Eyebrows are the most important facial feature in facial recognition with its shape rated to be more helpful than color or density for facial reconstruction or approximation. However, little extant research has estimated the position and morphological territory of the eyebrow from the orbit. Three-dimensional craniofacial models, produced from CT scans of 180 Koreans autopsied at the National Forensic Service Seoul Institute, were used to conduct metric analyses of subjects  between 19 and 49 (mean 35.1) years. We employed 18 craniofacial landmarks to examine the morphometry of the eyebrow and orbit with 35 pairs of distances per subject measured between landmark and reference planes. Additionally, we used linear regression analyses to predict eyebrow shape from the orbit for every possible combination of variables. The morphology of the orbit has more influence on the position of the superior margin of the eyebrow. In addition, the middle part of the eyebrow was more predictable. The highest point of the eyebrow in female was located more medially than the male. Based on our findings, the equations for estimating the position of the eyebrow from the shape of the orbit is useful information for face reconstruction or approximation. Moreover, this is especially true when evidence cannot be obtained from objective identifiable methods such as DNA, fingerprints, dental records, and non-dental radiographic comparison. Hence, facial reconstruction or facial approximation, a face recreation tool aimed to reproduce the face before death based on interpretation of the skull is employed, with the objective of recognition leading to an identification3. Furthermore, accuracy assessment also assists in the analysis of specific regions of the face, such as eyes, nose, mouth, and ears, which are critical to facial recognition by predicting the location, size, and morphology of facial features4.Identifying the remains of a missing person, especially those whose faces cannot be recognized due to decomposition or skeletonization is often difficult for law enforcement and investigative agencies5. The most frequently reported guidelines for facial feature properties is the study on the nose, such as nasal profile or projection9, followed by research on the eye, such as eyeball position or protrusion12, the mouth such as mouth width or lip morphology15, and ear shape estimation18. Conversely, Farkas et al. emphasized that facial morphology databases on various ethnic groups are still required19.Considerable data has been derived from craniofacial reconstruction studies in forensic identification. Fedosyutkin and Nainys, who summarized and described the relationship of skull morphology to facial features, showed general characteristics of how the skull morphology affects facial features21. Specifically, eyebrow shape is more helpful than color or density in facial recognition22. However, to our knowledge, no research has been conducted that estimates the position and morphological territory of the eyebrow from the orbit using 3D craniofacial reconstruction methods. There have been studies of eyebrows using 2D methods and their importance has been described in face restoration with suggestions that additional studies be conducted to the point of the most superior part of the eyebrow23. This study establishes the parameters that may help estimate the position and shape of the eyebrow from the orbit using 3D computed tomography (CT) imaging methods. The findings of this study can be applied in the field of forensic facial reconstruction and are expected to increase the possibility of recognition and identification of persons.Among these features, eyebrows are the most important facial feature in recognizing emotions under the influence of cognitive loadIn this study, 180 subjects were analyzed for each of the 35 measurements on both eyebrows and orbits. Descriptive statistics results are shown in Supplementary Table 24. Males had the larger mean value in the majority of measurements in this study. In males . On the other hand, the regression equation taking measurement L10 to predict measurement L19 showed the lowest power of explanation (R2 13%). On the right side, the equation predicting measurement R26 by measurement R9 showed the weakest power of explanation (R2 18%). In females (Supplementary Table 2 35%). The highest power of explanation on the right side was shown by the equation for R10 making prediction of measurement R20 (R2 43%). The measurement showing the lowest power of explanation on both sides was measurement 19 predicted by measurement 10 . The measurements of the highest power of explanation differ in each sex. Therefore, to reconstruct a face, applying different measurements according to a subject’s sex would lead to a better outcome.It has been reported that there is sexual dimorphism of eyebrows in primates2 13%; however, it was 31% on the right side. For the equation of measurement 19 by measurement 8, the power of explanation of the left side was 16% and the R2 was 34% on the right side. The discrepancies between sides were also observed in females. Regression equation of measurement 18 taking measurement 10 as a factor showed a R2 value of 28% on the left and 39% on the right. For the equation of measurement 21 estimated by measurement 9, the power of explanation was 21% on the left and 32% the right. R2 of the equation measurement 24 predicted by measurement 9 was 29% on the left side and 18% on the right side. Therefore, one might need to consider the side to apply the regression equation.Further, we observed that there is discrepancy between sides  has been applied to assess sex or ethnicity in other studies23. In Stephan’s study the mean horizontal distance between the superciliare and most lateral point of the iris showed a standard deviation greater than the mean. In this study, the R2 values ranged within 44%, suggesting a valid guideline, which provides a narrow-ranged figure, which can be worthwhile in facial reconstruction cases. However, caution should be taken in applying this method to other ancestries since this study was performed on Koreans.Every measurement except measurement 7 (LO-O), 16 (EBS-LO), and 25 (EB3I-O) was larger in males. The average of measurement 1 (MO-LO), a standard of measurement, is shorter in female. Therefore, EBS, the highest point of the eyebrow, is located more medial in females. The eyebrow of primates was reported to be sexually dimorphic, but the eyebrow solely as an indicator of sex has rarely been reported in humans. Stephan has suggested the average position of “superciliare,” the highest point of the eyebrow, for eyebrow reconstruction; however, he also noted that the application can be limitedThis study provided data to estimate the position of the eyebrow using the basic width and height measurement values of the orbit rim. The findings reveal that the morphology of the orbit had more influence on the position of the superior margin than the inferior margin of the eyebrow. In addition, the middle part of the eyebrow was relatively more predictable when the regression equations were used; however, the medial and lateral ends of the eyebrow were not. Therefore, both ends of the eyebrow are barely affected by the morphology of the orbital margin. Most of the male eyebrows had larger values than the female. The highest point of the eyebrow in female was located more medially than in the male. However, this fact is not clear enough to show sexual dimorphism. Through this study, it is expected that the equations for estimating the position of eyebrow from the shape of the orbit would be used as useful information for face reconstruction or approximation.All methods performed in this study complied with the Declaration of Helsinki and were approved by the Institutional Review Board (IRB) of the National Forensic Service (No. 906-170118-HR-004-01). This retrospective study was approved, and prior informed consent was waived by Ethical Committee for National Forensic Service. The study is in accordance with relevant guidelines and regulations. The subject of all figures included in this study were images of Dr. Lee (Won-Joon Lee), a co-author and one of study participants, and written informed consent was obtained from him. We used craniofacial samples from 180 Koreans autopsied between March 2017 and September 2018 at the National Forensic Service Seoul Institute (NFS Seoul Institute). We conducted metric analyses for 180 subjects  between the ages of 19 and 49 mean, 35.1) years to minimize the influence of changes in eyebrow morphology due to aging . Barium sulfate (BaSO4) solution, a contrast agent, was applied to the subject’s eyebrows before taking the CT scans to reveal the radioactive area on the CT images. During this process, subjects with severe hair removal traces were excluded from the study. 3D craniofacial data were created using Digital Imaging and Communications in Medicine (DICOM) data acquired from a 128-slice multidetector CT (MDCT) scanner  under the following properties; 120 kV, 175 mA, and slice-thickness 0.6 mm. 3D models built using soft and hard tissue images were imported into a biomedical image engineering program , to obtain measurements of distances for 18 anatomical landmarks of the eyebrows and orbits.The Frankfort horizontal plane, a plane passing through orbitale and auriculare, as well as coronal and sagittal planes, perpendicular to each other were adopted as reference planes for cranio-cephalometric analysis. In total, 18 craniofacial landmarks [12 cephalometric (eyebrow) and 6 craniometric (orbit)] were used to examine the morphometry of the eyebrow and orbit Table .Table 4Vp > 0.05) as independent t-tests; otherwise, Mann–Whitney U-tests were used to determine sex differences. We also conducted intra-class correlation coefficient analysis to verify reproducibility of measurements by assessing intra- and inter-observer errors. Finally, we performed linear regression analyses using the SPSS to predict eyebrow shape from the orbit for every possible combination of variables using the command syntax. All statistical results were considered significant if p values were less than 0.05.We conducted a statistical analysis using SPSS . Independent t-test and ANOVA were conducted after obtaining descriptive statistics for the samples, to verify significant differences between sex and age groups, respectively. We applied Levene’s test for homogeneity of variance under the assumption of equal variances (Supplementary Information.


Pathological aseptic calcification is the most common form of structural valvular degeneration (SVD), leading to premature failure of heart valve bioprostheses (BHVs). The processing methods used to obtain GA-fixed pericardium-based biomaterials determine the hemodynamic characteristics and durability of BHVs. This article presents a comparative study of the effects of several processing methods on the degree of damage to the ECM of GA-fixed pericardium-based biomaterials as well as on their biostability, biocompatibility, and resistance to calcification. Based on the assumption that preservation of the native ECM structure will enable the creation of calcinosis-resistant materials, this study provides a soft biomimetic approach for the manufacture of GA-fixed biomaterials using gentle decellularization and washing methods. It has been shown that the use of soft methods for preimplantation processing of materials, ensuring maximum preservation of the intactness of the pericardial ECM, radically increases the resistance of biomaterials to calcification. These obtained data are of interest for the development of new calcinosis-resistant biomaterials for the manufacture of BHVs. Heart valve defects are the main cause of disability and deterioration of quality of life and are the leading cause of cardiovascular morbidity and mortality worldwide . ConsideCurrently, the standard therapy for valvular diseases is the surgical replacement of dysfunctional native valves with artificial prostheses such as mechanical or bioprosthetic heart valves (BHV) . Over thAseptic calcification is the most common pathological form of SVD  and is cOver the past decade, an increasing amount of data has accumulated, convincingly indicating the decisive involvement of the immune response in the calcification and degradation of BHV ,12,13. SIt is also assumed that preservation of the native ECM structure during processing is highly desirable to reduce the recipient’s immune response to decellularized ECM biomaterials ,23,24. GThis article presents a comparative study of the effects of several processing methods on the degree of preimplantation damage of ECM-based GA-fixed biomaterials, as well as on the degree of their biocompatibility in vitro and aseptic calcification in vivo. Based on the assumption that the preservation of structural integrity during ECM processing will allow the creation of calcinosis-resistant materials, this study presents the authors’ method of manufacturing GA-fixed ECM-based biomaterials using gentle methods of decellularization and chemical stabilization.®  was purchased from JSC “Valenta Pharm” . Mayer’s hematoxylin, eosin H, Alizarin red S, toluidine blue, neutral buffered formalin, and other reagents for histological analysis were purchased from Labiko LLC . All other reagents used were of analytical reagent quality.MycoFluor™ mycoplasma detection kits were purchased from Thermo Scientific . The fetal bovine serum was from Gibco . Sodium dodecyl sulfate, Sodium Deoxycholate Monohydrate, Triton™ X-100 solution, Glutaraldehyde and Formaldehyde solution, Isopropyl and Ethyl Alcohol, Accutase cocktail, fluorescent dyes Calcein AM, Propidium iodide and Bisbenzimide Hoechst 33342, Culture media alfa-MEM and DMEM, L-glutamine, gentamicin sulfate, Alcian Blue 8GX, Biebrich scarlet, Fast Green FCF, Schiff’s reagent and other chemicals were purchased from Sigma-Aldrich . DioxidineThe freshly harvested pericardium of mature bulls was transported to the laboratory in a cold saline solution . Next, the pericardium was placed in 0.02% EDTA solution for 2 h with constant stirring on a shaker (40 rpm) at room temperature with a tissue-to-volume ratio of 1:50. The tissue was then mechanically cleaned of adipose tissue and treated as described in Reagents used by leading BHV manufacturers were used for the preimplantation treatment. The damaging effect of SDS on the ECM is well known ,29,30. An = 12) and expressed as a percentage of the total number of collagen fibers analyzed. A qualitative assessment of the degree of ECM damage was also performed based on the intensity of fuchsinophilic saturation of histological preparations stained with Alcyan blue-PAS.To assess the degree of damage to ECM materials after processing, native pericardium (group N) was used as a comparative control. The degree of damage to the collagen ECM was assessed by analyzing the histological preparations stained with Lillie’s trichrome. The number of damaged collagen fibers was calculated in the field of vision  was covered with gold by Q150R Quorum Technologies  at a pressure of 7.3 × 10−2 Pa in the column and 1.5 × 10−1 Pa in the chamber. The area of damage was calculated in the field of vision  using the non-commercial ImageJ software (https://imagej.nih.gov (accessed on 16 March 2023)) and was expressed as a percentage of the total area of the estimated surface.The morphological characteristics of all samples before and after the preimplantation treatment were studied using a Tescan VEGA III (scanning electron microscopy (SEM), Brno, Czech Republic) equipped with energy dispersive spectroscopy . The surface of the lyophilized samples  were obtained from the CLS Cell Lines Service GmbH . These cells were cultured in alfa-MEM medium  supplemented with 10% fetal bovine serum  and 40 µg/mL of gentamicin sulfate  at 37 °C in a humidified atmosphere of 5% COA cytotoxicity assay was carried out to study the contact toxicity (cultivation on the surface of samples) and non-contact toxicity (cultivation with extracts from samples) of the experimental materials.Before seeding the cells, the materials were subjected to standard washing in saline solution for 2 h, in accordance with the deadlines for washing chemically stabilized bioprostheses of heart valves, according to the manufacturer’s recommendations (standard from 3 min to 2 h). To determine the potential for re-endothelialization of the experimental materials, additional washing of samples was carried out in a nutrient medium of DMEM with 10% FBS for 4 days with a tissue-to-volume ratio of 1:30, simulating the leaching of stabilizing agents and capping of free aldehyde groups with blood plasma amino acids observed in the recipients after implantation of BHV into the systemic circulation. 2 for 72 h, with constant stirring on a shaker (40 rpm). The media samples were filtered through a sterile membrane with a pore diameter of 0.22 µm  and used in experiments.Extracts from the materials (non-contact toxicity assay) were carried out in an alfa-MEM medium with 10% FBS at 37 °C in a humidified atmosphere of 5% COp.serosum) using an Accutase cocktail .The calculation of living and dead cells during cultivation on the surface (contact cytotoxicity) of the experimental materials was performed 96 h after seeding. The cells were detached from the surface of the samples  and propidium iodide (1 µg/mL) for 25 min at 37 °C. Live and dead cells were analyzed using an Accuri C6 flow cytometer .The number of cells in suspension after their detachment with the Accutase cocktail was analyzed using a BD Accuri C6 flow cytometer . The mitotic cells were assessed by means of fluorescence of cells stained with nuclear dye H33342 at a concentration of 1 µg/mL and counting the number of mitotic cells using a DM 6000 fluorescence microscope  . The totSixty male Wistar rats weighing 180–200 g (age two months) were used. The animals were individually housed in a temperature-controlled room (22 °C) and fed a standard diet with full access to water and food. The experiments were performed according to the Regulations for Studies with Experimental Animals . The protocol was approved by the Commission on Biological Safety and Ethics at the Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences . For the experiments, the rats were divided into six groups (five in each group), and independent replicates were performed for each group. The generally accepted model of ectopic (subcutaneous) implantation of biomaterials was used  and zoletil 7 mg/kg . To implant the specimens, a 1.5-cm-wide transverse skin incision was made in the dorsal interscapular area, subcutaneous pockets were formed parallel to the skin using a smooth trocar, and the specimens were implanted at a depth of at least 2 cm from the incision line. The samples were implanted with full interstitial contact without restriction chambers or meshes. All the experimental samples  were used for implantation. The samples (leaflets) of PERIMOUNT RSR bioprosthesis  were used as a positive control (Edw group). For postsurgical recovery, the animals were exposed to a heating plate until awakening.Totals of 4–8-week implantation periods are most often used to assess the degree of calcification of GA-fixed biomaterials in ectopic implantation models ,42,43,44The animals from each group were randomly divided to be euthanized (carbon dioxide protocol) 8 and 24 weeks after the surgical procedure. To prevent autolysis, immediately after withdrawal, samples of implanted materials with surrounding tissues of the recipient bed were washed for 30 s with cold (14 °C) saline solution and fixed for 48 h in neutral buffered formalin (NBF) at a tissue-fixator volume ratio of 1:30.After the termination of fixation, the fragments of samples were washed with distilled water (3 × 3 min) to remove excessive phosphates and placed for no less than 12 h in medium Optimum Cutting Temperature (O.C.T.) Compound Tissue Tek . Cross slices of experimental samples (9 μm) were prepared by cryosectioning . The micrographs of stained histological samples were obtained on a Nikon Eclipse Ti-E microscope station  and processed using the software NIS Elements AR4.13.05 (Build 933).The qualitative content of acidic and neutral GAGS, as well as the number of residual aldehyde groups in the materials before and after treatment, were evaluated using Alcyan blue-PAS staining (the Mowry method in the Luna modification) . The biocompatibility of these materials was assessed using H&E staining (Mayer’s Hematoxylin and Eosin Y), the number of cells that migrated into the matrix of materials (primarily fibroblast-like cells with elongated nuclei) was calculated, and the intensity of the inflammatory response was determined by the number of leucocytes (cells with rounded nuclei) migrating into the matrix. To assess the degree of cellular-resorptive processes, the preparations stained with the collagen/non-collagen structures differential staining (using Lillie’s trichrome method) were also evaluated . The degree of calcification of the materials was assessed based on a study of histological preparations of explanted samples stained with different staining methods for calcium deposits using the Alizarin red S staining (by the McGee-Russell method . n = 16) using the non-commercial ImageJ software (https://imagej.nih.gov (accessed on 16 March 2023)) and expressed as a percentage of the total area of the assessed area. The areas of collagen resorption and calcification were determined in the field of view . The statistical significance of the differences was determined using one-way ANOVA, followed by multiple Holm–Sidak comparisons (p < 0.05). The results of in vivo studies are presented as mean ± standard deviation (M ± SD). Each experiment was performed at least four times (n ≥ 4). The Mann–Whitney U-test was used to compare two independent samples; the differences between the groups were considered statistically significant (p < 0.05). The design of the experiment and related statistics were performed using SigmaPlot™ 14.0 . Plots were created using SigmaPlot™ 14.0.The results of in vitro studies are presented as mean ± standard deviation (M ± SD). Each in vitro experiment was performed at least five times  in the development environment Spyder (v. 5.4.1) with libraries Pandas (v. 1.5.2), Numpy (v.1.24.2) and Scipy (v. 1.10.0). The graphical display of the obtained results was performed using Python 3 (version 3.10) with libraries Seaborn (v. 0.12.2) and Matplotlib (v. 3.7.0).p.serosum was studied in detail as the most important surface for evaluating the valve leaflets of BHVs experiencing the main hydrodynamic shock of blood flow. Considering that this surface should be as intact as possible in order to preserve the properties of thromboresistance, the surface was separately studied for the “exposure” of the collagen layer to be covered by the p.serosum lamina, as well as the presence or absence of areas of fibering and longitudinal micro-fractures. During the histological analysis of the matrix of materials after processing, the samples were examined for signs of structural disorganization of the ECM, as well as areas of micro-ruptures, fusion, and separation of collagen fibrils. The preservation of GAG in the matrix structure was separately investigated.For all groups, the surface of p.fibrosum. In addition, in p.fibrosum, signs of transverse cracking of ECM materials were observed, which is a characteristic sign of loss of flexibility of collagen fibrils as a result of their dehydration. The most pronounced signs of longitudinal and transverse cracking of matrix fibers were observed in Group A2, both from the side of the p.fibrosum and from the side of the p.serosum. In addition, the matrix of materials of Groups A2 and A1 differed in the compacted structure typical of hyper-fixed biomaterials. In the M2 group, the most pronounced signs of reduction in the ECM were observed, which were thinning and partial straightening of collagen fibers, as well as their strong separation.Morphological analysis of the ECM materials after processing (preimplantation treatment) showed that the greatest degree of preservation of the ECM structure was observed in the samples of Group S a,b. The It should be noted that in Groups A1, A2, M1, and M2, the reduction in collagen fibers was combined with a change in their affinity for the dye, which manifested itself in varying degrees of dye impregnation (Fast Green FCF) of individual fibrillary bundles or the entire collagen matrix. In addition, a particularly high degree of dye saturation of the entire matrix was observed in Group A2, which indicates obvious dehydration and damage to the matrix of the materials in this group.Fuchsinophilic saturation of collagen fibers was also characteristic of all the studied groups, which may be associated with both damage to the collagen matrix and GA concentration since the lowest degree of fuchsinophilia was observed for samples S and the highest for A1. In the samples of all studied groups after preimplantation treatment, acidic GAGs were not detected b.p.serosum for this group reached 80.9 ± 8.9%), which manifested itself in partial detachment of the p.serosum from the underlying layer and strong collagen fibers separation of the underlying fibers , all studied samples had a pronounced cytotoxic effect. After 96 h of cultivation, the number of living cells on the surface of the experimental biomaterials did not exceed 10.3 ± 6.2% a.Additional washing (four days) significantly suppressed the cytotoxic effects of the biomaterials b. The maA study of non-contact cytotoxicity showed that extracts from all groups of biomaterials had high toxicity to cells c, while When analyzing the proliferative activity and dynamics of the number of cell populations, it was found that the growth of cells cultured on samples from all experimental materials was completely absent. Simultaneously, a decrease in the number of cells was observed owing to the pronounced cytotoxic effect of the experimental samples a. FigureIt was revealed that extracts from biomaterials of all groups not only prevented cell proliferation but also contributed to a decrease in their number due to the pronounced cytotoxic effect c.The mitotic activity of hMSC-AT cultured on samples of all groups of biomaterials, both under the conditions of standard washing and additional washing, was completely absent. In the case of samples of experimental materials washed according to the standard procedure (2 h), the pronounced cytostatic effect was based on the direct toxic effect of the materials themselves. In the case of samples with additional 4-day washing in Groups A1, A2, and M1, the cytostatic effect was also mediated by the residual toxic effect of the materials themselves. In turn, the cytostatic effect of the biomaterial samples of Groups M2 and S may be due to the modification of the surface of the samples after processing and the weak residual toxic substances from the samples of materials.Morphological analysis of the samples after 8 weeks of implantation showed that fibroblast-like cells migrated most actively to the medial matrix of the materials in Groups M2 and S a. At thep.fibrosum. An analysis of the morphology of cells that migrated to the border zones of the matrix found that in groups A1 and M1, most cells (>80%) had small and rounded nuclei, indicating the development of focal leucocytic invasion .In Groups A2 and M2, the relative number of leucocytes was approximately 70%, whereas, in the Edw control group, the cell population was equally represented by leucocytes and fibroblast-like cells. In Group S, the number of migrated leucocytes did not exceed 32% . From thFurther analysis of the collagen matrix of the experimental materials showed that the areas of leukocyte invasion were co-localized with areas of resorption of VCM in all groups, which indicates the classical cellular nature of resorption (utilization) of damaged components of the extracellular matrix .After 8 weeks of implantation, the most pronounced cellular matrix resorption (utilization) was detected in Groups A1 and M1; the resorption area was 38.12 ± 2.39 and 29.66 ± 1.09%, respectively . SimultaIn Group A2 samples, pathological calcification was observed 8 weeks after implantation. Mineralized samples from this group were characterized by low rates of resorption of the collagenic matrix a. GroupsGroup S and the control group Edw showed no signs of calcification during any of the implantation periods.To identify the dependence of the calcification of materials on previously identified parameters , a correlation analysis was performed. Group A1 was excluded from this analysis because in vivo studies showed that the materials of this group underwent intensive cellular resorption, bypassing the calcification stage. The power (strength) of the correlation was determined, as shown in s > 0.5). However, a detailed study of these data presented in s = 0.88) (s = 0.69)  b and wit = 0.69) e.s > 0.5) and a moderate correlation between the indicators of cell migration and damage to the matrix of materials (rs > 0.3)  .Apparently, damage to materials during preimplantation treatment leads to a decrease in cell survival, which may be due to both modification of the surface in contact with cells and the increased leaching of cytotoxic substances from the materials. The damaged material also attracts more leukocytes to the implantation area and reduces the migration of fibroblast-like cells, which is caused by two factors: the death of the recipient’s cells in contact with toxic material and the initiation of cell-resorptive processes in response to the damaged collagen matrix. Together, these effects increase the likelihood of the development of intensive aseptic calcification of GA-fixed materials.Thus, general damage to the structure of materials by more than 40% leads to the development of intense calcification, whereas slightly damaged materials (20–40%) undergo less intense calcification. In addition, as shown earlier for the A1 group materials, damage to materials by more than 70% initiates the development of their rapid cellular resorption (utilization).A low degree of damage to the VCM significantly reduced the immunogenic properties of materials (cytotoxicity and leukocyte invasion), increased the potential of materials for re-endothelization, and contributed to its resistance to calcinosis, which was shown for group S.The proposed mechanisms of calcification initiation include two possible pathways: (a) the release of calcium ions from dying cells due to the cytotoxic effect of GA ,48,49 anp.serosum induce chemotaxis of macrophages and neutrophils and increase MMP activity [Our study confirmed the dependence of aseptic calcification on the immune response and the launch of cellular resorptive processes. The correlation between the degree of damage to materials after processing and the degree of calcification indicates that the immune response and subsequent calcification, in this case, developed in response to damage patterns of the implanted collagen matrix by the mechanism of the DAMP-associated immune response. Fragmented components of the extracellular matrix, such as GAGs or fibronectin, are among the most well-known DAMPs that are released due to rigid decellularization ,53. The activity . Furtheractivity ,61. ThisThus, violation of the ECM integrity of BHVs during preimplantation treatment can directly increase their immunogenicity. Simultaneously, it can be assumed that a high degree of matrix damage leads to direct proteolytic destruction of the BHVs matrix, bypassing the intermediate stage of calcification, which was demonstrated in Group A1, which had the greatest degree of damage, pronounced leukocyte invasion, and lack of calcification against the background of active cellular proteolytic resorption.In this study, the areas of the matrix of experimental materials that underwent active degradation were not mineralized, whereas the zones of calcification nucleation were observed exactly where active cellular migration was accompanied by a decrease in the intensification of the processes of ECM resorption. Thus, it was shown that in the case of free migration of cells into the entire thickness of the matrix, damaged areas that are not amenable to direct cellular and proteolytic degradation undergo intensive mineralization after eight weeks of implantation, as was observed for Group A2. Conversely, in the M2 group, which was also actively repopularized by the recipient cells in the early stages of implantation, there were no signs of calcification, but there were signs of active resorption of the border regions and even foci of resorption of the matrix thickness itself. However, at the later implantation dates (24 weeks), part of the extracellular fibrillar matrix that was apparently relatively well preserved and could not be directly disposed of due to the proteolytic activity of cells underwent an intermediate phase of calcification, which is essentially a form of disposal of a foreign body. Finally, the A1 group materials, where degradation and cellular resorption of the matrix of materials were observed even after six months of implantation, showed no signs of calcification since the severely damaged matrix could be resorbed directly by recipient cells and intercellular fluid proteases. Thus, the observed cell-resorptive processes preceding calcinosis  may indicate the utilization nature of this process; that is, calcification, in this case, maybe one of the stages of the process of utilization of the implanted matrix, perceived by the body as a foreign body that is not resorbed through proteolysis.Several researchers have assumed that GA can initiate calcification. There are theories that both residual aldehyde groups and changes in the structure and surface charge of collagen molecules can contribute to calcification ,62. At tp.serosum of the materials, which was observed for materials of the M1 group in comparison with M2. Such a “restoring” effect of storage solutions can lead to an incorrect assessment of the suitability of the finished material since the results of histological analysis of the collagen matrix showed the presence of a wide range of different types of damage, even in the M1 group.It should be noted that the use of fixing agents  as a component of storage solutions contributes to the visual restoration of the macrostructure of the At the same time, by analyzing the results obtained, we can safely assume that the use of gentle methods of preimplantation processing of materials that ensure maximum preservation of the intactness of the ECM makes it possible to radically increase the resistance of pericardial materials to calcinosis, as shown for Group S materials.Thus, the use of aggressive methods for processing biomaterials  leads to damage and changes in the structure of the matrix and opens up patterns of ECM damage, initiating an immune response aimed at the utilization of damaged matrix components by recipient cells. If direct proteolytic utilization by cells of individual sections of the GA-fixed matrix (insufficiently damaged) is impossible, an intermediate phase of calcification is initiated, preceding the intensive utilization of the mineralized fibrillar matrix by osteoclast-like cells. With this form of damage to the extracellular matrix of BHVs, signs of calcification appear mainly by late implantation (at least 20–24 weeks), after which it is necessary to use long implantation periods in laboratory animals to assess the true tendency of biomaterials to undergo calcification. A high degree of cytotoxicity of materials, which is not related to the concentration of GA used but is associated with the degree of damage to the surface of the biomaterial and the rate of release of toxic substances, can delay calcification, blocking access to the matrix of materials for cells, and thus inhibiting the development of calcification. At the same time, despite such a delay, calcification of damaged but chemically stabilized materials will still occur and will most likely occur in an avalanche, as is often observed in clinical practice.In summary, we can safely assume that the use of sparing methods for preimplantation processing of materials that ensure maximum preservation of the intactness of the ECM of the pericardium allows us to radically increase the resistance of biomaterials to aseptic calcinosis, as was shown for group S materials.


Covid-19 pandemic and its relative containment measures have affected populations' quality of life and psychological well-being worldwide. The fear related to the pandemic and the imposed containment measures has acted as a trigger causing a global increase in negative mental health states. Thus, we aimed to explore the relationship between fear of covid-19 and mental health via QoL .Through a quantitative cross-lagged path model research design, the study investigates people’s fear of Covid-19, quality of life, and negative mental states in a population of 444 Italian adults , in the period between the first and the second waves of the pandemic.Results show that participants’ Covid-19 fear decreased between waves, contributing to a decrease in negative mental states , thus improving the perceived quality of life. Furthermore, quality of life emerged as able to buffer the impact of fear of Covid on people’s psychological distress in short and medium terms, confirming its central role in regulating mental distress.The study suggests important guidelines for developing interventions to support the populations’ well-being and mental health. Over two years since Covid-19 began making headlines across the globe, many scholars are concerned about its impact on people's mental health and quality of life. To date, literature has globally documented different factors and aspects related to the pandemic outbreak – and the adopted measures to contain it – that have affected the quality of life and psychological well-being of the populations around the world  and the start of the second wave and lockdown (November 2020). In a recent meta-analysis of longitudinal studies, it was highlighted that the increase in symptoms of stress, anxiety and depression that characterized the first wave of the Covid-19 pandemic did not remain constant in the months that followed, showing significant decreases instead  with different restrictions on citizens' freedom of movement. Many schools and universities reintroduced the distance learning mode adopted during the quarantine, and a curfew was introduced. Within the still few longitudinal studies in the Italian territory  and whether this enabled a consequent decrease in negative mental states of anxiety, depression, and stress (H2), thereby producing an improvement in perceived quality of life (H3). Finally, we tested the function of the quality of life in buffering the effect of fear of the covid-19 on psychological distress  both in short and medium terms (H4).In this study, we went to investigate the longitudinal chain of relationships between fear of COVID-19, quality of life, and negative mental states in the period between the first and second waves of the pandemic in Italy. Thus, the purpose was to observe whether fear of COVID-19 (FCV-19) decreased between waves ; 89 (20%) were males and 355 (80%) were females. In terms of educational level, 2.5 % (n=11) have a lower secondary school diploma, 36.3 % (n=161) have an upper secondary school diploma, 54.5 % (n=242) have a university diploma, and 6.8 % (n=30) have a degree higher than a university diploma. Regarding the general level of health, 22.5% (n=100) of the sample report having some chronic disease . The criteria for inclusion in the study were (1) having more than 18 years of age, (2) having experienced the pandemic period in Italy, and (3) agreeing to participate in the study.We used a quantitative cross-lagged path model (CLPM) research design to investigate the longitudinal chain of relationships between fear of COVID-19, quality of life, and negative mental states . The participants' fear of the coronavirus was measured with the Fear of COVID-19 Scale  was administered; the reliability of the cumulate score , WHOQOL-BREF (α =.810), and DASS-21 (α =.936).CLPM was assessed by analyzing the regression and auto-regression coefficients they yielded. The regression coefficients were estimated via structural equation modelling  at Time 1 was evaluated. In line with the research aims, fear of COVID-19 was included in the model as an exogenous variable with direct effects on quality of life and negative mental states as measured at Time 1. Following that, a "stability model" (Model B) was estimated that included direct effects between measures at Time 1 (T1) and Time 2 (T2). Model B was critical in assuming that longitudinal regression paths accounted for causal mechanisms in the observed data  and T2 (r=-.12). This means that younger people tended to report lower scores of negative internal states than older participants. Looking at the quality of life, the correlations with negative internal states are statistically significant and inverse in both waves. The data indicate in this case that the higher the negative internal states, the lower the quality of life scores (and vice versa). Finally, concerning the stability of scores between T1 and T2, substantial stability can be found with statistically significant correlations ranging between r = .71  and r = .69 .The next step was to estimate the prospective relationship between fear of Covid-19, quality of life and negative mental states. We first tested the baseline model (Model A), then the model including the stability coefficients between measures gathered at Time 1 and Time 2 (Model B), and finally the cross-lagged model (Model C). All models were controlled for age and gender. The results are reported in Fig. p < .001; RMSEA=.122, SRMR = .040, NFI=.961, TLI=.966, CFI=.968). Model B's fit with the empirical data suggested that using longitudinal effects in the model resulted in a good representation of the covariances between scores at Time 1 and Time 2 with quality of life and negative mental states. As the last step, we tested the cross-lagged model (Model C), which included the stability coefficients between measures at Time 1 and Time 2 and the cross-lagged paths  and, all in all, it represented the best model among the three tested.Evaluation of goodness of fit indexes suggested the acceptance of the cross-sectional model (M1): (χ2(1)=12.09, p=.001; RMSEA=.158, SRMR = .042, NFI=.954, TLI=.956, CFI=.958). Concerning the fit indices, it should be noted that the only value that does not suggest adopting the model is RMSEA. In this regard, however, it should be remembered that the indicator tends to underperform in complex models with few degrees of freedom  were large, positive, and statistically significant.Similarly, fear of COVID-19 was also found to be less related to the quality of life  and negative mental states  at Time 2. In other words, the direct effect of COVID fear was more significant at T1, while the indirect effect at T2 (i.e. after six months) is attenuated. In both cases, the effects' statistical significance and direction were maintained. In addition, including the cross-lagged paths significantly increased the model's fit. In addition to the path values estimated in the null model, Model C suggested that quality of life and negative mental states were longitudinally interrelated. On the one hand, the results confirmed the stability of the scores across time; in fact, both direct effects on quality of life at Time 1 to quality of life at Time 2  and negative mental states at Time 1 and negative mental states at Time 2 , and negative mental states at Time 1 were not statistically related to the quality of life at Time 2 . In other words, the more participants displayed a good quality of life, the less they rated negative mental states six months later. On the contrary, the relationship between negative mental states at T1 and quality of life at T2 was less relevant in terms of effect size , highlighting the importance of quality of life in buffering the effect of being afraid of the COVID-19 pandemic on negative mental states both at short terms and medium terms (i.e. after six months) again.Finally, with regards to participants' demographic, in general, age and gender did not report any statistically significant effects, with the only exception of the direct path from age to negative mental states at T1 , whereas at T2 that effects did not report statistical significance.Our longitudinal study explored the relationship between fear of COVID-19 and mental health via QoL  over time. The decrease in fear seemed to augment the participants' mental health  and a better perception of QoL (H3), confirming our hypothesis. Furthermore, quality of life promoted better mental health in Italian adults during the second lockdown.QoL confirmed a central role in regulating mental distress and fear of COVID-19 in our sample (Ferreira et al. This paper showed how QoL might play a crucial role in mental health during the pandemic and the effect of fear on increasing psychological symptoms such as depression, anxiety, and stress (Ornell et al. Some limitations to this study must be discussed and addressed. First, the respondents were mainly women, showing a certain unbalancement in the sample recruitment. However, differences between gender were detected and did not show relevant gaps. Online recruitment should have limited access to the strip of the population technologically disadvantaged and, consequently, with lower QoL. Qualitative future research might help see differences and potentially aggravating factors in people belonging to the most socioeconomic disadvantaged part of the population.The Italian authority policies on COVID-19 contrast have been focused on two pillars. Response to the health emergency first, and mitigation of the psychological consequences. The two levels were considered dramatically imbricated (Pompili et al.


The rapid diagnosis of rice nitrogen nutrition is of great significance to rice field management and precision fertilization. The nitrogen nutrition index (NNI) based on the standard nitrogen concentration curve is a common parameter for the quantitative diagnosis of rice nitrogen nutrition. However, the current NNI estimation methods based on hyperspectral techniques mainly focus on finding a better estimation model while ignoring the relationship between the critical nitrogen concentration curve and rice hyperspectral reflectance.This study obtained canopy spectral data using unmanned aerial vehicle (UAV) hyperspectral remote sensing and determined the rice critical nitrogen concentration curve and NNI. Taking the spectrum at critical nitrogen concentration as the standard spectrum, the original spectral reflectance and logarithmic spectral reflectance data were transformed by the difference method, and the features of the spectral data were extracted by a Autoencoder. Finally, the NNI inversion models of rice based on Extreme Learning Machine (ELM) and Bald Eagle Search-Extreme Learning Machine (BES-ELM) were constructed by taking the feature bands of four spectral extractions as input variables.1) from the feature extraction results of the self-encoder, simple logarithmic or difference transformation had little effect on NNI estimation, and logarithmic difference transformation effectively improved the NNI estimation results; 2) the estimation model based on the logarithmic difference spectrum and BES-ELM had the highest estimation accuracy, and the coefficient of determination (R2) values of the training set and verification set were 0.839 and 0.837, and the root mean square error (RMSE) values were 0.075 and 0.073, respectively; 3) according to the NNI, the samples were divided into a nitrogen-rich group (NNI ≥ 1) and nitrogen-deficient group (NNI < 1).The logarithmic difference transformation of the spectrum can effectively improve the estimation accuracy of the NNI estimation model, providing a new approach for improving NNI estimation methods based on hyperspectral technology. Nitrogen is an important element affecting the growth, development, and yield of rice. A change in nitrogen content will affect the photosynthesis, protein production, and carbon and nitrogen metabolism of crops . NitrogeCurrently, research has mainly focused on obtaining nitrogen content data from rice. However, it remains difficult to provide quantitative guidance on nitrogen abundance and deficiency simply based on the nitrogen content of rice. The critical nitrogen concentration curve theory is the leading standard for rice nitrogen nutrition . Greenwo2) values of 0.913–0.949 and root mean square error (RMSE) values of 0.055–0.127  and Bald Eagle Search-Extreme Learning Machine (BES-ELM). Finally, the estimation accuracy of the models was compared, and the best estimation method of rice NNI based on hyperspectral data was determined to provide a new research approach for rapidly obtaining rice NNI.22.1-2, N1 = 75 kg.hm-2, N2 = 150 kg.hm-2, N3 = 225 kg.hm-2, and N4 = 300 kg.hm-2. In Experiment 2, five nitrogen gradients were designed, and the nitrogen application rates were N0 = 0 kg.hm-2, N1 = 50 kg.hm-2, N2 = 100 kg.hm-2, N3 = 150 kg.hm-2, and N4 = 200 kg.hm-2. The area of each plot in Experiment 1 was 5*8=40m2, and that in Experiment 2 was 660 m2. Except for the gradient of nitrogen fertilizer, the field management of the two experimental areas was consistent. The application rate of phosphorus and potassium fertilizer was the local standard, in which the standard application rate of phosphorus fertilizer was 144 kg.hm-2, the standard application rate of potassium fertilizer was 192 kg.hm-2, and the ratio of base to topdressing was 1: 1. The other field management practices were the same as for conventional high-yield management. Field sampling was conducted from the tillering stage to the heading stage, and the sampling interval was nine days. Each sampling in each experimental plot selected in each experimental plot randomly selected a 1 m × 1 m plot, and a plastic frame was used for subsequent ROI identification. Three representative holes were used to obtain nitrogen concentration and aboveground Biomass(AGB), and the average value was used to determine the nitrogen concentration and AGB in the plot. In Experiment 1, 120 groups of cell data were collected, and in Experiment 2, 88 groups of cell data were collected, totaling 208 groups of samples.The experiment was conducted from June to September, 2021 at the Precision Agriculture Aviation Research Base of Shenyang Agricultural University (40 58’ north latitude and 122 43’ east longitude) in Gengzhuang Town, Haicheng City, Anshan City, Liaoning Province. To avoid remote sensing data collection error caused by rainy and cloudy weather, data collection proceeded only on fine days. The collection was postponed if the cloud cover exceeded 20% or if the weather was unfavorable for remote sensing data collection. The rice variety used was ‘Beijing 1705’. The experimental field was divided into two large areas. In Experiment 1, five nitrogen gradients were set up, and the nitrogen application rates were N0 = 0 kg.hm2.22.2.1The UAV hyperspectral platform adopted the M600 PRO six-rotor UAV of Shenzhen DJI Innovation Company, and the hyperspectral imager used the GaiaSky-mini built-in push-sweep airborne hyperspectral imaging system of Sichuan Shuangli Hepu Company. The hyperspectral band range was 400–1,000 nm, the resolution was 3 nm, and the number of influential bands was 253. The hyperspectral imaging system is shown in 2.2.2When measuring the nitrogen concentration of the samples and the AGB, the rice in each plot was sampled destructively at first. The pieces were brought to the laboratory and placed into an oven to deactivate the enzymes at 105°C for 30 min and then dried to constant weight at 80°C. The AGB of the dried sample was then measured and converted into theAGB part according to the planting density. Finally, the dried samples were ground, and the plant nitrogen concentration was measured using the Kjeldahl nitrogen determination method . The cal2.32.3.12), and a and b are both curve parameters.Critical nitrogen concentration is the minimum nitrogen concentration needed by crops to reach maximum biomass, and so the nitrogen deficiency equation can be constructed based on the critical nitrogen concentration to determine the nitrogen deficiency of rice. According to the critical nitrogen concentration curve method proposed by ncN represents the actual nitrogen content (%) of the plant under different nitrogen application rates, and Nc is the critical nitrogen concentration (%) corresponding to the plant biomass.The N nutrition index is the ratio of the actual nitrogen concentration to the critical nitrogen concentration in rice, and the formula was as follows:2.3.2As the aboveground nitrogen nutrition status and canopy leaf structure of crops reach the best state under the critical nitrogen concentration, these are the direct factors affecting the spectral reflectance of the rice canopy . Therefoln, RD, RlnDR represent the log spectral reflectance, difference spectral reflectance, and log difference spectral reflectance, respectively; ncR represents the spectral reflectance of the sample under the critical nitrogen concentration state; and Rc represents the remaining spectral reflectance of the same period.In the equation, Due to the high similarity of data in adjacent bands of the full-band spectrum and a large amount of redundant information unrelated to the variables sought, modelling based on the full-band range often has some shortcomings, such as a slow running speed, high model error, and low inversion accuracy . To redu2.3.3In this study, two algorithms, namely ELM and BES-ELM, were selected for modelling. The ELM model was mainly used to test the optimization effect of BES. ELM is a new feed-forward type of neural network proposed by By imitating the hunting strategy or intelligent social behavior of vultures when searching for fish, the BES algorithm simplifies the process of searching for the optimal solution into three stages: selecting an area, searching for prey, and diving and hunting. In the first stage, eagles choose the space with the most significant game. In the second stage, the eagle moves to the selected area in search of prey. In the third stage, the eagle swings from the best position located in the second stage and determines the best hunting spot.The main flow of the Condor optimization algorithm is as follows: initialize population P, randomly generate the position information of each solution, and set the initial iteration number t to 0. After that, the following steps are performed for each key in the population P: evaluating new regions using formula (7), and searching and selecting parts using spiral movement. The new dive position is then assessed using Formula (8). Finally, the new work is used to dive into the prey, and formula (9) is used to evaluate the new solution. The iteration counter k is increased by two, and the previous step is repeated until the final solution in P is the best solution for solving the problem when the maximum number of iterations is reached.In the main work, the parameters in the above equations of the BES algorithm are defined and explained. The complete flow chart of BES algorithm optimization is shown in 2.3.42 and the root mean square error (RMSE) of the model’s prediction results for the training and test sets. The calculation procedure was as follows:To determine the estimation ability of the model for NNI, this study evaluated the estimation accuracy and robustness of the model by the coefficient of determination R33.13.1.1The basic information on plant nitrogen concentration and AGB during the whole growth period of rice is shown in According to the construction method of the critical N concentration curve proposed in 2.3.1, this study fitted the regression of N concentration and AGB obtained from eight sampling days in Experiments 1 and 2 to calculate the critical N concentration value of rice for each sampling day. The critical N concentration curve according to each critical N concentration value of Experiment 1 and the corresponding AGB was then constructed,  and nitrogen-rich group (NNI ≥ 1) according to the NNI of the training set and test set, and the estimation results of the rice NNI model based on logarithmic difference spectrum and BES-ELM model in two groups were analyzed, respectively. The results are shown in 44.1To construct the diagnostic criteria of rice nitrogen nutrition at the field scale, this study selected the critical nitrogen concentration curve crop rice nitrogen nutrition status evaluation criteria according to the studies of In the feature extraction method, this study used a self-encoder to extract the features of the transformed spectrum. The self-encoder can learn the spectral data and control the output dimension freely, which is a standard algorithm for feature extraction at present. At the same time, the self-encoder, as an unsupervised feature extraction method, can extract too many redundant features, which may affect the estimation of the NNI. In this study, MLR was used to analyze the linear relationship between the feature extraction results of the self-encoder and NNI. The extraction results with the best fitting degree were selected as the model input, which avoids the problem of unclear unsupervised learning objectives to a certain extent.4.2To improve the accuracy of the NNI estimation, two machine learning algorithms, ELM and BES-ELM, were selected to deal with four input spectra, and the combination with the highest estimation accuracy was chosen to construct the NNI estimation model. It can be seen from the results that in terms of spectral transformation, the estimation accuracy of the model based on the logarithmic difference spectrum was better than that of the other transform spectra. However, the difference transformation and logarithmic transformation alone had a slight improvement on the accuracy of the model, which may be because a single logarithmic transformation only narrows the relative differences in spectra of various bands, while the simple difference transformation amplifies the characteristic difference between the transformed spectrum and the critical nitrogen concentration spectrum. The dimensional differences of rice spectral reflectance in different bands are not considered, which blurs the spectral difference characteristics of different bands due to different dimensions, therefore making the actual characteristics difficult to see. The logarithmic spectrum makes the spectral characteristics of different spectra more prominent by reducing the dimensional differences between different bands, thus improving the estimation accuracy of the model. In the estimation model, BES-ELM based on the estimation model accuracy was significantly better than ELM based on the estimation model. This may be because the BES algorithm has the advantages of global solid search ability and fast convergence speed compared with traditional optimization algorithms. In this study, the RMSE of the test set was used as the fitness function of the BES algorithm. By optimizing the initial parameters of the hidden layer of ELM, the convergence speed and prediction accuracy of the algorithm were improved. The inversion effect of BES-ELM was then optimized.2 of 0.735 and 0.545, respectively, which indicated that the model was able to estimate the NNI of rice better for rice with less N fertilizer application, while the model was less able to estimate the NNI of rice with sufficient or excessive N fertilizer application. Considering that the current N nutrition diagnostic method is mainly used for precise fertilization operations for rice in N deficit state rather than N rich state, this result has less impact on actual field N fertilization management. From the research point of view, a more detailed study of rice spectra in N deficit and N enriched states could be considered in the follow-up study to investigate the response principles with canopy spectra.At the same time, considering that based on the nitrogen dilution effect, the developmental status of rice canopy in the nitrogen-rich state does not change significantly with the elevated nitrogen nutrient status, these phenomena may lead to the reduction of correlation between spectral features and NNI and enhance the difficulty of NNI estimation, which does not occur in the rice canopy spectra in the nitrogen-deficient state. To test this conjecture and explore the analysis of model predictive ability in different NNI intervals, this study divided the data into the N deficit group (NNI< 1) and the N enrichment group (NNI ≥ 1) based on the NNI of the training and test sets, and analyzed the estimation ability of the best estimation results within the 2 groups. The results showed that the estimation ability of the N deficiency group was significantly better than that of the N enrichment group, with the R4.3Several aspects of the present study can be further optimized. The first is the selection of the standard spectra. Due to the complex relationship between rice growth and development and canopy spectra, even crops with the same NNI may exhibit differences in spectra, which may also cause some interference in the selection of the standard spectra. Second, this study took the sample set size and the stability of the machine learning algorithm into account, and chose to mix the fertility samples together for model construction. Although this process satisfies the sample set requirements for subsequent modeling, the differences in canopy structure between rice at different fertility stages can cause some interference in the estimation of NNI by the model. These uncertainties also directly or indirectly reduce the accuracy and stability of the model results. Future research should combine the relationship between NNI and rice canopy structure with the existing research methods to further evaluate the principle of log-difference spectroscopy to improve the estimation results of the NNI and explore the influence of different fertility periods on the log-difference spectra of rice. A more generalized spectral transformation method should be sought to enhance the practical value of model N nutrition diagnosis.52 values of 0.839 and 0.837 and RMSEs of 0.075 and 0.073 for the training and validation sets, respectively. This study provides a new feasible approach for estimating the NNI spectra of rice.In this study, rice canopy spectra were obtained by unmanned aerial remote sensing technology to estimate the NNI of rice. We determined the NNI by constructing the critical nitrogen concentration curve of rice, and performed log, difference, and log-difference transformations on the canopy spectra based on the critical nitrogen concentration spectra, and then used ELM and BES-ELM algorithms for modeling after-feature extraction by a self-encoder. Comparison of the final results indicated that the estimation model based on log-difference spectra and BES-ELM algorithm worked best, with RThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.YF, BJ, and TX designed the experiment and performed the final critical review. YF, BJ, ZJ, HZ, and YJ performed experiments and data analysis. YF, BJ, and HZ wrote the manuscript. YF, BJ, and TX provided critical review. All authors contributed to the article and approved the submitted version.


This research studies the regularities of changes, depending on the synthesis regimes used, in the chemical composition of ion-implanted SiO2/Si and Al2O3/Si samples. It has been shown that the formation of Ga-O chemical bonds occurs even in the absence of thermal annealing. We also found the conditions of ion irradiation and annealing at which the content of oxidized gallium in the stochiometric state of Ga2O3 exceeds 90%. For this structure, the formation of Ga2O3 nanocrystalline inclusions was confirmed by transmission electron microscopy.The ion-beam synthesis of Ga However, the creation of these devices on the basis of silicon is difficult or fundamentally impossible [Current microelectronics technologies that use silicon as the basic material can no longer meet the ever-increasing requirements for electronic devices. Current trends in the development of electronics suggest the use of wide bandgap semiconductors, such as SiC, GaN, and especially Gapossible .2O3) occupies a leading position in terms of promising applications among wide bandgap semiconductors for a number of vital fields: power electronics, the detection of hard ultraviolet radiation and hazardous gases, high-power computing, nuclear and space industries, medicine, ecology, etc. [2O3 has an ultrawide bandgap (~4.5–5.3 eV depending on the polytype) and high breakdown voltage associated with it. It is also radiation and chemically resistant, among other important features. There are five well-known polytypes of gallium oxide: α -, β (monoclinic)-, ε(κ) (orthorhombic)-, δ (cubic)-, and γ (defective spinel)-phase Ga2O3, the formation of which strongly depend on the synthesis conditions [Gallium oxide GaO3 occupinditions . Each ofnditions ,8,9,10. 2O3 is experiencing a period of rapid progress [2O3 synthesis and modification methods with traditional microelectronic technologies. Despite the existence of single-crystal substrate growth technologies (including large diameters), the abovementioned methods remain quite expensive, and thin-film growth technologies  have a large number of unsolved problems that limit their practical application [The research and development of Gaprogress . However2O3-based materials can be the use of nanostructured materials; in particular, the creation of nanosized gallium oxide inclusions of various polymorphic modifications [2O3 inclusions could considerably expand the application of this material, particularly for heterogeneous catalysis problems [2O3 nanostructures requires the application of various chemical methods , which 2O3 nanocrystals (nc-Ga2O3) in solid matrices. Ion implantation—a foundation of this method—has been one of the main operations of CMOS technology for many years, and it has already proved its effectiveness for materials based on gallium oxide [2O3 [2O3 nanocrystals achieved by using this method has not been previously reported, except for our recent works [One of the promising methods for creating structures with gallium oxide nanocrystals is the method of ion synthesizing Gaum oxide . The ionum oxide , SiC [17um oxide , GaN [19ide [2O3 . Howevernt works ,22.2O3 nanocrystals in dielectric matrices, showed the promise of using this method to create “solar-blind” photodetectors [2O3 nanocrystalline inclusions, as well as the influence of the post-implantation annealing atmosphere—have not been studied yet.Previously, we demonstrated the possibility of ion-beam syntheses of Gaetectors , and stuetectors . However2O3 nanocrystalline inclusions in the dielectric matrices of SiO2 and Al2O3 on silicon are considered. The article also features data on the change in the structural and chemical composition, depending on the conditions of post-implantation thermal treatment, of the synthesized samples.In this paper, the regularities of the physical and chemical processes occurring during the ion synthesis of Ga2 and Al2O3 films were deposited by electron-beam deposition. The thickness of the films was ~350 nm and ~200 nm, respectively. Ion-beam synthesis includes two consecutive stages: irradiation with ions of phase-forming elements and post-implantation annealing (16 cm−2) and O2 ions  were implanted with variation in the irradiation order. The implantation of only gallium ions was also used since it was expected that oxygen from the oxide matrix would be involved in the creation of the Ga-O chemical bonds, which in turn form Ga2O3 nanocrystalline inclusions. The implantation of molecular oxygen ions instead of atomic ones was used to reduce the total irradiation time. When colliding with the sample surface, the molecular O2+ ions decomposed to form two ions with half the energy. The irradiation regimes were selected based on the condition of maximum coincidence of the ion distribution profiles, as calculated using SRIM-2013 software . In the calculations, the density of SiO2 and Al2O3 was taken as 2.32 and 3.98 g/cm3, respectively. The displacement energies were 15, 25, and 28 eV for Si, Al, and O atoms, respectively. The maximum of the implanted atoms (Rp) was at a depth of ~60 nm for SiO2/Si structures and ~35 nm for Al2O3/Si structures (2 and O2 atmospheres.Silicon n-Si (100) wafers were used as the initial samples, on which SiOnnealing . In the ructures . At the s, C 1s, Ga 23/p2, and Si 2s photoelectronic lines were registered. The acquisition area was ~3 mm2. Spectra were taken at the transmittance energy of the analyzer of 50 eV and an energy discretization of 0.2 eV/step. Ar+ ion etching with an energy of 1 keV and an etch region diameter of 20 mm was used to obtain chemical element depth distribution profiles. The angle of incidence of the ion beam to the sample surface was 45°. The experimental procedure and data analysis are described in detail in [The composition and chemical state of the initial samples were investigated by X-ray photoelectron spectroscopy (XPS), and implemented on the basis of an Omicron MultiprobeRM  ultrahigh-vacuum complex. O 1etail in .The structure of the samples was investigated by a transmission electron microscopy (TEM) of the cross-section on a JEM-2100F  microscope using a 601.07000 TEM Specimen Preparation Kit  for preparation, which was conducted according to the Gatan method (USA).2/Si samples that were not subjected to post-implantation annealing were investigated. It was established that, even in the absence of annealing, all samples contain not only gallium in the elemental state, but also oxidized gallium in the stoichiometric Ga2O3 and the oxygen-deficient Ga2O states. The distribution profiles of gallium , where the energy density released in elastic collisions is the highest, hence the local temperature is higher; on the other hand, the Ga located closer to the surface is partially preserved in the elemental state and forms the near-surface maximum observed in + before Ga+ implantation, the degree of Ga oxidation becomes even higher. Moreover, since the unbound gallium can undergo radiation-stimulated desorption, the pre-implantation of O+ weakens it due to a more complete binding of gallium to oxygen in the SiO2 matrix. Oxygen ions, being lighter, create less dense cascades compared to Ga+ and, for them, the effect of “thermal spikes” is expressed to a much lesser extent; therefore, O+ implantation after Ga+ implantation does not contribute to the oxidation of Ga, and it even leads to an additional radiation-accelerated desorption .This can be explained as follows. It is known that ion implantation is characterized by the appearance of so-called “thermal spikes” during the introduction of ions . This ph2O3/Si samples, the situation is different  was in the oxidized state with different degrees of oxidation. This is additional evidence that, in the absence of annealing, the participation of oxygen from the matrix for oxidation is hindered. This process also requires the matrix, into which gallium atoms are implanted, to be initially oversaturated with oxygen.In the case of oxygen implantation prior to gallium, two maxima appear on the depth distribution profiles of gallium: the first one mainly consists of gallium in the elemental state, while the second one is due to the presence of oxidized gallium in the Ga program . In the 2O3/Si matrix, which is associated with a significantly greater impurity depth compared to the calculated one, shows that this effect is observed for all variants of implantation. However, it has its peculiarities in each case. In the case when only gallium atoms are implanted, the shift of the maximum is ~10 nm. Upon implantation of Ga+ → O+, this shift increases. In the case of oxygen implantation before gallium ions, the first maximum approximately coincides in position with the previous ones, and the second maximum relates mainly to gallium bound with oxygen, which is located at much greater depths. The nature of this phenomenon is associated with the effect of radiation-accelerated diffusion. The fact that the increase in the depth of gallium distribution is more pronounced in the case of additional oxygen implantation can be explained as follows. If it is assumed that gallium diffuses by the interstitial mechanism, then the presence of excess oxygen vacancies in the irradiated film leads to their capture of diffusing gallium atoms, which slows down diffusion. In the case of preliminary irradiation with oxygen ions, some of the oxygen vacancies are filled; as a result, the diffusion flux of gallium atoms increases. The preferential capture of gallium atoms by oxygen vacancies in the absence of preliminary irradiation with oxygen ions is also confirmed by the low concentration of Ga-O bonds , but also by the possible diffusion of oxygen atoms from this region. This assumption is supported by the following facts. First, according to the results obtained for the case of implantation of Ga ions only, it can be noted that there is a limited number of oxygen atoms from the matrix that can participate in the oxidation process. Second, it can be expected that the oxidation of gallium in the elemental state as a result of annealing can be determined not only by the processes of chemical interaction at elevated temperatures, but also by the influx of oxygen atoms into the near-surface region from the depth.Finally, for the O2O3 was observed for this variant of ion synthesis, an attempt to increase it was made by annealing the samples in an oxygen atmosphere, while all other annealing conditions (temperature and duration) remained the same. The study of the chemical composition revealed an interesting effect—namely, the almost complete oxidation of implanted gallium to the stoichiometric Ga2O3 state  were formed after irradiation. These bonds serve as the centers for the nucleation of a new phase. During annealing, the process of nanocrystal formation goes through several stages. First, small non-phase inclusions containing Ga and O atoms are formed as nuclei, which can later be enlarged due to the addition of new atoms. With an increase in annealing temperature, the formation of nanoinclusions in the metastable γ-Ga2O3 phase that contain a large number of structural defects is possible. A further increase in annealing temperature and time leads to the preferential transition of nanoinclusions to the stable β-phase. However, in the annealing conditions used in this work (oxygen-containing atmosphere), this transition did not complete; therefore, the presence of the nanocrystals of both phases was observed in the samples. Comparing these data with the previously obtained results for the case of step-by-step annealing in a nitrogen atmosphere [2O3 under various heat treatment conditions requires separate investigations.The high-resolution image obtained in the region of darker contrast b shows tas found . One of as found , during mosphere , it can 2/Si and Al2O3/Si samples during the ion synthesis of Ga2O3 nanocrystalline inclusions were studied depending on the conditions of irradiation with Ga and O ions and the subsequent heat treatment. It was shown that the formation of Ga-O bonds occurs even in the absence of post-implantation annealing, and their formation significantly depends on the order of ion irradiation. For the Al2O3/Si matrix, the loss of implanted gallium was observed as early as at the irradiation stage, and subsequent heat treatment led to a further decrease in the Ga concentration. Therefore, the implantation and annealing conditions used made the ion synthesis of Ga2O3 nanocrystals in the Al2O3/Si matrix very difficult.The regularities of changes in the chemical composition of the SiO2/Si samples leads to a decrease in the fraction of gallium in the elemental state, with a simultaneous increase in the concentration of oxidized gallium in the oxygen-deficient states (Ga2O) and in the stoichiometric state (Ga2O3). The use of oxygen instead of nitrogen in the annealing atmosphere leads to an increase in the content of gallium in the stoichiometric state of Ga2O3 above 90%. The study of the structure of the sample obtained, by transmission electron microscopy, under such synthesis conditions revealed the formation of the nanocrystalline inclusions of γ- and β-Ga2O3, with an average size of 4–9 nm.Annealing implanted SiO2O3 nanocrystalline inclusions by varying the parameters of synthesis. This feature of the ion synthesis method makes it possible to create structures with repeatable and controllable properties using ion implantation and thermal annealing processes that are fully compatible with traditional electronic technology. Further development of the technology for creating gallium oxide nanocrystals using the method of ion synthesis in silicon-compatible dielectric matrices opens up prospects for the wide application of this technique in the fabrication of a new generation of electronic devices based on gallium oxide.Thus, this study demonstrates the possibility of controlling the chemical composition and properties of the ion-synthesized Ga


The Schlafen5(SLFN5)gene belongs to the third group of the Schlafen protein family. As a tumor suppressor gene, SLFN5 plays a pivotal role in inhibiting tumor growth, orchestrating cell cycle regulation, and modulating the extent of cancer cell infiltration and metastasis in various malignancies. However, the high expression of SLFN 5 in some tumors was positively correlated with lymph node metastasis, tumor stage, and tumor grade. This article endeavors to elucidate the reciprocal relationship between the SLFN5 gene and malignant tumors, thereby enhancing our comprehension of the intricate mechanisms underlying the SLFN5 gene and its implications for the progression, invasive potential, and metastatic behavior of malignant tumors. At the same time, this paper summarizes the basis of SLFN 5 as a new biomarker of tumor diagnosis and prognosis, and provides new ideas for the target treatment of tumor. Thr4.2Currently, there is limited research on the role of SLFN5 in gastric cancer (GC) . Xu et a4.3Fischietti et al.  inferred4.4in vivo growth of GBM cells. SLFN5 can be induced by type I interferon (IFN) in malignant brain tumor cells and acts as an inhibitor of gene transcription driven by STAT1 through direct protein interactions  is highly sensitive to interferon treatment . Sassano4.6SLFN5 represents a promising biomarker for early-stage non-small cell lung cancer (NSCLC) patients . The dow4.7Due to the dependency of SLFN5 protein on I-type interferon (IFN) , it is w4.8Mark et al. , employi4.9in vitro and in vivo  is a crucial step in the metastatic process, facilitating the detachment of tumor cells from the primary site and their adhesion to secondary sites . Xu et ain vivo . Howeverin vivo , 64. Conin vivo , 66.5In conclusion, the research on the regulatory mechanism of SLFN5 expression has achieved good results in recent years, such as the breakthrough in the study of the pathway through which SLFN5 affects tumor development, the proposed inhibition of MT3-MMP expression by SLFN5 through the AKT/GSK-3β/β-catenin pathway to inhibit the migration and invasion of cancer cells, and SLFN 5 regulation of LAT 1-mediated mTOR activation in depot resistant prostate cancer regulates LAT 1-mediated mTOR activation, among other findings. More convincing studies have been carried out at affecting reversible epithelial and epithelial-mesenchymal transition; and research ideas have been clarified in studying the effects of SLFN 5 on different tumors. However, it cannot be ignored that there are still a large number of problems to be solved in the study of SLFN5, such as the lack of research on the regulatory mechanism and effect of SLFN5 in gastric cancer and glioblastoma, and the lack of research on the effect of SLFN5 as a biomarker of chemotherapy and a potential target of antitumor drugs in the clinic, on the one hand, it is because the effect of SLFN5 is not universal, and has not the same effect on different types/subtypes of cancers, on the other hand, it is not universal. On the one hand, SLFN5 is not universal, and has different mechanisms of action for different types/subtypes of cancers and different effects on cancers, and part of the mechanism of SLFN5 has not yet been clarified and is in the early stage of research; on the other hand, there is no clear research that can show whether SLFN5 is easy to mutate and whether it is stable when put into the production of drugs.TT wrote the manuscript. YYu and YYa designed and supervised the study. XL provided critical suggestions for the manuscript and revised the revised manuscript. XXL revised the manuscript. XY revised the manuscript and supervised the study. All authors contributed to the article and approved the submitted version.


Sperm cryopreservation is an important part of maintaining the genetic diversity of chicken breeds. However; a significant percentage of spermatozoa lose their viability and motility when frozen/thawed. Cell membranes are the most vulnerable in this process; and their cryoresistance largely depends on their lipid composition. Based on the study of the lipid composition of the plasma membranes of the spermatozoa of roosters of two breeds; a cryoprotective diluent was developed containing biocryoprotective trehalose with a concentration of 9.5 mM; which allows the optimal ratio of cell lipids and the kinetic ability of frozen/thawed spermatozoa (motility) to be maintained at a high level—52.4%.The plasma membrane of spermatozoa plays an important role in the formation and maintenance of many functions of spermatozoa, including during cryopreservation. As a result of chromatographic analysis, the content of lipids and fatty acids in the membranes of spermatozoa of roosters of two breeds was determined under the influence of cryoprotective media containing trehalose LCM-control (0 mM), Treh20 (9.5 mM), and Treh30 (13.4 mM). The use of the cryoprotective diluent Treh20 made it possible to maintain a dynamic balance between the synthesis and degradation of phospholipids and sterols in the plasma membranes of frozen/thawed spermatozoa, close to that of native spermatozoa. This contributed to an increase in the preservation of frozen/thawed spermatozoa membranes from 48.3% to 52.2% in the egg breed and from 30.0% to 35.1% in the meat- and-egg breed. It was also noted that their kinetic apparatus (mobility indicators) remained at the level of 45.6% (egg breed) and 52.4% (meat-and-egg breed). An increase in the concentration of trehalose to 13.4 mM in a cryoprotective diluent for rooster sperm resulted in a decrease in the morphofunctional parameters of frozen/thawed spermatozoa. The plasma membrane plays an important role in the formation and maintenance of many functions of spermatozoa. There are three main components of the plasma membrane: phospholipids, sterols, and glycolipids. Many studies have determined the special role of phospholipids containing a significant amount of polyunsaturated fatty acids (PUFAs), which provide the necessary degree of membrane fluidity, but at the same time are most susceptible to the harmful effects of reactive oxygen species and peroxidation .When developing new protocols for cryopreservation of avian reproductive cells, it is important to specify the deep mechanisms of formation of the adaptive capabilities of the cell to new conditions. One of the mechanisms of such adaptation may be the modulation of the plasma membrane of the cell that, since it is the plasma membrane, as a cell organelle maintaining the integrity of the cell, is the first to be exposed to external factors. The use of diluents for semen cryopreservation makes it possible to prevent structural changes in the lipid and protein molecules of the plasma membrane of spermatozoa during freezing/thawing of sperm, which is of key importance for the functioning and metabolism of cells.The decrease in the functionality of spermatozoa in the cryopreservation protocol has been noted by numerous studies. In today’s reproduction technology, there are widely presented methods for determining the decline in the functionality of spermatozoa in terms of their motility, viability, membrane integrity, acrosome integrity, mitochondrial activity, apoptosis status, lipid peroxidation, and fertility .It is known that 70% of the plasma membrane of spermatozoa is composed of lipids and sterols . The matLipids are important structural components of the cell plasma membrane and are also a source of energy, being oxidized in mitochondria and peroxisomes . MembranThe lipids of the inner and outer layers of the plasma membrane differ in composition and perform different functions. Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are characteristic of the inner layer of the membrane and together account for up to 37% of the total composition of phospholipids, with phosphatidylserine present in a smaller amount . PE is tThe outer layer of the plasma membrane of the cell is characterized by a significant amount of phospholipids represented by phosphatidylcholines (PC) and sphingomyelins (SM). Phosphatidylcholines make up 40–50% of the total amount of membrane phospholipids. In this case, the ratio of phospholipids may vary depending on the type of cell or its individual organelles .It is known that trehalose disaccharide is able to protect lipids and proteins of cell plasma membranes from oxidative stress caused by exposure to reactive oxygen species and prevent the oxidation of unsaturated fatty acids. Trehalose protects biological molecules by triggering three mechanisms: replacing water in the cell cytosol, minimizing the formation of ice crystals, and ensuring the chemical stability of biomolecules. Together, these mechanisms make trehalose a highly effective biocomponent of cryoprotective media that ensure the morphological integrity of spermatozoa and their functionality at ultralow temperatures ,13The aim of our study was to determine the composition of lipids in the plasma membranes of rooster spermatozoa (egg and meat-and-egg breeds) and its changes during cryopreservation of male reproductive cells using trehalose disaccharide as a component of cryoprotective media.The experiment used roosters—Tsarskoselskaya (TS) (meat-and-egg breed) and Russian White (RW) (egg breed)—kept in individual cages according to the technology of keeping and feeding adopted in the Centre of Collective Usage “Genetic collection of rare and endangered chicken breeds” .n = 10 for each breed) aged 52–56 weeks. Each ejaculate was evaluated individually and selected according to the following criteria: volume using a graduated pipette; spermatozoa concentration using a photometer ; total and progressive sperm motility using computer-assisted sperm analysis (CASA)  and software . The assessment was carried out in triplicate. The resulting semen from each breed was divided into 4 aliquots. The first aliquot of the native semen was left unchanged, stored at the temperature 5 °C. The other three aliquots were diluted with a separate dilution medium in a 1:1 ratio with a control medium LCM-control [Semen was collected from TS and RW breed roosters (-control  and specThe viability of spermatozoa (dilution 1:20) was studied on the basis of histological smears stained with nigrosin-eosin (3% aqueous solution of eosin and 10% nigrosin). The viability of spermatozoa (dilution 1:20) was studied on the basis of histological smears stained with nigrosin-eosin (3% aqueous solution of eosin and 10% nigrosin). Cells were assessed at 1000-fold magnification  by the presence of color. Pink cells were considered dead; unstained (white) cells were considered alive. The results were expressed as percentages of individual categories of spermatozoa . The assessment was carried out in triplicate.To prepare samples for assessing the lipid composition of spermatozoa, semen was collected from 10 males of each breed. Each aliquot  was processed according to the protocol: Centrifuged for 10 min at 3000 rpm; the supernatant was removed, 0.9% sodium chloride solution was added, thoroughly mixed and centrifuged again under the same parameters. The procedure was repeated three times. At the next stage, the centrifuged sperm were collected in small portions on a nylon filter and precipitated for another 30 min. The prepared samples of centrifuges and supernatants were frozen and stored at −25 °C.A portion of raw biomass (700–1000 mg) was homogenized in isopropanol, after which lipid extraction was carried out for 30 min at 70 °C and the supernatant liquid was decanted . The res4OH (65:30:10:6) in the first direction (Vaskovsky system with modification [Separation of polar lipids was performed using 2D TLC on Silica gel 60 glass plates (Merck) in the following systems: chloroform: methanol: toluene: 28% NHfication ) and chlfication ). A sampfication . A samplfication   was used as the standard for determining the amount of phospholipids, a mixture of glycoceramides  was used for sphingolipids, and ergosterol  was used for sterols. Quantitative lipid analysis was performed by densitometry using the Dens computer program  in the linear approximation mode using calibration curves based on standard solutions.The fatty acid composition was determined according to the following protocol. The polar fraction of lipids was isolated by one-dimensional TLC. The polar spots of lipids were scraped off, eluted with a mixture of chloroform:methanol (1:1), and the extract was evaporated. Fatty acid methyl esters were obtained by keeping lipids in a 2.5% solution of sulfuric acid in methanol at 70 °C for 2 h .Determination of the composition of lipid fatty acids was carried out on a Kristall 5000.1 ZAO gas-liquid chromatograph  on an Optima −240, 60 m, 0.25 µm, 0.25 mm capillary column . For chromatography, a temperature program from 130 to 240 °C was used. Identification was carried out using Supelco 37 Component FAME Mix .The degree of unsaturation of phospholipids (DU) was determined by the formula :DU = 1.0p < 0.05.For statistical data processing, the software applications Excel 2013  and Statistica 7.0  were used. Data are presented as mean values standard error (SE) and were considered significant at As a result of chromatographic analysis of the lipids composition in the spermatozoa plasma membranes, interbreed differences were established in the ratio of membrane lipids of native and frozen/thawed spermatozoa . In bothThe ratio of phospholipids/sterol in spermatozoa membranes, which reflects membrane fluidity, was best preserved in frozen/thawed semen using Treh20 cryoprotective diluent compared to native semen .The number of phospholipids of the inner (PE + PS) and outer (PC + SM) layers of the plasma membranes of native spermatozoa in both breeds differ little—6.2% and 6.6% (TS) and 7.6% and 7.6% (RW), respectively a,b. It sThe use of the cryoprotective diluent Treh20 made it possible to maintain the dynamic balance between the phospholipids of the inner and outer membranes of frozen/thawed spermatozoa (TS) at the level of 7.2% and 7.5%, respectively. When using the LCM-control medium, an imbalance of phospholipids of the inner and outer lipid layers of frozen/thawed spermatozoa membranes was noted. The ratio of phospholipids of the outer and inner layers of the plasma membrane of spermatozoa at an increased concentration of trehalose (Treh30) compared with a lower concentration (Treh20) shifted towards the phospholipids of the outer layer. In the Russian White breed, an increase in the content of polar phospholipids of the inner and outer layers of the plasma membrane of spermatozoa was also observed when using the cryoprotective diluent Treh20—8.0% and 10.1%, respectively, compared with native semen.A significant variety of fatty acids was determined in the phospholipids of spermatozoa membranes (native and frozen/thawed)  in both A total of 15 fatty acids have been identified in the lipid composition of the plasma membranes of spermatozoa. In native rooster semen, the predominant fatty acids are the saturated fatty acids palmitic acid (C16:0) and stearic acid (C18:0). The main unsaturated fatty acids in plasma membrane lipids of rooster spermatozoa are docosatetraenoic acid (C22:4), oleic acid (C18:1n9c), and arachidonic acid (C20:4n6) .The use of the cryoprotective diluent Treh20 increased the content of polyunsaturated fatty acids in the membrane lipids of the plasma membranes of spermatozoa of both breeds after freezing/thawing. In the Tsarskoselskaya breed, this value reached 46.5% in native semen and 50.1% in frozen/thawed semen. A similar trend was observed in the Russian White breed in the content of polyunsaturated fatty acids in the lipids of the plasma membranes of spermatozoa during freezing/thawing, and the content of polyunsaturated fatty acids was higher than in the Tsarskoselskaya breed.In frozen/thawed spermatozoa, the best degree of unsaturation of fatty acids of plasma membrane lipids was determined using the cryoprotective diluent Treh20 in both breeds—1.71 (TS) and 1.81 (RW) .p < 0.05) higher rates of overall motility, progressive motility, and viability of spermatozoa compared to alternative cryoprotective diluents , lipid peroxidation, a decrease in the level of ATP, etc. ROS-induced damage to spermatozoa in the freeze/thaw protocol is mediated by oxidative ROS attack on polyunsaturated fatty acids that are part of cell membrane phospholipids. This leads to lipid peroxidation and loss of sperm motility and viability .The lipid composition of spermatozoa membranes is quite stable, but under the influence of temperature and the composition of the cryoprotective diluent, the ratio of phospho- and glycolipids, as well as sterol, changes. Sperm plasma membranes of native semen contain a large amount of major phospholipids inherent in the internal layer (phosphatidylethanolamine—22.7% (TS) and 23.9% (RW); phosphatidylserine—18.6% (TS) and 17.6% (RW)) and the external layer (phosphatidylcholine—31.4% (TS) and 37.1% (RW); sphingomyelin—12.9% (TS) and 10.9% (RW)). In addition, one of the major elements of the lipid layer of the rooster spermatozoa plasma membrane for both breeds is sterol, which in our study amounted to 9.7% (TS) and 6.9 % (RW) of the total lipid fraction for native spermatozoa. It is known that cholesterol performs many functions in the biological cycle of the cell: it stabilizes membranes, reduces their permeability, contributes to the stabilization of morphological characteristics and affects the phase transition of the lipid matrix of the membrane at low temperatures, and provides a suitable microenvironment  for membrane proteins . In expeTo ensure the integrity of spermatozoa membranes during cryopreservation, it is necessary to maintain the optimal ratio of phospholipids/sterol. The best cryoprotective effect was achieved when using the Treh20 diluent , which ensured the ratio of phospholipids/sterol in frozen/thawed spermatozoa in both breeds at the level of native semen indicators of 8.6/9.1 (TS) and 12.7/12.6 (RW).It is known that the level of sphingomyelin, as well as phosphatidylcholine and phosphatidylethanolamine in the plasma membranes of spermatozoa is associated with the percentage of motile spermatozoa and can serve as an indicator of spermatozoa motility and fertility ,30. The The refined composition of minor lipids in spermatozoa membranes  was not subject to significant changes either under the influence of the composition of the cryoprotective diluent or under the influence of low-temperature stress.An analysis of changes in the lipid ratio was carried out taking into account the proven differentiation of phospholipids in the outer and inner layers of membranes ,32. The The study of the morphological and functional features of the frozen/thawed spermatozoa confirms the positive effect of the combination of mono- and disaccharides in the cryoprotective diluent Treh20 on the indicators of total motility 52.4% (TS)/45.6% (RW) and progressive spermatozoa motility 32.6% (TS)/ 32.4% (RW), respectively, compared with LCM-control total motility 44.5% (TS)/42.4% (RW) and progressive motility 28.3% (TS)/30.0% (RW).Trehalose, as an antioxidant additive in the Treh20 diluent at a dose of 9.5 mM, had a positive effect on the morphofunctional quality of frozen/thawed spermatozoa, but an increase in the dose to 13.4 mM (Treh30) significantly reduced these parameters compared to Treh20 and LCM-control, which is consistent with the research results of J.-H. Hu et al., 2010 . AccordiThe data obtained agree positively with the results of studies of the effect of sugars under cold stress on the liquid crystalline lamellar phase of cell membrane phospholipids  and lipiIn our study on two breeds of roosters (egg and meat-and-egg breeds), a dose-dependent effect of trehalose disaccharide, as part of a cryoprotective medium for freezing semen, on the lipid composition of the plasma membrane of spermatozoa, its integrity, and motility of spermatozoa, was revealed. It was found that the optimal concentration of trehalose in the cryoprotective diluent is 9.5 mM (Treh20). The use of the cryoprotective diluent Treh20 made it possible to maintain a dynamic balance between the synthesis and degradation of phospholipids and sterol in the plasma membranes of frozen/thawed spermatozoa, which is close to that of native spermatozoa.Changes in the composition of cell membrane lipids under the influence of low-temperature stress are a universal natural mechanism for many organisms ,38. The Apparently, the use of the cryoprotective diluent Treh20 contributed to the maximum preservation of the membrane fluidity of frozen/thawed spermatozoa due to the balance between the amount of phospholipids and sterols and the high content of polyunsaturated fatty acids. This contributed to the preservation of the kinetic apparatus of spermatozoa (motility indicators) at the level of 45.6% (egg breed) and 52.4% (meat-and-egg breed). An increase in the concentration of trehalose to 13.4 mM in a cryoprotective diluent for rooster sperm resulted in a decrease in the morphofunctional parameters of frozen/thawed spermatozoa.