemic groups, differing from the results obtained in other studies.35 When physical training was performed, the values were decreased in the control group, but in the dyslipidemic group, they presented an increase. Interestingly, this marker seems to have no association with the groups proposed in this experimental model.Aging, menopause and dyslipidemia are factors that also result in oxidative stress.The data of this research indicate that physical exercise beneficially influenced the control and dyslipidemic groups in the volume density parameter of type I collagen fibers and the control group in relation to oxidative stress.


The Wisconsin Upper Respiratory Symptom Survey-21 was used to determine URTI. The study endpoints included WURSS-21 scores, number of URTI events, and proportion of medication  usage. We found that the physical symptom scores for “Runny nose,” “Sneezing,” and “Head congestion” were significantly lower in the 25OHD group than in the placebo group; for all items except “Breathe easily, “the quality of life” scores were significantly improved in the 25OHD group. There was no significant difference in the number of URTI events or the proportion of medication use between the groups. Collectively, the findings of this study indicate that a sufficient 25OHD intake can reduce physical symptoms at the onset of upper respiratory tract infection, particularly nasal symptoms, and may improve the quality of life at the time of onset.To evaluate the effects of 25-hydroxyvitamin D The common cold is a group of diseases that present with various upper respiratory symptoms, including nasal discharge, nasal obstruction, sneezing, sore throat, and cough. In the United States, it is estimated that children catch colds on average six to eight times a year, whereas adults are affected by colds two to four times a year, missing 22 million days of schooling, and 20 million days of work, respectively. Moreover, the economic loss due to colds is estimated to be US$25 billion per year ,2.The common cold is caused by a variety of viruses and bacteria, including rhinoviruses, coronaviruses, adenoviruses, RS virus, and parainfluenza viruses. Although vaccines may be effective in preventing the common cold, the multiple pathogenic viruses that cause these infections make complete prevention with vaccines difficult, and thus symptomatic treatment at the onset of the disease is the mainstay of treatment . MoreoveThe intake of micronutrients such as vitamins A, C, and E, and minerals, such as zinc, stimulates the immune system by inducing epithelial barrier function and cellular immunity and promoting antibody production . The effVitamin D has been extensively studied in relation to respiratory tract infections and in the prevention and management of these infections. It has been reported that a reduction in the concentration of 25-hydroxyvitamin D in the blood is associated with an increased risk of respiratory tract infections ,7,8, and2 or D3. Regardless of whether it is synthesized or ingested, vitamin D is mainly hydroxylated at position 25 in the liver by CYP2R1 or CYP27A1, members of the cytochrome P450 superfamily of enzymes, to yield 25-hydroxyvitamin D [Vitamin D is synthesized by the skin through exposure to ultraviolet rays and is obtained from dietary sources, such fish, dairy products, and mushrooms, which contain vitamin Ditamin D .2 vitamin D [2 vitamin D may play an important in the body’s initial defense against pathogens [While, 25-Hydroxyvitamin D binds to vitamin D-binding protein and circulates in the blood. On reaching the kidneys, 25-hydroxyvitamin D is subsequently hydroxylated at the 1α position by CYP27B1 to form 1α, 25 (OH)itamin D ,13, whicitamin D ,15,16. Cathogens .3 on upper respiratory tract infection (URTI) in Japanese subjects with insufficient or deficient 25-hydroxyvitamin D levels in the blood, and the results indicated that 25-hydroxyvitamin D3 is effective in improving overall symptoms, and quality of life of subjects with URTI and shortening the duration of the URTI [3 intake, nor has it been established what effects it might have on the efficacy of other medications.We have previously studied the effects of 25-hydroxyvitamin Dthe URTI . However3 intake on the number of symptoms and events at the onset of URTI and the proportion of medication (antibiotics and antipyretics) use.In this study, we conducted a post hoc analysis of data obtained from our previous clinical trials to evaluate the effects of 25-hydroxyvitamin D3 (25OHD) per day. Double-blind indicates that randomization to the placebo or 25OHD group was blinded to all relevant parties, including subjects, researchers, and physicians. Details of the characteristics of the subjects and the inclusion and exclusion criteria can be found in the original paper [We performed a post hoc analysis using data obtained from a previous clinical trial . The stual paper . In thisSubjects completed a Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) and a diary of changes in physical conditions and medication status during the test food intake.In this study, physical examination and clinical tests were conducted at the community hospital clinics, and a system was established for the management of subjects and the implementation of the study. The investigator responsible of all aspects of the study, gave instructions to the subjects, provided necessary explanations, obtained their written informed consent, interviewed them, checked for and assessed adverse events, and managed the study system. The original study was conducted from December 2015 to September 2016. https://www.umin.ac.jp/ctr/) prior to commencement. The purpose and details of the study were fully explained to the subjects by the investigators, and their voluntary consent was obtained in writing. The original study was conducted having initially been reviewed and approved by the HUMA R&D Ethical Review Committee, which consisted of third parties who were not involved in the study . The study was conducted in accordance with the Declaration of Helsinki (adopted in June 1964 and revised in October 2013) and the Ethical Guidelines for Medical Research Involving Human Subjects . The content of the study was registered in UMIN Clinical Trials Registry .The Japanese version of the WURSS-21 was used to assess the incidence and severity of physical symptoms of upper respiratory infections, as well as subject quality of life . QuestioThe date of URTI onset was set to the date when the subject selected a score values from 1 to 7 after selecting 0 for the first two consecutive days, and the date of disappearance was set to the date 2 days before  the subjects selected 0 for the second consecutive day after the onset. In this study, URTI events were defined as those with a duration of illness (the period from the date of onset to the date of resolution) of more than 2 days and a maximum physical severity score of more than 10 during the period of illness. As outcome measures, we used the following: The WURSS-21 question scores and proportion of incidence of each symptom, the number of events during the period of URTI, the proportion of antibiotic and antipyretic analgesic (acetaminophen and NSAIDs) use, and the proportion of medication use at the time of URTI. The symptom score and proportion of incidence rate, as well as the proportion of medication use at the time of URTI onset, were evaluated only in subjects with URTIs. According to the task team guidelines described by Holick et al., the risk of osteomalacia and rickets is increased in a deficiency state where the serum 25-hydroxyvitamin D level is 50 nmol/L = 20 ng/mL) or lower, and that the state where the serum 25-hydroxyvitamin D level is between 50 nmol/mL and 75 nmol/L (= 30 ng/mL) is an insufficiency state where the risk of falling is increased  ng/mL or. We condt test was used to assess the WURSS-21 score of each subjects. The proportion of incidence of each symptom and the proportion of drug use were assessed using Fisher’s exact test, and the Mann–Whitney U test was used to assess the number of URTI events. Due to the exploratory nature of this study and the small number of subjects analyzed, 10% on both sides was considered marginally significant, in order not to overlook the efficacy of 25OHD.Student’s ® 14.1.0 . Owing to the exploratory nature of this analysis, we did not adjust for the multiplicity of the tests.All analyses were performed using JMPBackground information pertaining to the subjects participating in this study is presented in Overall, there was a significant gender difference between the 25OHD and placebo groups, with a larger number of males and fewer females in the 25OHD group than in the placebo group. Otherwise, there were no significant differences between the groups.Among the PPS, 43 subjects in the placebo group and 41 in the 25OHD group developed URTIs. In the 25-hydroxyvitamin D-insufficient group, 17 subjects in the placebo group and 15 in the 25OHD group developed URTIs, whereas 26 subjects in the 25-hydroxyvitamin D-deficient group and 26 in the 25OHD group developed URTIs. The scores for physical symptoms and quality of life, and the number of subjects with each symptom, are shown in With the exception of “Breathe easily,” all quality of life-related items were significantly lower in the 25OHD group than in the placebo group . We specIn the 25-hydroxyvitamin D-insufficient group, individuals in the 25OHD group had marginally significantly lower levels of “Plugged nose,” “Sneezing,” and “Head congestion” than those in the placebo group , whereasThe scores of the 25-hydroxyvitamin D-deficient group were generally higher than those of the 25-hydroxyvitamin D-insufficient group, thereby indicating that a deficiency of 25-hydroxyvitamin D in the blood tended to be associated with URTI of greater severity. In addition, 25OHD supplementation in the 25-hydroxyvitamin D-deficient group was considered to be effective. Given that each score for the 25OHD-deficient group approached that recorded for the 25-hydroxyvitamin D-insufficient group, we take this to be indicative of an effect of 25OHD supplementation.A summary of the number of URTI events identified during the study period is shown in For both the 25-hydroxyvitamin D-insufficient and -deficient groups, there were no significant differences between the 25OHD and placebo groups with respect to the proportions of antibiotic and antipyretic analgesic usage. In contrast, in the 25-hydroxyvitamin D-insufficient group, there was a trend toward a higher proportion of acetaminophen use for URTI in the 25OHD group, which was higher than that observed in the 25-hydroxyvitamin D-deficient group. The proportion of NSAIDs use of 25OHD was lower than that of the placebo group, thus indicating an anti-inflammatory effect of 25OHD intake. There was also a trend for higher acetaminophen use in individuals receiving 25OHD in the 25-hydroxyvitamin D-insufficient group.In Japan, numerous common cold medications available in pharmacies contain acetaminophen, and given that the severity of URTI was lower in the 25OHD group than in the placebo group, more subjects in the former group used over-the-counter cold medications, and accordingly, the proportion of acetaminophen use in the 25OHD group was higher than that in the placebo group.Given that respiratory tract infections are caused by a diverse range of viruses and bacteria, it has been difficult to develop effective treatments for such infections. A number of studies have been conducted to investigate the preventive and control effects of vitamin D and other micronutrients on respiratory tract infections , among wThe results indicated that 25OHD intake can generally alleviate various symptoms of URTI, such as runny nose, sneezing, and head congestion, and can also reduce the incidence of URTI events and the proportion of NSAID usage. Both these effects can be attributed to an increase in 25-hydroxyvitamin D concentrations in the blood following the intake of 25OHD. Indeed, we found that individuals in the 25-hydroxyvitamin D-deficient group had a higher severity score for each symptom at the onset of URTI, thereby indicating that lower blood levels of 25-hydroxyvitamin D may be correlated with the severity of URTI, and that an increase in blood 25-hydroxyvitamin D following 25OHD intake contributes to reducing disease severity.2 vitamin D. It is assumed that most of the functions of vitamin D are vitamin D receptor mediated. Both the affinity of 25OHD for vitamin D receptors and its concentration in blood are approximately 1,000 times greater than those of 1α, 25 (OH)2 vitamin D, indicating that 25OHD may also directly exert a variety of functional effects.It is conceivable that these reductions in the severity of URTI in response to 25OHD intake are associated with a reduction in the levels of cytokines and chemokines, which have been investigated with respect to vitamin D metabolism, modulation of the renin-angiotensin system, regulation of neutrophil activity, maintenance of pulmonary epithelial barrier function, and promotion of epithelial repair ,23,24. ARecently, a deficiency in vitamin D has been reported to be associated with an increased risk of COVID-19 infection, and clinical trials on the efficacy of vitamin D and 25OHD for COVID-19 have been conducted and are underway  . If indeThe findings of this study indicate that 25OHD intake in individuals with both insufficient and deficient levels of 25-hydroxyvitamin D in the blood can be effective in reducing the severity of nasal and other symptoms at the onset of URTI, and also improve the associated quality of life. However, owing to the small number of cases assessed in the present study, our findings should be interpreted with caution.


Tautomerism of nucleic acid (NA) bases is a crucial factor for the maintenance and translation of genetic information in organisms. Only canonical tautomers of NA bases can form hydrogen-bonded complexes with their natural counterparts. On the other hand, rare tautomers of nucleobases have been proposed to be involved in processes catalysed by NA enzymes. Isocytosine, which can be considered as a structural fragment of guanine, is known to have two stable tautomers both in solution and solid states. The tautomer equilibrium of isocytosine contrasts with the remarkable stability of the canonical tautomer of guanine. This paper investigates the factors contributing to the stability of the canonical tautomer of guanine by a combination of NMR experiments and theoretical calculations. The electronic effects of substituents on the stability of the rare tautomers of isocytosine and guanine derivatives are studied by density functional theory (DFT) calculations. Selected derivatives are studied by variable-temperature NMR spectroscopy. Rare tautomers can be stabilised in solution by intermolecular hydrogen-bonding interactions with suitable partners. These intermolecular interactions give rise to characteristic signals in proton NMR spectra, which make it possible to undoubtedly confirm the presence of a rare tautomer. Nucleic acid bases are responsible for maintaining and translating genetic information in organisms. Hydrogen bonding is the key interaction for the recognition of nucleobases during the replication and translation processes.In their seminal work, Watson and Crick emphasised the importance of the tautomerism of nucleic acid (NA) bases for DNA structures; only canonical tautomers of NA bases can form hydrogen-bonded complexes with their natural counterparts . A tautoH forms, which are not possible for the 9-substituted guanine found in nucleic acids. Despite numerous experimental studies, there is no clear evidence of the presence of a rare tautomer of the 9-substituted or 9H guanine base in solution; see, e.g., References 18 and T 1H-NMR spectra were recorded on a Bruker 500 MHz NMR spectrometer (1H at 500.0 MHz) in a 3:1 mixture of dimethylformamide (DMF)-d7 and CD2Cl2 . The assignment of the signals was based on analogy with previously published data [Variable-temperature hed data .T was substituted by a methyl group. The vibrational frequencies and free energies were calculated for all of the optimised structures, and the stationary–point (minimum) character was thus confirmed. The Gaussian16 program package was used throughout this study [The studied structures were subjected to geometry optimisation at the DFT level using the B3LYP functional ,29 with is study . No corr2 and CF3 groups, electron-donating NH2 and CH3 groups and the sterically demanding tert-butyl group were used in the series. The 2,3-I tautomer has the lowest energy in all cases. However, the relative energy and relative order of the remaining tautomers are strongly substituent-dependent.We have calculated the relative energies of the four low-energy tautomers  of a series of isocytosine derivatives with various substituents in positions 5 and 6 . Electro4) stabilises the 1,2-I tautomer (with the relative energy 7.4 kJ/mol lower than for isocytosine 1), while the electron-accepting nitro group destabilises this tautomer (with the relative energy 11.0 kJ/mol higher than for isocytosine 1). Interestingly enough, the 1,2-I tautomer of the derivatives substituted in position 6 is destabilised by both electron-accepting and electron-donating groups, except alkyl groups. These calculations agree with the previous observation that the 2,3-I tautomer of compound 9 is strongly preferred in cocrystallisation with compounds containing complementary functional groups, whereas both 1,2-I and 2,3-I tautomers of compound 7 are formed in cocrystals with suitable partners [The second-most stable tautomer is always either 1,2-I or 2,4-I (enol). The relative energies of the 1,2-I tautomer substituted in position 5 strongly depend on the electronic nature of the substituent; the electron-donating amino group . The imino tautomer is particularly unstable for derivatives substituted with electron-accepting or strongly electron-donating groups in position 6.The enol tautomer  is relatively stable in the case of isocytosine 12–17) that includes guanine methylated in position 7 or 9, its 7-deaza and 9-deaza derivatives and benzo and tetrahydrobenzo derivatives  of isocytosine . Here, w18 is added to the solution of isocytosine derivative 16, a new signal in the high chemical shift range appears. This clearly indicates the formation of a hydrogen-bonding complex between these two molecules  might stabilise the imino tautomer  of compound 7 by the formation of an intermolecular complex with three hydrogen bonds. However, we have only observed the formation of isocytosine dimers in the mixture of compound 1 with T, which means that the energy gain produced by the complex formation of the enol tautomer of isocytosine with thymine could not overcome the energy barrier of the enol 2,4-I tautomer  tautomer of isocytosine , becausetautomer . Similarwith DAP .Estabil) of these dimers and complexes. These energies were calculated as the difference between the energy of the dimer/complex and the energies of the most stable tautomer of its components. For comparison, the table also includes complexation energies (Ecomplex), which were calculated as the difference between the energy of the dimer/complex and the energies of its components in the same tautomeric form as in the dimer/complex. The complexation energies thus reflect the strength of intermolecular hydrogen-bonding interactions, whereas the stabilisation energies include the energy penalty needed for the formation of a less stable tautomer. Additionally, note that solvent effects are only implicitly involved in the calculations using a polarisable continuum model. The real solvation of the molecules in solution will disfavour the dimer/complex formation, because dimethylformamide solvates the monomers well.To rationalise the observations of intermolecular complexes further, we have performed geometry optimisation of selected dimers of isocytosine and guanine derivatives  and of complexes of selected minor tautomers with the hydrogen-bonding partners used in the NMR experiments. 16, followed by the dimers of compounds 7 and 1, which means that the stabilisation energies have the same order as the appearance of the dimer signals in the spectra. The dimer of compound 10 is 20 kJ/mol less stable than the dimers of compounds 1, 7 and 16, which is in agreement with the fact that we did not observe the formation of this dimer at all. The lowest magnitude of stabilisation energy was found for the dimer of guanine derivative 12 .The computations of dimer stabilisation energies nicely reflect the experimental findings described above. The greatest stabilisation was calculated for the dimer of compound 7 or 16. On the other hand, the stabilisation of the complex of the isocytosine enol tautomer  with thymine is almost 20 kJ/mol lower than the stabilisation of the isocytosine dimer, which explains why only the dimer has been observed experimentally and no traces of the 2,4-I tautomer have been found. Similarly, the stabilisation of the complex of the isocytosine imino tautomer 1,3-I with diaminopyridine (DAP) is too low to enable the observation of this complex.The computations have also confirmed that guanine in its canonical tautomer can stabilise 1,2-I tautomers of compound We have studied the tautomeric equilibria of isocytosine and guanine derivatives. Guanine has a strong preference for its canonical tautomer that is found in DNA. Isocytosine is a structural fragment of guanine, but it has significantly lower relative energy of its minor tautomeric forms, particularly the 1,2-I tautomer. We have investigated the factors contributing to the remarkable differences between the stability of minor tautomers of guanine and isocytosine.12) have very high-calculated relative energies. Interestingly, 7-methylguanine (13) has significantly lower relative energy of the 1,2-tautomer than 9-methylguanine.The DFT calculations have revealed that the 1,2-I tautomer is stabilised by electron-donating groups in position 5 of isocytosine and destabilised by electron-accepting groups in this position. The same tautomer is destabilised by both electron-accepting and strongly electron-donating groups in position 6. All minor tautomers of 9-methylguanine . The DFT calculations have revealed that although the suggested intermolecular complexes also have three intermolecular hydrogen bonds, the magnitude of the complexation energy is significantly lower than in the complexes containing the 1,2-I tautomer.We also speculated that other minor tautomers of isocytosine might be stabilised by intermolecular interactions with suitable hydrogen-bonding partners. However, we have not observed any intermolecular complexes between thymine derivative 18 in any of the experiments. Furthermore, the relative energies of all minor tautomers of guanine are very high ; they are, in most cases, the highest among all the studied compounds. The instability of rare tautomers may have played a role in the natural selection of nucleobases suitable for the reliable storage of genetic information. The extraordinary stability of the canonical guanine tautomer is a key factor in high-fidelity replication, transcription and translation of the genetic code. Furthermore, in view of these results, the involvement of rare guanine tautomers in processes catalysed by nucleic acids seems to be improbable.Importantly, we have not observed any noncanonical tautomer of guanine derivative


Aronia melanocarpa L. berries (AME), characterized by their strong antioxidative potential, may have a beneficial impact on the oxidative-reductive status of the submandibular gland in an experimental model of low-level and moderate human environmental exposure to cadmium. The main markers of the antioxidative status ), total oxidative status (TOS), oxidative stress index (OSI = TOS/TAS), and lipid peroxides, as well as cadmium concentration, were evaluated in the submandibular gland tissue of female Wistar rats who received a 0.1% aqueous AME and/or a diet containing 0, 1, and 5 mg Cd/kg for 3 and 10 months. The treatment with cadmium decreased the activities of antioxidative enzymes (29%–74%), reduced glutathione concentration (45%–52%), and TAS and increased TOS, resulting in the development of oxidative stress and enhanced concentration of lipid peroxides in the submandibular gland. The administration of AME at both levels of exposure to cadmium offered significant protection against these actions of this xenobiotic. After the 10 month exposure to the 1 and 5 mg Cd/kg diet, TAS was decreased by 77% and 83%, respectively, TOS, OSI, and lipid peroxides concentration were increased by 50% and 52%, respectively, 11.8-fold and 14.4-fold, respectively, and 2.3-fold and 4.3-fold, respectively, whereas, in the case of the extract co-administration, the values of these parameters did not differ compared to the control group. The results indicate that the consumption of aronia products under exposure to cadmium may have a beneficial impact on the oxidative-reductive status of the submandibular gland and prevent oxidative stress development and enhanced lipid peroxidation in this salivary gland.Oxidative stress underlies the pathomechanisms of toxic action of cadmium (Cd), including its damaging impact on the oral cavity. This study investigated whether the administration of an extract from  It is well known that oxidative stress underlies the pathomechanisms of the development of various diseases, including diseases of the oral cavity ,2, as weThe harmfulness of cadmium to the human’s and animals’ organism is well known and widely reported ,9,11. ThBecause current environmental exposure to cadmium creates a threat to the health of the general population ,16,17,18Aronia melanocarpa L.  Elliott, Rosaceae; black chokeberry), which are one of the richest sources of polyphenols (719–6902 mg/100 g)  provided by the Laboratory Animal House in Brwinów . Throughout the experiment, the rats were kept in stainless steel cages  in standard conditions . The animals had unlimited access to drinking water and food, the consumption of which was monitored throughout the study. The submandibular salivary glands used in the current study were collected and secured during an experiment conducted at the Department of Toxicology at the Medical University of Bialystok (Poland), which was approved by the Local Ethics Committee for Animal Experiments in Bialystok . The experimental model was described in detail previously ,31,32,34Control group—the rats throughout the experiment (3 or 10 months) received redistilled water (containing < 0.05 μg Cd/L ) and theAME group—the rats received as the only drinking fluid a 0.1% aqueous AME and the standard Labofeed fodder;1 group—the rats throughout the experiment (3 or 10 months) received the Labofeed fodder containing 1 mg Cd/kg and redistilled water for drinking;Cd1 + AME group—during the whole period of maintaining on the diet containing 1 mg Cd/kg (3 or 10 months), the rats received the 0.1% aqueous AME as the only drinking fluid;Cd5 group—the rats received fodder containing 5 mg Cd/kg for 3 or 10 months and redistilled water for drinking; Cd5 + AME group—during the feeding with the diet containing 5 mg Cd/kg (3 or 10 months), the rats received the 0.1% aqueous AME as the only drinking fluid. CdThe animals were randomized into the following six experimental groups of 16 rats each:Throughout the experiment, no differences in the consumption of drinking water and food, as well as body weight gain, were noted among the experimental groups . Moreove®, Nanikon, Switzerland; accuracy to 0.0001 g). All submandibular glands showed proper macroscopic picture and there were no differences in their weight (each gland weighted about 0.2 g) among the experimental groups.After 3 months of the experiment, eight rats of each group  were sacrificed, whereas all remaining animals were necropsied after 10 months. The animals (after overnight deprivation of food) were subjected to necropsy under barbiturate anaesthesia with Morbital  and, after the blood collection (by cardiac puncture) and macroscopic examination, both submandibular glands (the left and the right one) were dissected . Immediately after collection, the salivary glands were rinsed thoroughly in ice-cold 0.9% sodium chloride  and gently dried on filter paper. Next, they were weighed with an automatic balance  at 700 × g for 20 minutes in 4 °C and the separated aliquot was used to the assay of CAT, GSH, TAS, TOS, and LPO. The second one was centrifuged at 20,000 × g for 30 minutes in 4 °C and the received aliquot was subjected to GPx and SOD assay [In order to perform the planned assays of markers of the oxidative/antioxidative status, 20% homogenates of the submandibular gland tissue were prepared. The right submandibular gland and half of the left one (of known weight) were homogenized in cold 50 mM potassium phosphate buffer (pH = 7.4) with the use of a high-performance homogenizer . In order to prevent autoxidation of the salivary gland tissue, 0.01 cmThe activities of GPx and SOD and the concentrations of GSH and LPO were assayed with the use of commercially available kits . The precision of these measurements, expressed as a coefficient of variation (CV), was < 4.5%, 5%, 3.5%, and 6%, respectively. The activity of CAT was determined by the spectrophometric method according to Aebi  and the TAS and TOS were measured using the ImAnOx (TAS) Kit and PerOx (TOS) Kit. The values of TAS determined in the control samples included in the kit reached 195.3 ± 12.3 and 306.4 ± 17.5 µmol/L  and were within the manufacturer’s range of values . Similarly, the values of TOS assayed in the control samples included in the kit  were within the ranges of values stated by the producer . The precision of TAS and TOS assay (CV) was < 6% and < 2%. The value of OSI was mathematically calculated as the ratio of TOS and TAS.The measured parameters of the oxidative-reductive status were adjusted for protein concentration. The assay of total protein was performed with the BioMaxima Kit  according to the manufacturer’s instruction. All the above-mentioned assays performed with the use of commercial kits were conducted according to the producers’ instructions . The measurements were done using the spectrometer UV VIS SPECORD 50 PLUS  or MULTISCAN GO microplate reader . Moreover, an automatic Wellwash 4 washer for microplates  was used. Known-weight halves of the left submandibular glands were wet-digested with a trace-pure concentrated nitric acid using UniClever II microwave system  and then the wet-digests were diluted with ultra-pure water. The concentration of cadmium in such preparations was determined by the graphite furnace AAS method (GF AAS) using HITACHI Z-5000 atomic absorption spectrophotometer  equipped with a graphite cuvette  and a hollow cathode lamp for this element assay . The concentration of cadmium measured in the simultaneously analysed reference material  was consistent with the value provided by the producer . The CV was < 6%. p < 0.05), the Kruskal–Wallis post hoc test was performed to compare individual groups and determine which two means differed statistically significantly. The possible impact of AME administration under exposure to cadmium on the values of estimated parameters was evaluated based on the statistical analysis of differences between the Cd1 + AME group or Cd5 + AME group and the respective group treated with cadmium alone , as well as differences between the groups co-administered with cadmium and AME (Cd1 + AME or Cd5 + AME groups) and the respective control group.The obtained results were analysed statistically using Statistica 10 software . The data are presented in figures in the form of mean ± SE for eight rats in each experimental group. In order to assess the statistical significance of differences between the experimental groups, the nonparametric Kruskal–Wallis test was performed. In cases when statistically significant differences occurred among the six experimental groups  was conducted with the aim to discern the possible independent and/or interactive impact of these agents on this parameter. F values having p < 0.05 were recognized as statistically significant. Moreover, in the case when the ANOVA/MANOVA analysis revealed an interactive effect of cadmium and AME, the possible character of the interaction was described based on the comparison of the effect of the co-administration of cadmium and AME to the sum of effects noted as a result of their separate administration. The effect of cadmium or/and AME was expressed as a percentage change or a factor of change in a measured parameter compared to the control group. Based on the obtained results it was estimated whether the interaction had an antagonistic (Cd + AME effect < Cd effect + AME effect), additive effect (Cd + AME effect = Cd effect + AME effect) or another character [When the Kruskal–Wallis test revealed any influence of the co-administration of cadmium and AME on the investigated parameter, a two-way analysis of variance  having p < 0.05. In the case of r having a negative value and p < 0.05, the correlation is recognized as negative, whereas, in the case of positive r value (and p < 0.05), the correlation is positive in character.Spearman rank correlation analysis was carried out to estimate mutual dependences between the measured markers of the oxidative-reductive status of the submandibular gland, as well as between these parameters and cadmium concentration in this salivary gland. A correlation is considered statistically significant at a correlation coefficient  except for an increase in the activity of SOD and the value of TAS after 10 months .In the rats maintained on the diets containing 1 and 5 mg Cd/kg for 3 and 10 months, the activities of antioxidative enzymes  in the submandibular gland and TAS were decreased at each time point  was the result of the independent action of the extract and/or its ingredients interaction with this heavy metal, which seemed to be antagonistic in character . Howeverd by 65% .The administration of AME alone for up to 10 months had no influence on the estimated markers of the oxidative status of the submandibular gland , except for a decrease in TOS after 10 months . The 3 month treatment with the 1 and 5 mg Cd/kg diet had no influence on TOS of the submandibular gland; however, after 10 months of the experiment, the value of this parameter was higher  compared to the control group . In all The exposure to the 1 and 5 mg Cd/kg diet resulted in an increase in OSI (3.4- to 14.4-fold) and the concentration of LPO (2.1- to 4.3-fold) in the submandibular gland, whereas the co-administration of AME entirely prevented these changes. The value of OSI and the concentration of LPO in all groups of rats co-administered with AME were within the ranges of values noted in the respective groups that did not receive the extract under the treatment with cadmium .The two-way analysis of variance showed that the beneficial impact of the administration of AME under exposure to cadmium on the estimated markers of the oxidative status of the submandibular gland was the result of the independent impact of the extract and its interactive action with cadmium, which was antagonistic in character .The administration of AME alone throughout the experiment had no effect on the concentration of cadmium in the submandibular gland of rats . Cadmium concentration in the submandibular gland of the rats feed with the 1 and 5 mg/kg diet in each time point was higher  than in the control animals .1 + AME group was higher than in the control group (2.2-fold) and maintained within the range of the Cd1 group  than in the Cd5 group; however, it was higher (2.3-fold) than in the control animals  and interactive action with this xenobiotic . The clear protection against cadmium accumulation in this salivary gland due to the 10 month administration of AME at the treatment with the 5 mg Cd/kg diet was an effect of an independent action of this extract .The ANOVA/MANOVA analysis revealed that the impact of the co-administration of AME under the 3-month exposure to the 1 mg Cd/kg diet on cadmium concentration in the submandibular gland resulted from its independent , except for the activity of SOD, and positively with TOS, OSI, and the concentration of LPO . The present study is a part of a wide research project designed to investigate the possibility of using AME in protection from the unfavourable health outcomes of low-level and moderate chronic exposure to cadmium, and it provided new relevant and promising data in this regard. The study not only revealed that the extract improved the oxidative/antioxidative status when it was administered both alone and under exposure to cadmium, but it also allowed for better understanding of the effect of this toxic metal on the salivary glands.In the available literature, some data showing that cadmium has a damaging impact on the salivary glands can be found ,34,37,38The factors of increase in the values of TOS in the submandibular gland tissue of the animals exposed to cadmium, compared to the respective controls, show that the extent of the intensity of this xenobiotic-induced oxidative stress depended on the level of exposure, and that at the low-level treatment it markedly intensified with its duration. The intoxication with cadmium first weakened the enzymatic antioxidative barrier , but also decreased the concentration of the main non-enzymatic antioxidants, such as GSH, leading, as a result, to the decrease in TAS. Detailed analysis of changes in particular markers of the antioxidative status, together with the fact that the value of TOS in the submandibular gland at both levels of exposure to cadmium for 3 months did not differ compared to the proper value, allow for the conclusion that the primary mechanism of the pro-oxidative action of this xenobiotic on this salivary gland consists of weakening the antioxidative protection. A key role in preventing oxidative stress in living organisms is played by antioxidative enzymes such as GPx, glutathione reductase (GR), SOD, and CAT . The cad3) may be higher than in the blood and urine. Abdollahi et al. [It is important to underline that Gonzalez et al.  have sugi et al.  have notThe results of the current study regarding the submandibular gland, together with our more recent findings on the parotid gland , show thOur results on the impact of cadmium on the submandibular gland confirm that cadmium may be dangerous for health even at a low exposure and, at the same time, it enhances the significance of the present study focused on the protective impact of AME regarding the outcomes of cadmium action and the importance of the findings of the study. Moreover, the study allowed us to evaluate the influence of aronia extract on the oxidative-reductive status, not only under the low-level and moderate repeated exposure, but also at very low exposure resulting from the trace, but unavoidable, presence of this xenobiotic in the standard diet (0.0584 ± 0.0049 mg/kg diet in our study ). DetailThe most important finding of the present study is revealing that the administration of AME under exposure to cadmium almost completely prevented the development of oxidative stress and enhanced lipid peroxidation in the submandibular gland. In the available literature there are few data on the impact of some polyphenolic compounds  or polyphenol-rich products (green and black tea) on the oxidative-reductive status of the salivary glands and saliva ,41,42. N2+) which in this way influences the body turnover of this element [Detailed analysis of the results of the present study show that the administration of AME offered protection from cadmium-induced oxidative stress irrespective of the intensity of intoxication with this xenobiotic, and that the beneficial impact of the extract was accompanied by the improvement of the enzymatic and non-enzymatic antioxidative barrier. Taking into account the results of the ANOVA/MANOVA analysis, the beneficial influence of AME on the submandibular gland during cadmium intoxication may be explained by the direct impact of the chokeberry extract, as well as an indirect action related to interactions between the extract ingredients and this toxic heavy metal. The direct effect may be explained by the high antioxidative properties of the ingredients of aronia berries ,27, refl element ,30, inclBecause the beneficial effect of aronia extract on the oxidative/antioxidative status of the submandibular gland has been reported by us for the first time, wider discussion of the results is impossible. However, it is important to note that, in these animals, the protective impact of the administration of AME has also been revealed regarding the parotid gland . AlthougIn sum, the present investigation has revealed for the first time that even low-level repeated exposure to cadmium may weaken the enzymatic and non-enzymatic antioxidative barrier of the submandibular gland, leading to the destruction of the oxidative/antioxidative balance and development of oxidative stress and enhanced lipid peroxidation. The fact that cadmium may induce oxidative stress and lipid peroxidation in an experimental model of human exposure shows that even low intoxication with this xenobiotic may create a risk of damage to the salivary gland. However, the most important and practically useful finding of this study is revealing that administration of the AME, both alone and under exposure to cadmium, improves the oxidative/antioxidative status of this salivary gland. The extract administration under low-level and moderate exposure to cadmium almost completely prevented xenobiotic-induced oxidative stress and lipid peroxidation. A. melanocarpa may be effective in the protection of the organism under the conditions of exposure to cadmium.The beneficial effect of AME may result from an independent antioxidative impact of its ingredients, as well as from their interactive action with cadmium, resulting in a decrease in its accumulation in this salivary gland. The findings of this study, together with our more recent results on the beneficial influence of AME on the parotid gland, show that the extract may offer protection against the unfavourable impact of cadmium on the organs of the oral cavity. Moreover, these results provide further evidence that products from the berries of