In this research, we identified 162 bHLH proteins (HhbHLHs) from the genomic and transcriptomic datasets of H. hamabo, which were phylogenetically divided into 19 subfamilies. According to a gene structural study, the number of exon-introns in HhbHLHs varied between zero and seventeen. MEME research revealed that the majority of HhbHLH proteins contained three conserved motifs, 1, 4, and 5. The examination of promoter cis-elements revealed that the majority of HhbHLH genes had several cis-elements involved in plant growth and development and abiotic stress responses. In addition, the overexpression of HhbHLH2 increased salt and drought stress tolerance in Arabidopsis.The basic helix-loop-helix (bHLH) family of transcription factors is one of the most significant and biggest in plants. It is involved in the regulation of both growth and development, as well as stress response. Numerous members of the bHLH family have been found and characterized in woody plants in recent years. However, no systematic study of the  Arabidopsis thaliana around 100 TFs have been characterized, and these are crucial for the plant [bHLH genes are involved in metabolic regulation, plant growth, and production, and responsiveness to environmental signals in plants. The maize R gene, which is required for anthocyanin production, was the first member of the bHLH family to be identified [bHLH genes have been found in various plants. A growing amount of evidence suggests that bHLH TFs are important for engagement in plant defense responses, particularly in response to abiotic stress responses such as drought, low temperature, high temperature, and salt stress [MfbHLH38 gene from Myrothamnus flabellifolia to Arabidopsis greatly improved the resistance to drought and salt stresses in transgenic plants [TabHLH49 gene controls the expression of the dehydrin WZY2 gene, improving drought tolerance [bHLH gene ZjICE2 in Zoysia japonica rice resulted in increased chlorophyll content and photosynthesis ability, as well as high cold tolerance [bHLH gene PebHLH35 in Populus euphratica was shown to act as a positive regulator of the drought stress response [Transcription factors (TFs) are protein molecules with distinct structural and functional characteristics that influence gene expression. In the model plant, he plant . It is che plant ,4,5. An entified . Since tt stress ,8. For ec plants . Wheat Tolerance . By contresponse .bHLH gene family has been identified in several organisms so far, and has been separated into 15–26 classes in plants based on sequence similarity [Arabidopsis thaliana, for example, 162 bHLH genes were discovered and divided into 21 subfamilies [bHLH genes, divided into 21 subfamilies, were identified in spine grapes [bHLH genes were discovered, which were split into 15 subfamilies [bHLH genes were discovered and split into 21 subfamilies [bHLH gene family has been conducted in Hibiscus hamabo Sieb. et Zucc.The milarity . In Arabfamilies ; 115 bHLfamilies . HoweverH. hamabo is a shrub plant in the genus Hibiscus, family Malvaceae, and an important semi-mangrove plant [H. hamabo is also a good material for investigating the mechanism of salt tension in woody plants. Therefore, it is very important to research the H. hamabo bHLH gene family to understand the semi-mangrove plant biological processes in response to stress.ve plant . It is cve plant ,20. At tbHLH genes that may be involved in the salt and drought stress biosynthesis pathways in H. hamabo. The study aimed to increase our awareness of the bHLH functions involved in the salt and drought stress responses of H. hamabo, while, our comprehensive research laid the groundwork for additional mechanisms of salt and drought tolerance for bHLH genes in H. hamabo, with particular emphasis on identifying potential genes implicated in H. hamabo salt and drought stress responses.In this study, bioinformatics was utilized to identify members of the bHLH family and to assess their relevant features using genomic sequence and full-length transcriptome datasets. Additionally, we screened for bHLH genes in the H. hamabo genome, we used a hidden Markov model (HMM) file (PF00010) to conduct a genome-wide HMM-search. There were 167 putative bHLH proteins discovered. The bHLH domains were confirmed by using Pfam online database, and redundant sequences were deleted. Eventually, we obtained 162 sequences from the H. hamabo bHLH family, which we named HhbHLH1–HhbHLH162 based on their chromosomal position. The 162 HhbHLHs gene protein sequences are shown in HhbHLHs genes, we examined their physicochemical properties. The 162 HhbHLHs proteins differ in Coding sequence (CDS) length, protein length, molecular weight, and theoretical isoelectric point (pI) (HhbHLH137) to 2169 (HhbHLH133), while their pIs were between 4.35 (HhbHlH67) and 10.03 (HhbHLH43). Furthermore, the 162 HhbHLH genes were spread at random across 46 chromosomes. The remaining 42 chromosomes, except chromosomes 28, 30, 35, and 46, had varying numbers of HhbHLH genes. Among them, chromosome 24 contained the largest number (10 HhbHLHs) (To find int (pI) . SpecifiHhbHLHs) .Arabidopsis bHLH proteins from each of the subgroups were chosen at random for further comparative studies. As shown in Arabidopsis.Multiple sequence alignment of bHLH domains was used to determine the phylogenetic relationship of HhbHLH proteins, which span across 40–50 amino acids. The bHLH domains of 19 different Arabidopsis and H. hamabo bHLH domains. The result revealed that the 162 HhbHLHs gene can be classified into 19 distinct clades. Among them, the VIIa+b subfamily had the most members (20 HhbHLHs), whereas Groups Ib, II, IVc, and X subfamily had the fewest members (one HhbHLH). In addition, the H. hamabo bHLH subfamily was extended or contracted to different degrees in comparison with Arabidopsis. The VIIa+b subfamily of Arabidopsis, for example, had 15 members, which grew to 20 in H. hamabo. In subfamilies Ia and Iva, a similar condition existed, while in the X subfamily there was a substantial reduction in the number of H. hamabo genes, from nine to one in Arabidopsis (We studied and drew the unrooted phylogenetic tree applying the neighbor-joining (NJ) method based on the similarities between bidopsis .The motif type and composition can determine the function of the protein, and an assessment of its preserved motifs can also be used to determine the evolutionary relationship between the HhbHLH proteins. MEME software was used in this analysis to explore the motif of 162 HhbHLH proteins, yielding a total of 20 motif structures. Other than motifs 1, 4, and 5, which are broadly dispersed bHLH domains, HhbHLH members belonging to the same classes typically share a similar motif composition. b, such aHhbHLH genes, as seen in H. hamabo. Exon numbers ranged from 1 to 17 in 162 HhbHLH genes. There were no introns in 15 of the genes. These 15 intron genes were divided into two subfamilies, with 13 belonging to the IIId+e subfamily and two to the VIIb subfamily. In addition, the results revealed that the HhbHLHs have not only a large number of exons but also have genetic structure diversity. Exon distribution patterns in the IVd and VIIIc subgroups, for example, are relatively conservative. The number of exons, their distribution, and the length of each exon is all very similar, whereas the exon distribution in the XI and XII subgroups is not. Overall, the phylogenetic study findings, along with the motif compositions and related gene structures of the bHLH members of the subfamily, could support the group classifications reliability.We analyzed the structure of all HhbHLH family, 162 HhbHLH genes were divided into three repetitive events, of which 150 (92.59%) genes were assigned to WGD/segmental events, and 11 (0.067%) genes belonged to the dispersed type. The results indicated that the HhbHLHs gene family expanded primarily as a result of WGD/segmental events. /segmental duplication, tandem duplication, and transposition, are the primary causes of gene family expansion and have a significant effect on the evolution of gene families ,23. Thro events. .HhbHLH genes, we used intragenomic synteny analysis to identify conserved chromosome blocks within H. hamabo. We mapped 162 HhbHLH genes on 46 chromosomes using annotation information of the genome-wide data. Except for chromosomes 28, 30, 35, and 46, the remaining 42 chromosomes had different numbers of HhbHLH gene distributions, of which chromosome 24 contained the most (10 HhbHLHs). Furthermore, there was no positive relationship between the number of genes on a chromosome and its length  and others occurred in a relatively recent period (Ks < 0) (HhbHLH genes was less than one in the HhbHLH gene family. This result suggests that HhbHLHs evolved primarily under purifying selection (To better understand the evolutionary process of the s length . We usedtionship . The Ks (Ks < 0) . The Ka/election .H. hamabo and Arabidopsis and Populus to get a better grasp of the phylogenetic mechanisms underlying the H. hamabo bHLH family. In total 60 HhbHLH genes showed a syntenic relationship with those in Arabidopsis, and 26 HhbHLH genes showed a syntenic relationship with those in Populus . Additionally, several bHLH collinear gene pairs identified between H. hamabo and Arabidopsis were linked to highly conserved syntenic blocks spanning over 100 genes. This phenomenon suggested that an evolutionary relationship may exist between the bHLH gene family of H. hamabo and that of Arabidopsis.We created comparative syntenic maps between  Populus . Some ofcis-acting elements in the upstream 2000 bp sequence of the HhbHLH gene were analyzed and, finally, a mass of potential cis-elements was obtained  and salt (400 mM NaCl) stress conditions in this study. Of the 162 HhbHLH genes, ten genes were not expressed in the transcriptome data, while the remaining genes were expressed (FPKM > 0)  . The expapparent a.HhbHLH genes at random to study their function further by quantitative real-time PCR (RT-qPCR). The expression trends of these genes under NaCl and PEG treatment for 0 h, 6 h, and 24 h were shown to be largely compatible with the transcriptome data, demonstrating the reliability of the transcriptome data. At the same time, three time points  were added to the analysis of the expression profiles of the genes under drought and salt stresses to further understand the changes of gene expression level in different treatment periods of stress. Compared with the transcriptome data, the expression trend of some genes was more abundant after the new time point was added. For example, HhbHLH51, HhbHLH78, HhbHLH82, and HhbHLH84 showed a significant up-regulation after 1 h of drought stress and were then significantly down-regulated. A similar expression was observed in HhbHLH57, HhbHLH78, HhbHLH136, and HhbHLH78 under salt stress. This phenomenon indicates that these genes may play different roles in different periods of stress. At the same time, some genes, such as HhbHLH20 and HhbHLH26, were significantly up-regulated during drought and salt stresses after 12 h, while there was no obvious differential expression in other periods. In addition, some genes have different expression trends under drought and salt stress. For example, HhbHLH8 is up-regulated after 1 h of drought stress, but down-regulated after 1 h of salt stress. HhbHLH20 is up-regulated after 12 h of drought stress and down-regulated after 12 h of salt stress. These results show that under different treatment times, some genes may respond to the two stresses through different regulatory ways. In conclusion, the results of RT-qPCR effectively supplement the transcriptome data and provide a reference for the future exploration of the function of the genes , and salicylic acid (SA) treatments. In general, some genes with higher expression levels under drought and salt stresses showed similar expression patterns under abiotic and hormonal stress. For example, HhbHLH2, HhbHLH8, and HhbHLH26 had similar expression patterns under the four stresses. Both had a significant trend in up-regulation. This result is similar to their expression under drought and salt stresses, indicating that these genes may play a very important role in the response of H. hamabo to stress. Some HhbHLH genes can be significantly induced or inhibited by a variety of treatments. For example, HhbHLH2 and HhbHLH82 have a significant response to low temperature, high temperature, ABA, and SA treatments. One treatment can also induce the expression of multiple HhbHLH genes at the same time. For example, cold treatment simultaneously induces the expression of HhbHLH2/20/26/29/57/84 and other genes. In addition, some genes had the opposite expression patterns under different treatments. For example, HhbHLH20 is significantly up-regulated under SA stress but exhibits a significant down-regulation under ABA stress. This phenomenon indicates that HhbHLH family genes can participate in multiple conditions, and their regulation is different under different stresses  via Agrobacterium-mediated transformation, and homozygous T3 generation plants  were obtained through screening. Under normal conditions, there was no obvious growth difference between wild-type and transgenic Arabidopsis  genes were assigned to WGD/segmental events, and 11 (0.067%) genes belonged to the dispersed type, which indicates that WGD/segmental events occur in H. hamabo. In addition, with synteny analysis, it was found that 60 and 26 HhbHLH genes have a syntenic relationship with Arabidopsis and poplar bHLH families, respectively. Some of the HhbHLH genes are connected to at least three syntenic gene pairs , such as HhbHLH11 and HhbHLH141, these genes may have played a key role in the bHLH gene family.Gene duplication events are very common in the evolution of plants, and they frequently play a critical role in the growth of gene families . The 162bHLH gene family plays a critical role in plants, especially in plant response to drought and salt stress. For example, overexpression of the MdSAT1 gene in apples increases the resistance of apple callus and transgenic Arabidopsis to drought and salt stresses [CsbHLH041 gene in cucumber enhances the tolerance of transgenic cucumber seedlings to salt and ABA [H. hamabo under drought and salt stresses and revealed the expression pattern of the HhbHLH genes under these stresses. Except for a few genes, the results indicated that the expression of the majority of HhbHLHs changed significantly. After that, 15 highly expressed genes under drought and salt stress were screened and further analyzed by RT-qPCR. The results showed that the gene expression was consistent with the transcriptome data at the same time, which proved the reliability of the transcriptome data. In addition, three time points  were added to the analysis of the expression profiles of these genes under drought and salt stress. These results effectively supplemented the transcriptome data and provided a basis for the exploration of functional genes in the future. After that, we analyzed the expression of these 15 genes under heat, cold, ABA, and SA treatment, and screened a HhbHLH2 gene with significant differences under various stresses for further study. Previous studies have found that the Arabidopsis IX subfamily AtbHLH122 gene can be expressed in large quantities under drought stress, and compared with wild plants, the plants with the AtbHLH122 gene overexpressed have stronger tolerance to drought stress [HhbhLh2 can improve the drought and salt stress tolerance of transgenic plants. In conclusion, the present study represents the first analysis of the bHLH gene family of the semi-mangrove plant H. hamabo, and the isolated identification of these transcription factors may help elucidate the molecular genetic basis of H. hamabo in response to drought and salt stress as well as reveal the importance of bHLH genes in response to abiotic stress.Substantial evidence proved that the stresses , and the and ABA . In thist stress . In thisH. hamabo, and isolation and identification of these transcription factors help clarify the molecular genetic basis of H. Hamabo response to drought and salt stress and reveal the importance of bHLH genes in response to abiotic stress. The expression analysis of HhbHLHs genes also lays a strong foundation for future studies on the regulatory functions of bHLH proteins during abiotic stress response in H. Hamabo and will be able to further help understand this gene family in other plants. In addition, the functional identification of the HhbHLH2 gene also provides a basis for further study on the regulatory network of H. Hamabo in response to drought and salt stress in the future. In conclusion, this is the first comprehensive and systematic analysis of bHLH transcription factors in the semi-mangrove plant bHLH gene in response to abiotic stress. This work may help to clarify the functions of bHLH family genes in protein interaction, signal pathway regulation, and defense response under different stress conditions, and provide basic resources for the future study of semi-mangrove plant tolerance to drought and salt stress. In conclusion, the results of this study reveal the importance of the http://pfam.sanger.ac.uk/, accessed on 16 March 2021) [bHLH gene in the H. hamabo genome database using default parameters and a cutoff value of 0.001 [HhbHLH is shown in http://web.expasy.org/protparam/, accessed on 16 March 2021).The Pfam protein family database was queried for the HMM file corresponding to the bHLH domain (PF00010) (ch 2021) . The HMMof 0.001 . Some gewww.psc.edu/biomed/genedoc, accessed on 16 March 2021) software was used to manually adjust the amino acid sequence in the bHLH domain. MEME SUITE  software was used to analyze the conserved motifs in the HhbHLH protein. The following parameters were used: any number of repetitions; the maximum number was twenty, and the optimal width was between six and one hundred residues [HhbHLH gene structure was determined through the program GSDS  [The default parameters of ClustalW  were used to align the protein sequence of the bHLH domain of the HhbHLH protein, and then GeneDoc (residues . The Hhbch 2021) . Tbtoolsch 2021) .AtbHLH in Arabidopsis and the comparison of the bHLH conserved domains of HhbHLH and AtbHLH proteins, the identified HhbHLHs were divided into different subgroups. The bHLH proteins of Arabidopsis were downloaded from the TAIR  A phylogenetic tree was constructed using the NJ method in MEGA 7.0 [According to the classification of MEGA 7.0 .HhbHLH gene’s chromosome location information. The same procedure was used to analyze the synteny among the HhbHLHs in PGDD . Primarily, local all-vs-all BLASTP searches were performed on identified HhbHLH genes. Following that, using the BLASTP result and gene location information as input files, MCScanX was used to determine syntenic gene pairs [HhbHLH family genes. We analyzed the Ka and Ks values using the KaKs Calculator 2.0 [HhbHLH genes. Tbtools  were used to visualize the results.The GFF file was used to extract the ne pairs . We usedator 2.0 . To estiH. hamabo seeds were collected from Nanjing’s Sun Yat-Sen Memorial Botanical Garden. The seeds were soaked in concentrated sulfuric acid for 10 min and then cultured in a greenhouse under aseptic conditions for 3 weeks . Then, the seedlings with the same taproot length were transferred to a 50 mL centrifuge tube and cultured in half-strength Murashige and Skoog (1/2 MS) nutrient solution at pH 5.8 for 1 week before treatment. The seedlings were renewed every 2 days during the growth period. For drought or salt stress treatments, the two groups of seedlings were placed in 1/2 MS solution containing either 15% PEG6000 or 400 mM NaCl. For hormone treatment, H. hamabo seedlings were treated with 200 μM SA and 400 μM ABA. Plants were exposed to 4 °C and 42 °C in an incubator with the same photoperiod, respectively, for low- and high-temperature stressors. After stress treatments, leaves were sampled at 0, 1, 2, 6, 12, and 24 h. As controls, leaves sampled at 0 h were used. Each treatment was replicated biologically three times. After collecting the samples, they were promptly frozen in liquid nitrogen and then stored at –80 °C for future analysis.® RT kit  was used to synthesize cDNA, following the product manual, and 1 μg RNA was used each time to synthesize the first-strand cDNA. Genscript online design software  was used to design primer pairs  values. HhbHLH gene expression was analyzed according to the FPKM value of the transcriptome data of the H. hamabo under drought and salt stresses [2 (1+FPKM) value.A Plant Rneasy Mini Kit  was used to extract total RNA according to the operating instructions. A spectrophotometer  was used to measure the RNA concentration at OD = 260 and 280 nm, and the threshold standard for 260/280 nm was 1.80–2.20. A PrimeScripter pairs . The RT-nce gene . Three rstresses , and TbtHhbHLH2 was cloned into the pCAMBIA1305 vector. Then HhbHLH2-1305 was transformed into GV3101, and Arabidopsis thaliana Col-0 was transformed by the inflorescence infection method [Arabidopsis was identified by hygromycin (150 mg/L) selection for subsequent experimental analysis. To treat Arabidopsis thaliana with salt stress and drought, the seeds of the transgenic Arabidopsis and Col-0 (WT) were sterilized and cultured on 1/2 MS medium containing 200 mM D-mannitol and 50 mM NaCl. The phenotypes were observed and the root length was measured after 7 days of cultivation at 22 °C.The ORF sequence of n method . The T3 HhbHLHs in H. hamabo. Then the 162 HhbHLH genes were split into 19 subfamilies. Gene structure and motif analyses corroborated the phylogenetic analysis results. The cis-elements found in the promoters of the HhbHLHs were found to be associated with phytohormones and abiotic stresses. Transcriptomic profiles and RT-qPCR analysis revealed that HhbHlH57, HhbHlH78, HhbHlH136, and HhbHlH78 were significantly up-regulated in response to salt and drought stress. In addition, the overexpression of HhbHLH2 increased salt and drought stress tolerance in Arabidopsis.Using the HMMER software, we identified 162


This study was determined to see the effects of secretome on cellular production of testosterone and androgen-binding protein (ABP) using immunohistochemistry in testicle dysfunction due to cisplatin.Forty-eight rats were divided into four groups: the healthy group, the group with testicular dysfunction, the secretome-treated group with 0.2 ml/kg body weight (BW), and the secretome-treated group with 0.3 ml/kg BW. The immunohistochemistry staining method is used to find out testosterone and ABP reactivity in tissue organs.Very strong testosterone and ABP immunoreactivity were found in Leydig cells of normal testes. While in the Leydig cells of cisplatin-induced testicles, testosterone and ABP immunoreactivity were not observed. Testosterone and ABP were observed 1 week after the second secretome injection. The number of testosterone-immunoreactive cells in the low dose group from week 1 to 3 was 0, 19, and 32, respectively. From week 1 until week 4, the high dose group was 0, 29, 33, and 65, respectively. The number of ABP-immunoreactive cells from the first week until the third week in the low dose group was 0, 28, and 34, respectively. The high dose group from the first week until the fourth week was 0, 26, 58, and 83, respectively. The number of cells that produce testosterone and ABP increased from week 2 to 4.The administration of secretome could increase the number of immunoreactive cells toward testosterone and ABP in testicular dysfunction. Testicular dysfunction can affect fertility and result in an insufficient supply of androgen (testosterone). Numerous environmental and internal factors have an effect on testicular function; one of the most significant is exposure to potentially dangerous drugs such as cisplatin . CisplatSecretome is a potential regenerative agent that has recently attracted the attention of a huge number of researchers. Secretome injection hastened wound closure and increased fibroblasts, collagen fibers, and blood vessels in the treated group compared to the control group . In anotNumerous clinical trials have been carried out to demonstrate the secretome’s usefulness in the treatment of a variety of degenerative diseases. Additionally, our team has shown regeneration of spermatogenesis in mice with testicular failure. However, the testosterone and androgen-binding protein (ABP) production processes required for spermatogenesis have never been verified. This work demonstrated testosterone and ABP synthesis in rats with cisplatin-induced testicular impairment following secretome injection.The Ethical Committee of Universitas Gadjah Mada approved this study with reference number 00035/04/LPPT/V/2017.ad libitum water consumption were used. Cisplatin was given in a volume according to the weight of the rat. To induce testicular dysfunction, cisplatin was injected intraperitoneally three times every 3 days. Rats were randomly allocated to one of four groups: healthy, testicular dysfunction, testicular dysfunction treated with secretome at a 0.2 ml/kg BW dose, or testicular dysfunction treated with secretome at a 0.5 ml/kg BW dose. Cisplatin was administered intraperitoneally three times at a dose of 3 mg/kg BW with a 3-day interval between each dose to induce malfunction. Secretome was injected four times into rats in the secretome-treated groups at 7-day intervals. Rats were slaughtered every week after secretome was injected with ketamine (75 mg/kg BW).To acclimatize mice, 7 days of basic meals and One week after secretome injection, testes tissue samples were collected each week. To determine testosterone and ABP distribution in the testes, testes tissues were fixed in Bouin’s solution for 24 h, processed using the paraffin method, and stained using the immunohistochemical method. A light microscope was used to examine immunohistochemical preparations, and the results were assessed descriptively.Testicular testosterone and ABP immunolocalizations were seen using a streptavidin–biotin complex detection kit . The tissue samples were deparaffinized, dehydrated, and rinsed for 10 min in running water. At room temperature, the tissue sample was incubated in a blocking endogenous peroxidase solution for 20 min and then washed three times with phosphate buffer saline (PBS) for 5 min each. Following that, the tissue slides were incubated for 20 min at room temperature in Biocare’s background sniper solution. The tissue slides were incubated overnight at 4°C with testosterone 19, polyclonal antibody (1:10) from Mybiosource®, San Diego, CA, and ABP/sex hormone binding globulin polyclonal antibody (1:500) from BIOSS®, Massachusetts, USA. One slide was kept as a negative control where no antibodies were given.The second day begins with the slides being removed from the refrigerator and left at room temperature for 10 min before being soaked in PBS three times for 5 min each. The antibody was then incubated with one drop of Trekkie Universal Link antibody and covered with parafilm for 25 min. Additionally, the slides were soaked in PBS three times for 5 min each. For 25 min, a drop of Trek Avidin horseradish peroxidase (HRP) was given and covered with parafilm. Three times for a total of 5 min, slides were soaked in PBS. To visualize the reactive component of the tissue, slides were dropped with 30 μl of 3,3’-diaminobenzidine substrate and observed under a microscope. For 1 min, counterstain was carried out with hematoxylin. Then, they were rinsed for 10 min under running water and the object was mounted.The testosterone and ABP histochemistry staining findings were examined semi-quantitatively to determine the color intensity distribution. Four levels of color intensity are defined as follows: extremely strong, strong, medium, and weak. Establishing intensity standards requires comparing all photographs and then categorizing them according to their color intensity. The staining results were viewed under a 40× magnification light microscope. Each week, the number of testosterone- and ABP-reacting cells was determined and examined. Manual counting of immunoreactive cells was carried out using a light microscope. Calculation of five field views at a magnification of 40× for each sample was carried out.Testosterone immunoreactivity was extremely strong in normal testes, particularly in Leydig cells situated between seminiferous tubules. Negative controls lacked immuno-reactivity (brown) to testosterone. There was a shift in the intensity of testosterone immunoreactivity following secretome induction; testosterone reactivity increased and remained stable 1 week after the second secretome injection, at both high and low doses.One week after the initial secretome injection, testosterone immunoreactivity remained unchanged. However, 1 week after the second induction of the secretome, testosterone immunoreactivity increased in both low and high doses. After the third and fourth secretome injections, the immunoreactivity persisted for up to 1 week. Unfortunately, testicular samples taken during the fourth secretome induction at low levels could not be studied. A semi-quantitative analysis of testosterone immunoreactivity was carried out. The immunohistochemical labeling of testosterone in each group of mice is shown in p < α), but in the third week, the data were normally distributed (asymp. sig. 0.63 (p > 0). The findings of the testosterone immunoreactive cell count are shown in Quantitative analysis revealed an increase in the number of cells in the secretome-treated group. Along with the intensity variations, the number of testosterone immunoreactive cells increased. In the first to third low dosage secretome inductions, the number of immunoreactive testosterone cells was 0, 19, and 32 in five fields of view at 40× magnification. Whereas the number of immunoreactive testosterone cells at the induction of the first to fourth high dose secretome was 0, 29, 33, and 65 in the five fields of view at 40× magnification, the number of immunoreactive testosterone cells at the induction of the first to fourth high dose secretome was 0, 29, 33, and 65, respectively. In the second week, there was a significant difference between low and high doses  and high doses (moderate immunoreactivity). After the third and fourth secretome injections, the immunoreactivity persisted for up to 1 week. Unfortunately, testicular samples taken during the fourth secretome induction at low levels could not be studied. The result of ABP immunohistochemistry staining in each group of mice can be seen in p > 0), but in the third week, the difference between low and high dosage was significantly different with asymp. sig. 0.046 (p < α). Quantitative analysis revealed an increase in the number of cells in the secretome-treated group. Along with the intensity variations, the number of ABP immunoreactive cells increased. In the first to third low dose secretome inductions, the number of ABP immunoreactive cells was 0, 28, and 34 in five fields of view at 40× magnification. Whereas in the five fields of view at a magnification of 40×, the number of ABP immunoreactive cells was 0, 26, 58, and 83 at the time of induction of the first to fourth high dose secretome, respectively. In the second week, the difference between low and high dosage was normally distributed with asymp. sig. 0.197 (No cells were immunoreactive against testosterone or ABP in the group with testicular dysfunction. Cisplatin-induced testicular dysfunction results in the death of germ cells, shrinkage of the testicles, Leydig cell dysfunction, infertility, decreased libido, and decreased testosterone production ,10. CispOne week following the second secretome injection, tissue repair occurred as indicated by the intensity of the brown color of the Leydig cells found in the interstitial part of the testis, meaning that an androgen hormone synthesis process occurred. Secretome administration has not been shown to have chemoattractive, anti-apoptotic, anti-tumorigenic, anti-fibrotic, anti-inflammatory, pro-angiogenic, neuroprotective, or antibacterial properties ,12. The In vitro experiments have shown that these cells secreted growth factors into polymorphonuclear cell cultures, establishing it as a possible repair agent via paracrine signals and secretome as a protein secreted by cells, organs, or organisms that can respond to heal injured tissue [Quantitative investigation revealed an increase in the number of testosterone-reactive cells in both secretome dosage groups. This is because the secretome regenerates spermatogenic cells in the injured seminiferous tubules caused by cisplatin injection, allowing the hormone-producing and spermatogenesis processes to resume. d tissue ,14,15. Dd tissue . Wound hd tissue . Additiod tissue .Tissue repair occurred 1 week after the second secretome injection; the intensity of the brown color of the Leydig cells suggested the presence of an ABP production process. Not only did the color intensity change, but there was also an increase in the number of reactive cells to ABP in both secretome dosage groups, as shown by quantitative analysis. Sertoli cells generated ABP and were found in the seminiferous tubules; immunohistochemical labeling revealed that cells immunoreactive with ABP were Leydig cells, as ABP bound testosterone with high affinity and specificity and sustained high intratesticular testosterone concentrations. Interstitial testosterone is the amount of testosterone linked with Leydig cells that are bound to ABP.The results indicated that testicular tissue had improved, as evidenced by the presence of cells immunoreactive to ABP. Spermatogenesis occurs as a result of a complicated interaction between spermatogenic cells and testicular interstitial cells, which is facilitated by an appropriate environment. These include endocrine, paracrine, and autocrine systems . The secTGFα was found in the secretome, and it played a role in enhancing Sertoli cell activity . The secThe extracts of MSC growth media were able to regenerate testes that had suffered from cisplatin-induced dysfunction, as determined by testosterone and ABP immunolocalization in testicular tissue.ABP, Androgen-binding protein; BW, Body weight; MSC, Mesenchymal stem cell; IL, Interleukin; HRP, Horseradish peroxidase; PBS, Phosphate buffer saline; ROS, Reactive oxygen species; TGFα, Transforming growth factor beta; BCL-2, B-Cell Lymphoma-2.


Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura  and Caudata , the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian  Among amphibians, the skin performs, in addition to physical protection, several other vital functions such as gas exchange, ion and water transport, temperature control, chemical and mechanical sensory reception, reproduction and defence against predators and microorganisms ,2,3,4,5.Siphonops annulatus is widely distributed and adapted to different humidity levels ranging from dry (semi-arid) regions to tropical forests [Amphibians belonging to the order Gymnophiona, commonly referred to as caecilians, are vertebrates primarily adapted to a fossorial life . They co forests .Siphonops annulatus, and presumably in all other caecilians, the skin glands show a polarised distribution, with mucous glands predominating in the head, and the poison glands being much larger and numerous in the tail region [S. annulatus, although the poison glands have been associated with a defensive role for almost a century [The skin of caecilians is very well adapted to a fossorial lifestyle, including the external homogeneity of the moist and slippery body surface facilitating movement in the soil . The skil region ,22. Anurl region ,18,23,24l region . The morl region ,26,27 anl region ,29. Like century , until t century ,32 and,  century .Siphonops annulatus skin [Given so many open questions, the present study aims to complement and extend our previous findings about tus skin , going iSiphonops annulatus  , m1b cells show granules with hollow appearance  A. Both cand m1b) A. While  stained A. Scannipearance B, in addlescence C. In addpparatus D.2 glands are smaller when compared to the M1 glands. They are composed of three cell types   A. The m2ine blue A. Cells ine blue B. Cells ll types A, with all types A,B. At Sy aspect C. In m2bm matrix C. With TM images D. At thell types E. Withinll types E. The m21 glands were positive to PAS (periodic acid-Schiff) and alcian blue pH2.5 , we revealed significant morphological and histochemical differences. The M1 type presents granules with lipid content and flocculent appearance, especially in one of the cell types. Jared and co-authors [S. annulatus. In addition to their function in head and body lubrication, helping the animal to access a system of tunnels in a process similar to “diving” into the earth, such glands may also produce toxins able of repelling predators attacking the head underground. When considering defence, it is important to emphasise that in some amphibians the mucous glands are related to the secretion of toxic molecules such as tetrodotoxin [2 gland, with homogeneous body distribution and a secretion consisting basically of glycoprotein and mucopolysaccharide compounds, may have a role mainly related to the general body moistening, participating in the animal’s gas exchange and homeostasis, similarly to other amphibians [S. annulatus, rebutting ideas that they can merely represent different stages of the same glandular type. However, studies on the ontogenetic development of the skin glands would be welcome to add new evidence in this respect.Based on the acinar structure and the presence of syncytia, the morphological analysis of on gland . Compari-authors  showed aodotoxin , one of odotoxin . The M2 phibians . Moreovephibians ,6,22,35.Siphonops annulatus, particularly in relation to the defence and reproductive behaviour in the field and in captivity [S. annulatus remains in the soil, under the thick and moist leaf litter covering the forest floor that forms a constant and favourable mantle and prevents water loss. Our intensive observation of this caecilian over the years allows some considerations about its cutaneous secretion. The animal is highly slippery with intense mucous secretion. Manual manipulation stimulates the release of tiny squirts of secretion that can be observed even with the naked eye. When the animal is handled, the handler (and those close to him) react with sneezing and a runny nose [The cutaneous secretion from the poison and mucous glands is understudied among caecilians. In recent decades, our group has been studying the biology and behaviour of aptivity ,17,37,44nny nose .Siphonops annulatus does not form bulges as seen in other amphibians. This may be due to the fossorial environment where a cylindrical and smooth body favours the movement inside the tunnels [In moments of defence, the skin secretion, besides mucus, may also contain toxins released from the poison glands. In addition, the accumulation of hypertrophied poison glands at the animal’s rear end was once more confirmed in this work by the examination of three different regions along the body. It seems clear that the tail glands form a type of macrogland precisely in the body region where the animal is most vulnerable , reinfor tunnels .Siphonops annulatus, as in other caecilian species [S. annulatus poison glands consist of a set of juxtaposed syncytial compartments with a low level of cohesion between their external membranes, forming a structure similar to a three-dimensional puzzle. The poison release may consist in the detachment of part of the compartments (or even of entire compartments) through the glandular duct to the skin surface.Among amphibians the cutaneous poison glands of anurans are best known . Anuran  species ,35,36, aSiphonops annulatus was the cluster of cells located in a latero-apical position, next to the region that gives rise to the glandular duct . This cell cluster is mainly constituted of mucous content morphologically resembling a conventional cutaneous mucous gland, with a narrow lumen and apparently connected to the main duct of the poison gland. This unusual structure had already been mentioned in early studies carried out by [Another unusual feature detected in some of the poison glands in d out by  and, latd out by , who refd out by  postulatd out by ,19,49, ad out by ,36,39.Siphonops annulatus, just below the duct, that we termed “differentiated syncytial compartments” due to their syncytial morphology and the granular content essentially consisting of mucus. So far, we have not been able to obtain an indication of a possible function for such glandular units. However, in the salamander Pleurodeles waltl, Heiss and co-authors [Taricha granulosa [In addition to the append gland, we detected some glandular units typically located in apical position in -authors  interpreranulosa  revealedWhen the structure of the poison glands is analysed within the context of the different amphibian orders, marked differences can be identified. Concerning complexity, the poison glands of anurans, composed of a single syncytium ,24,29,38Although the phylogenetic affinity between the currently recognised orders of Lissamphibia has been extensively investigated ,56,57,58Siphonops annulatus has several cutaneous glandular types that, as reported for several species of amphibian, participate mainly in the chemical defence against predators and microorganisms. Our study shows that, particularly in S. annulatus, defence must be very active since the skin contains numerous glands with a characteristic distribution possibly related to its adaptation to the fossorial environment. Although our work makes a valuable contribution to the comparative morphological knowledge of the skin glands of the three orders of amphibian, the functioning mechanism of such structures in caecilians and salamanders remain unknown, especially in respect to the secretory dynamics. Further studies are necessary to understand the mechanism of poison release out of the syncytial structure and posterior glandular refilling process.Siphonops annulatus  of nnulatus A were coSelected specimens were euthanised using a lethal dose of sodium thiopental (70 mg/kg) with the addition of Lidocaine (10 mg/mL). 2 of dorsal and ventral skin from three body regions  were collected and fixed for 24–48 h in buffered 4% paraformaldehyde pH 7.2, or Bouin’s solution. The samples were transversely and longitudinally embedded in historesin and paraffin. The material embedded in historesin was sectioned in a semi-automatic microtome Microm® HM340 E  (2 µm–3 µm) and stained with toluidine blue-fuchsin. The samples processed in paraffin were sectioned in the same microtome (5 µm–6 µm), and the sections were stained with haematoxylin-eosin. The sections were also subjected to the following histochemical reactions: bromophenol blue, to indicate the presence of proteins in general, PAS, to identify neutral glycoconjugates, Sudan black B, for detection of lipids, and alcian-blue pH 2.5, for distinguishing acid glycoconjugates [Fragments around 1 cmnjugates . The imaDorsal and ventral skin fragments were fixed in Karnovsky  fixativeDorsal and ventral skin fragments were fixed in Karnovsky  solutionThe 3D reconstruction was performed using the free software Reconstruct . After a


As a result of the quantum confinement effects, and the dependence on material’s composition, atomic arrangement, and layer thickness, these 2D nanomaterials display an array of exceptional electronic, optical, physical, and chemical properties that are quite unique. Therefore, by developing advanced, functional 2D nanomaterial-based membranes, we may achieve a new level of efficiency, selectivity, sensitivity, and mechanical stability, all of which are critical for environmental remediation applications.Our environment desperately needs creative solutions to limit the effect of industrialization’s fast rise and, consequently, to remediate vast amounts of harmful by-products and toxic exhausts. Day by day, nanomaterials are becoming a more viable and eco-friendly option for efficient environmental remediation. Due to their large surface area, high reactivity, enhanced redox, and photocatalytic properties, a variety of nanomaterials are identified as ideal adsorbents, catalytic and antibacterial agents, as well as functional membranes. Among the emerging nanomaterials, 2D nanomaterials have attracted increasing attention recently following the discovery of graphene, which quickly placed among the most promising materials due to its distinctive structures and novel properties. Moreover, the discovery of graphene has paved the way for the synthesis of various 2D materials. Around 20 different types of 2D nanomaterials have been reported to date, including graphene, MXenes, graphitic carbon nitride , deep electrical conductivity, and very rich surface chemistries  have piqued researchers’ attention for water purification applications. Berdiyorov et al.  have use2+, have been evaluated against brackish water and produced water. Following 72 h of pervaporation, the examined membranes revealed a drop in Zn2+ content, indicating partial hydrolysis of the Zn2+ crosslinker that held the GO sheets together. While salt was rejected in all instances, soluble organic components  were rejected at a rate of 40–50%.Desalination via pervaporation is one of the membrane-based separation techniques that require membranes with exceptional chemical and thermal stability. In the paper by Almarzooqi et al. , the staAdditionally, 2D nanomaterials have also been employed in the fabrication of 3D porous structures. N’Diaye et al. [In the work of Chergui et al. , an rGO–Due to their unique structural features and precise molecular sieving ability, 2D nanomaterials are intriguing candidates for effective organic solvent nanofiltration (OSN) membranes. In the review by Nam et al. , the focUsing advanced 2D nanomaterials (2DNMs) in membranes manufacturing is comprehensively summarized in the review of Shahzad et al. . SeveralIn the research presented in these six papers, 2D nanomaterials have been shown to have an essential role in improving the performance of different membrane-based separation processes. The writers of these contributions have dived into the major features of 2D nanomaterials and addressed several constraints. Greater knowledge of the recent advancements of various 2D nanomaterial-based membranes and the potential for filling research gaps has been gained. This special issue is expected to provide some useful recommendations and future directions for creating more fascinating and high-performance 2D nanomaterial-based membranes.


Sexuality is defined as a multidimensional experience that involves genital, mental, and bodily components. It is also assumed as a basic condition inherent to the human existence that encourages the search for love, intimacy, sex, and proximity to others. The main objective of this study is to assess the relationship between cues of sexual desire and sexual attitudes in Portuguese women. This is a cross-sectional study with 804 Portuguese women to whom the protocol was applied. It included an informed consent, a sociodemographic questionnaire, a questionnaire related to intimacy, a scale of sexual attitudes, and the scale of cues of sexual desire. The protocol was applied via Google Forms due to the current pandemic situation (COVID-19). Differences were found in sexual attitudes and the cues of sexual desire in terms of sociodemographic characteristics, as well as in terms of women’s intimacy. Significant correlations were found between the brief sexual attitudes scale (BSAS) and the cues of sexual desire scale (CSDS). Age, sexual orientation, relation nature, sexual practices, visual proximity cues, erotic explicit cues, and sensory explicit cues explain, altogether, 25% of the total sexual attitudes. Additionally, age, sexual orientation, the relation’s nature, sexual practices, visual proximity cues, emotional bonding cues, romantic implicit cues, erotic explicit cues, and sensory explicit cues explain, altogether, 30% of the permissiveness. Sexual attitudes are developed under the influence of sociodemographic variables, variables related to women’s intimacy, and cues of sexual desire, which are new data in the study of sexual attitudes and have implications at the level of gender issues. Sexuality is a central organizing construct that includes attitudes, behaviours, and values aligned with one’s sex, gender, and sexual identity, eroticism, pleasure, intimacy, reproduction, desire, and the like . SexualiThe World Health Organization  defines Sexual attitudes refer to the attitudes one has toward sexuality or sexual behaviours ,14. ThesSexual desire motivates individuals to engage in sexual activity  and it iAs natural selection requires variation in psychological traits, women naturally differ relating this specific aspect . ConcernWith regard to the relationship between sex and commitment, there is the so-called “casual sex”, whose main characteristic is the absence of a relational commitment between people or just the existence of a friendship. In this regard, Alvaréz et al.  concludeAccording to Rosemary Basson’s Non-Linear Model of Sexual Response , most woConsidering that the assumptions of good psychometric qualities of the instruments are guaranteed (described below), the following hypotheses were established: Hypothesis 1 (H1).It is expected that the cues of sexual desire and sexual attitudes vary according to age, marital status, and educational qualifications, as well as according to sexual orientation, the duration of the relationship, and the nature of the relationship.Hypothesis 2 (H2).It is expected to find significant and positive correlations between cues of sexual desire scale (CSDS) and sexual attitudes (BSAS) and respective subscales.Hypothesis 3 (H3).It is expected that the sexual attitudes of Portuguese women are explained in a multifactorial way: by sociodemographic characteristics, by characteristics of intimacy, and by the cues of sexual desire scale (CSDS).This study has a descriptive, correlational character, since it intends to describe the relationships found between variables. It is also transversal, i.e., single application, and quantitative  in nature. min = 18; max = 71)). For the purposes of statistical analysis, age was converted into a variable divided into four groups: 1–18 to 20 years, n = 102 (12.7%); 2–21 to 30 years, n = 325 (40.4%); 3–31 to 40 years, n = 197 (24.5%); 4–41 to 71 years, n = 180 (22.4%).Based on the analysis of questionnaires obtained from 851 individuals aged between 17 and 70 years old, and after applying the inclusion criteria, i.e., being female, being aged between 18 and 74 years old, and having Portuguese nationality, the sample of this study is composed by 804 Portuguese women with a mean age of approximately 26 years (26.05 ± 10.19 (n = 612 (76.1%), is married/in an unmarried union: n = 158 (19.7%), and divorced: n = 34 (4.2%)). Concerning education, the majority of the sample is concentrated in higher education, n = 464 (57.7%). With regard to their professional status, 481 women (59.8%) are students and the remaining work.Most of the sample is not in a relationship . Participants are at the two extremes in terms of relational duration, i.e., 322 (40%) have been in a relationship for more than 24 months and 282 (35.1%) are not in a relationship, while 59 (7.3%) women are in a relationship for about six months, 51 (6.3%) for 6 to 12 months, and 90 (11.2%) for 12 to 24 months.Considering the relational nature, the following data were obtained: 483 (60.1%) women are in a committed relationship, 282 (35.1%) are not in a relationship, and 39 (4.9%) are in a non-committed relationship. In regard to the practice of sexual relations in the last month, there were 516 (64.2%) negative responses and 288 (35.8%) positive ones. Finally, 533 women (66.3%) stated that they only maintain current sexual practices with a single partner, in contrast to 16 women (2%) who admit practices with multiple sexual partners. In addition, 115 women (14.3%) report current sexual practices without a partner, e.g., masturbation, and 140 (17.4%) report not having any type of sexual activity.Considering the most up-to-date version of the Declaration of Helsinki  and all The protocol was made available online through Google Forms due to the current pandemic period (COVID-19) and was posted on a page on a social network created specifically for this purpose. Its dissemination was made mainly through personal contacts, using social networks and other digital means, obtaining a sample of snowball effect, non-probabilistic for convenience. The collection of the data was carried out during the month of November 2020. The data obtained were processed using the Statistical Package for the Social Sciences (SPSS 27).Sociodemographic questionnaire and questionnaire on women’s intimacy. A sociodemographic questionnaire was constructed, including sociodemographic issues ; the questionnaire on women’s intimacy includes issues related to the intimacy of women . Regarding the latter, the sexual orientation variable was open and was later recoded as 1—heterosexual and 2—other orientations, given the scarcity of answers for this option. The variable duration of the current relationship included the following answer options as: 1—I am not in a relationship; 2—less than 6 months; 3—6 to 12 months; 4–12 to 24 months; 5—more than 24 months. The relational nature variable allows the following response options: 1—I am not in a relationship; 2—no commitment; 3—with commitment. The variable practice of sexual intercourse in the last month allows answering yes or no. The variable current sexual practices and sexual partners allows choosing one of the following response modalities: 1—no; 2—yes, no sexual partner; 3—yes, with only one sexual partner; 4—yes, with more than one sexual partner.Sexual Attitudes Scale (SAS). The SAS was developed by Hendrick and Hendrick  = 37.851; p < 0.001) , erotic explicit cues (CSDS), and sensory explicit cues (CSDS), altogether, explain 25% of the BSAS  variance (< 0.001) , being tF = 39.215; p < 0.001) , emotional bonding cues (CSDS), romantic implicit cues (CSDS), erotic explicit cues (CSDS), and sensory explicit cues (CSDS), altogether, explain 30% of the permissiveness (BSAS) variance (< 0.001) , being tβ = −0.149; t = −4.742; p < 0.001), CSDS  , and visual proximity cues (CSDS) , altogether, explain 3%  of the sexual practices (BSAS) variance , being that visual proximity cues is the variable that contribute the most.Age , relation nature , visual proximity cues (CSDS) , and sensory explicit cues (CSDS) , altogether, explain 10%  of the instrumentality (BSAS) variance  = 22.390; p < 0.001), being that visual proximity cues is the variable that contribute the most.Relation duration , sexual practices , emotional bonding cues (CSDS) , romantic implicit cues (CSDS) , erotic explicit cues (CSDS) , and sensory explicit cues (CSDS) , altogether, explain 11%  of the communion (BSAS) variance  = 17.826; p < 0.001), being that romantic implicit cues is the variable that contributes the most.Education  is used to examine the internal consistency or reliability of summated rating scales [The instruments used to assess the cues of sexual desire (CSDS) and sexual attitudes (BSAS) in Portuguese women presented good psychometric qualities. In fact, the Cronbach’s alpha values found in this study are very close to the values found in the original versions and in the validation versions for the Portuguese population, and, in some cases, these values are even higher in this study. Cronbach’s alpha  predicted that cues of sexual desire and sexual attitudes would vary according to age, marital status, and educational qualifications, as well as according to sexual orientation, the duration of the relationship, and the nature of the relationship. This hypothesis was also confirmed, which is in line with the perspective proposed by Laumann et al. [The first hypothesis  and erotic explicit cues (CSDS) subscale. However, women who had no recent sex had consistently less well-being, especially for women with more permissive attitudes . Women wH2) predicted finding significant and positive correlations between cues of sexual desire (CDSD) and sexual attitudes (BSAS) and respective subscales, which was confirmed. In fact, the results of this study showed multiple significant correlations, although mostly moderate and weak. The positive correlations between the CSDS total, sensory explicit cues, erotic explicit cues, visual proximity cues, and the BSAS total are highlighted, as well as the positive correlations between the sensory explicit cues, visual proximity cues, and the BSAS permissiveness subscale. This is in line with the literature that suggests that a positive sexual attitude can be responsible for the positive assessment of one’s own sexual experience and for the presence of various cues of sexual desire [The second hypothesis  predicted that the sexual attitudes of Portuguese women would be explained in a multifactorial way: by sociodemographic characteristics, by characteristics of intimacy, and by the cues of sexual desire. This hypothesis was also confirmed, especially in relation to the total BSAS and the BSAS permissiveness subscale, as in relation to the other dimensions of the BSAS. The values explained by the regressions were equal to or less than 11%, which is clearly not significant. Thus, age, sexual orientation, the relation’s nature, sexual practices, visual proximity cues (CDSD), erotic explicit cues (CDSD), and sensory explicit cues (CDSD) explain, altogether 25% of the BSAS . These results are in line with the literature. In fact, several authors have reported the impact of sociodemographic variables on sexual attitudes [Finally, the third hypothesis , digital means were used to apply the questionnaire, through Google forms, which in turn made it impossible to clarify doubts. Due to the cross-sectional nature of the study, the relevance of conducting a longitudinal investigation is highlighted, as data collected in more than one moment and over a longer period of time will make it possible to establish stable and reliable relationships between the variables without the interference of biased information and verify possible changes in the participants’ answers. The application of this research to an older female population is also suggested. Finally, a new measurement of both scales aimed at the Portuguese population is recommended, considering the social and behavioural changes that have taken place in recent decades, thus allowing for more up-to-date and truthful results.


Training intensity monitoring is a daily practice in soccer which allows soccer academies to assess the efficacy of its developmental interventions and management strategies. The current systematic review’s purpose is to: (1) identify and summarize studies that have examined external and internal training intensity monitoring, and to (2) provide references values for the main measures for young male soccer players. A systematic review of EBSCO, PubMed, Scielo, Scopus, SPORTDiscus, and Web of Science databases was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. From the 2404 studies initially identified, 8 were fully reviewed, and their outcome measures were extracted and analyzed. From them, the following range intervals were found for training: rated perceived exertion (RPE) 2.3–6.3 au; session-RPE, 156–394 au; total distance, 3964.5–6500 m and; distance >18 km/h, 11.8–250 m. Additionally, a general tendency to decrease the intensity in the day before the match was Found. This study allowed to provide reference values of professional young male players for the main internal and external measures. All together, they can be used by coaches, their staff, or practitioners in order to better adjust training intensity. Training intensity monitoring is a daily practice within the soccer training context . TraininThe monitoring is commonly organized in two dimensions : (i) intFrom such instruments, the typical variables extracted are heart rate-bases scores, RPE-based scores and distances covered at different speed thresholds, or changes of velocity (accelerations and decelerations). The measures are daily quantified in training and match scenarios. Using absolute intensity per session and per week, the sports scientist has some possibilities of understanding the within- and between-weeks variations . The analysis of intensity impact on players is highly relevant. One of the reasons is justified by the gap between coaches perception and the actual intensity imposed on the players . Based oAlthough the increase in intensity monitoring reports in young soccer, most of the articles are from the same team. This reduces the possibility of generalization of the evidence. However, is also known that is not expectable to reach different teams at the same time over long periods of the season. Thus, one of the strategies can be to summarize the available evidence about intensity values of young soccer players by conducting a systematic review. Although a possible idea, no systematic review was found about that. Although there is pertinence of looking into both males and females, summarizing both within the same article would not be useful since the huge differences related with a number of publications and a logical difference between sexes produces a different approach to the consequences for the article logic. Thus, a more focused contribution as a systematic review may help coaches to compare values of training intensity in young male soccer and provide some benchmarks. Thus, the aim of this systematic review is to identify and summarize studies that have examined external and internal training monitoring and to provide references values for the main measures for young male soccer players.The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed to write this systematic review  and guidThe inclusion and exclusion criteria can be found in The screening process related to analysis of the title, abstract and reference list of each study to locate potentially relevant studies was independently executed by two of the authors (A.M.V. and M.R.G.). Moreover, both authors also reviewed the full version of the included papers in detail to identify which article met the inclusion criteria. Additionally, a search within the reference lists of the included records was performed to add additional relevant studies. In the cases of discrepancies, a discussion was performed with the participation of a third author (R.O.). Possible errata for the included articles were considered.The following electronic databases were used to search for relevant publication on the 3 July 2021, after protocol registration: FECYT , PubMed, and Scopus. A manual search was also conducted after search in electronic databases to retrieve additional studies that could fit our eligibility criteria. Keywords and synonyms were entered in various combinations in the title, abstract or keywords which means that the following research content was applied:  AND . Search results were exported to EndNote 20.0.1 for Mac . No filters or limits were applied.A specific spreadsheet was designed in Microsoft Excel  for process the data extraction. The design followed the recommendations of the Cochrane Consumers and Communication Review Group’s data extraction template . In thisThe methodological quality was assessed using the methodological index for non-randomized studies (MINORS) by two authors (A.M.-V. and M.R.-G.) . MINORS A total of 2404  original articles were initially retrieved from the mentioned databases, of which 834 were duplicates. Thus, a total of 1570 original articles were found. After this, a total of 1558 articles checked by title and abstract were excluded. The remaining 12 articles were checked in full text, leading to the exclusion of 3 articles according to criterion nº 1, and 1 according to criterion nº 2. Additionally, 1 article was included from additional sources. A total of 8 articles met all the inclusion criteria and were finally included in the qualitative synthesis. All the steps followed for the selection of the articles are available in The overall methodological quality of the cross-sectional studies can be found in In addition, three studies analyzed both internal and external measures ,19,24, tOnly one study presented data for starters and non-starters. Since non-starter presented lower values than starters, we provided a range interval with lower values related to non-starters and higher values related to starters . The last line of training duration of 79–117 min ,21,22,24total distance of 364.5–6500 m ,21,22,24distance > 18 km/h of 11.8–250 m ,21,22;distance > 25 km/h of 0–30 m ,21,22,24The aim of this systematic review was to identify and summarize studies that have examined external and internal training intensity monitoring in young male soccer players and to provide references values for the main training intensity measures. The main results showed the following range intervals by overall teams that include (U12 to U19):The importance of prescribing appropriate training intensities to improve player performance is well recognized in the current literature ,28. HoweWhen analyzing the mean of the total distance per training session for overall team of the selected studies, there was a pattern of increasing distance until older ages ,21,22,24When considering, the accumulated weekly, total distance did not show that pattern from under U12 ~18.6 Km), U13 ~19.3 km), U14 (~19.5 km), U15 ~21.0 km), U16 (~20.8 km), U17 (~16.0 km), U18 (~21.2 km) to U19 (~16.0 km) age-groups [.6 Km, U1.3 km, U1.0 km, U1Perhaps technical-tactical methodologies, namely game model, can explain these data. There was an age-related increase in the training intensity and to a greater extent in the training volume . Due to The sprints number reported by the studies showed a high dispersion. In the study by Abade et al.  11 ± 6 sRegarding internal training intensity, the main results showed a range interval range of 2.3 to 6.3 au for RPE between U14, U15, U16, and U18 ,25 and 1Moreover, Wrigley et al.  noted a There were only one study analyzed Banister TRIMP and player status and found no differences between starters and non-starters  which waThe main findings showed that young players usually training between 3 to 4 days per week. The higher intensity was found on MD-4, MD-3, and MD-2 while the lower intensity was found on MD-1, although only two studies used this approach for data analysis ,24. The The decrease in high-speed running distance and sprint distance in training sessions before match day was also evident  but it wRegarding internal intensity analysis, two studies analyzed s-RPE with different scales, CR-10  vs. CR-2In youth soccer, the positional role has been analyzed in constrained training tasks ,42,43, h−1) [−1) [The influence of playing position on physical and physiological performance during competition is well documented ,45,46, w−1) . Other s−1) . On the −1) [−1)  in mesoc−1) [−1) . In futuIn our literature review, only two studies reported values of the internal training intensity between playing positions ,24. TeixThis study presents some limitations that influenced the results and the reference values provided. First, the small number of studies proves that much more research is needed in young players, even more considering the different age-group categories. Additionally, the studies included only analyze one soccer team which limit their results. Consequently, few studies analyzed contextual variables, such as player status, player positions, and MD- approach. In addition, few studies included full seasons. Moreover, the different speed thresholds and scales used makes difficult to generalize the results and compare them between studies. In addition, training intensity and reference values provided came from general training, but this information was not revealed by the studies. Only one study identify data from the field and from the gym training . FinallyDespite the limitations, the present study constitutes a relevant tool in the field of training intensity quantification of young male soccer players that can be used for coaches, their staff and practitioners as a reference for future studies in order to replicate such values or even to increase the numbers presented.In future studies, the comparison between elite and sub-elite soccer academies is an important research gap which should be considered. Additionally, information on what type of training and exercises could provide further knowledge on training intensity. At last, future studies can analyze values of training intensity for injury prevention, since the general aims of the studies in this field are to improve performance and avoid injuries.The standard microcycle from U12 to U16 included three training sessions, while U17 and U19 included four training sessions per week. The duration of training sessions for all age-groups was between ~70 and 117 min.Specifically, the following range intervals were found for training: RPE 2.3–6.3 au; s-RPE, 156–394 au; total distance, 3964.5–6500 m and; distance > 18 km/h, 11.8–250 m.Even though the internal training intensity did not present significant differences for weekly inter-day analysis, there was an inverted U-shaped curve in the distribution of the weekly external training intensity. In the older age-group players, tapering is evidenced. On the other hand, the U14 to U16 players seems present relatively high training intensities across the weekly microcycle. Indeed, it could be suggested that coaches opt for different tapering strategies when the age and competition focus increases.The playing position seems to have a minimal effect on the weekly training intensity of young players.


Prior research suggests that psychiatric disorders could be linked to increased mortality among patients with COVID-19. However, whether all or specific psychiatric disorders are intrinsic risk factors of death in COVID-19 or whether these associations reflect the greater prevalence of medical risk factors in people with psychiatric disorders has yet to be evaluated.We performed an observational, multicenter, retrospective cohort study to examine the association between psychiatric disorders and mortality among patients hospitalized for laboratory-confirmed COVID-19 at 36 Greater Paris University hospitals.p < .01). When adjusting for age, sex, hospital, current smoking status, and medications according to compassionate use or as part of a clinical trial, this association remained significant . However, additional adjustments for obesity and number of medical conditions resulted in a nonsignificant association . Exploratory analyses after the same adjustments suggested that a diagnosis of mood disorders was significantly associated with reduced mortality, which might be explained by the use of antidepressants.Of 15,168 adult patients, 857 (5.7%) had an ICD-10 diagnosis of psychiatric disorder. Over a mean follow-up period of 14.6 days (SD = 17.9), 326 of 857 (38.0%) patients with a diagnosis of psychiatric disorder died compared with 1276 of 14,311 (8.9%) patients without such a diagnosis (odds ratio 6.27, 95% CI 5.40–7.28, These findings suggest that the increased risk of COVID-19–related mortality in individuals with psychiatric disorders hospitalized for COVID-19 might be explained by the greater number of medical conditions and the higher prevalence of obesity in this population and not by the underlying psychiatric disease. Prior studies , 9, 10 sThis issue is key in the context of the worldwide infectious disease crisis , 15, 16,et al.  University hospitals from the onset of the epidemic in France, i.e., January 24, 2020, until May 1, 2020 , 20, 21.This observational study using routinely collected data received approval from the Institutional Review Board of the AP-HP Clinical Data Warehouse . AP-HP Clinical Data Warehouse initiatives ensure patient information and consent regarding the different approved studies through a transparency portal in accordance with European Regulation on Data Protection and Authorization No. 1980120 from the National Commission for Information Technology and Civil Liberties.AP-HP Health Data Warehouse (Entrepôt de Données de Santé) contains all available clinical data on all inpatient visits for COVID-19 to 36 Greater Paris University hospitals. The data include patient demographic characteristics, vital signs, laboratory test and reverse transcriptase polymerase chain reaction test results, medication administration data, current medical diagnoses, and death certificates.Data for each patient were obtained at the time of the hospitalization through electronic health records . The assPsychiatric disorder diagnoses were also obtained through electronic health records and recorded at the time of hospitalization for COVID-19, based on the ICD-10 diagnosis codes (F01–F99) as determined by the practitioners in charge of the patients. Patients with at least one ICD-10 diagnosis of mental, behavioral, or neurodevelopmental disorder (F01–F99) were considered as having a psychiatric disorder.The study baseline was defined as the date of hospital admission for COVID-19. The outcome was all-cause mortality from the study baseline until the end of the hospitalization for COVID-19 or until the time of data cutoff on May 1, 2020.We calculated the frequencies of all baseline characteristics described above in patients with and without a diagnosis of psychiatric disorder and compared them using standardized mean differences.To examine the crude, unadjusted association between a diagnosis of psychiatric disorder and all-cause mortality, we performed a logistic regression model. Patients with a diagnosis of psychiatric disorder were compared with a reference group without a diagnosis of psychiatric disorder. To reduce the effects of confounding, the primary analysis was a multivariable logistic regression that included sex, age, hospital, current smoking status, medications according to compassionate use or as part of a clinical trial, obesity, and the number of current medical comorbidities.We performed several additional exploratory analyses. First, we reproduced these analyses for different psychiatric diagnostic categories  instead of three classes  to distinguish the group of patients without any medical comorbidity. Fourth, we performed additional multivariable logistic regression models including interaction terms to examine whether the association between any psychiatric disorder and mortality significantly differed by age and sex. Fifth, we examined whether having at least two diagnoses of psychiatric disorders was associated with a significantly greater risk of death than having only one diagnosis of psychiatric disorder. Sixth, we examined, among patients with a diagnosis of any psychiatric disorder, whether the mortality risk differed between those with and without this diagnosis confirmed during a prior hospitalization in Greater Paris AP-HP hospitals in the past 2 years. Finally, we reproduced the main analyses while successively adjusting additionally for time to follow-up and individual medications prescribed as part of a clinical trial or according to compassionate use.p value < .05. We planned to perform additional exploratory analyses as described above only if a significant association was found. All analyses were conducted in R software .For all associations, we assessed the fit of the data, checked assumptions, and examined the potential influence of outliers. We followed the recommendations of the Strengthening the Reporting of Observational Studies in Epidemiology Initiative . BecauseOf the 17,131 patients hospitalized for laboratory-confirmed COVID-19, 1963 patients (11.5%) were excluded because of missing data or their young age . Of these 15,168 patients, 857 (5.7%) had a diagnosis of psychiatric disorder and 14,311 (94.3%) did not . Of thesZ = 1.93; p = .053).Reverse transcriptase polymerase chain reaction test results were obtained after a median delay of 1.2 days (SD = 12.7) from the hospital admission date. This median delay was not significantly different between patients with and without a diagnosis of a psychiatric disorder .Over a mean follow-up of 14.6 days , 1602 patients (10.6%) died at the time of data cutoff on May 1. Among patients who had a diagnosis of a psychiatric disorder, the mean follow-up was 12.7 days , while it was 14.7 days  in patients without a diagnosis of a psychiatric disorder  patients with a diagnosis of psychiatric disorder and in 1276 of 14,311 (8.9%) patients without this diagnosis. Rates of mortality by psychiatric diagnosis category ranged from 20.0% in patients with an alcohol-induced psychiatric disorder to 47.0% in patients with an illness-induced psychiatric disorder .Figure 2p < .001), the multivariable regression analysis adjusted for age and sex , and the multivariable regression analysis adjusted for age, sex, hospital, current smoking status, and medications according to compassionate use or as part of a clinical trial  showed significant association between a diagnosis of a psychiatric disorder and increased mortality. However, this association was not significant after additional adjustments for obesity and the number of medical conditions   .p = .045 and AOR 0.64, 95% CI 0.42–0.99, p = .046, respectively)  . Additioctively) . After sWhen examining these associations in the population of patients with a diagnosis of a psychiatric disorder, we found that mortality rates were significantly higher in patients with diagnoses of anxiety disorders and intellectual disabilities than in those diagnosed with other psychiatric disorders . These tResults of additional analyses are detailed in In this multicenter, retrospective, observational study involving 15,168 patients hospitalized for laboratory-confirmed COVID-19, we found that individuals with a diagnosis of a psychiatric disorder had a sixfold increased risk of mortality than those without this diagnosis. All individual psychiatric diagnoses were significantly associated with increased mortality when adjusting for age, sex, hospital, current smoking status, and medications according to compassionate use or as part of a clinical trial. However, after additionally adjusting for obesity and the number of medical comorbidities, no diagnosis of psychiatric disorder was significantly associated with mortality as compared with individuals without psychiatric disorders. A notable exception was that a diagnosis of mood disorders was significantly associated with reduced mortality, which might be explained by antidepressant use. Our analyses suggest that increased mortality in patients diagnosed with a psychiatric disorder compared with those without this diagnosis was mainly explained by the higher rates of medical risk factors, including a greater number of medical conditions and the higher prevalence of obesity, in this population.Of the 15,168 patients hospitalized for COVID-19, we found that 857 (5.7%) had a diagnosis of a psychiatric disorder, of which 398 (46.4%) had a primary psychiatric disorder, 511 (59.6%) had a substance-induced or illness-induced psychiatric disorder, and 52 (6.1%) had both types of disorders. Although no direct comparison can be performed in this study, the rate of any current psychiatric disorder in our sample was about twofold lower than that observed in the European or French general population, estimated at 11.5%  and betwIn line with prior evidence , 9, 10, Our results indicate that this increased risk of mortality associated with psychiatric disorders could be explained by the higher rates of medical risk factors, including a greater number of medical conditions and higher prevalence of obesity, which importantly increase the risk of severe COVID-19 ,17,39. TNo category of psychiatric disorder was significantly associated with increased mortality compared with patients without psychiatric disorders. This finding supports the idea that it is not psychiatric disorders per se that increase this risk but rather specific determinants of health, including social, economic, and environmental factors, which may influence both mental and physical health and mortality risk in COVID-19. For example, obesity and medical diseases, a number of which are due to alcohol or tobacco consumption, may constitute shared risk factors of both psychiatric disorders and COVID-19–related mortality.One notable exception was that patients diagnosed with mood disorders, specifically those taking an antidepressant during the visit, showed a reduced risk of death than patients without psychiatric disorders, which was not explained by differences in medical risk factors or clinical severity at baseline. The hypothesis previously advanced, that of potential antiviral and anti-inflammatory effects of several psychotropic medications, especially those of certain antidepressants such as fluoxetine or fluvoxamine, which are taken by a substantial proportion of people with mood disorders, could be a promising avenue to explore further ,40. For Several mechanisms have been proposed to explain the potential beneficial effect of certain antidepressants against COVID-19 ,45. FirsAmong patients with a diagnosis of psychiatric disorder, mortality rates may be significantly higher in those with diagnoses of anxiety disorders or intellectual disabilities. These results are in line with prior findings , 57, 58.Our study has several limitations. First, an inherent bias in observational studies is unmeasured confounding. We tried to minimize the effects of confounding and performed the analyses while adjusting for numerous potential confounders. Second, there were missing data for some baseline characteristic variables , which might be explained by the overwhelming of all hospital units during COVID-19 peak incidence, and different results might have been observed during a lower COVID-19 incidence period. However, sensitivity analyses using imputed missing data reached similar results. Third, because of the cross-sectional design, correlation does not imply causation . Fourth,Findings from this multicenter retrospective observational study suggested an increased risk of mortality in individuals diagnosed with a psychiatric disorder than in those without this diagnosis, which could be explained by higher rates of medical risk factors, including a greater number of medical conditions and higher prevalence of obesity, in this population. Future studies including data on outpatients and inpatients with and without psychiatric disorders and COVID-19 and taking into account main medical risk factors of severe COVID-19, i.e., age, medical comorbidities, and obesity, will be important to determine whether the risk of hospitalization and mortality due to SARS-CoV-2 infection is similar or different across psychiatric diagnoses and psychotropic medications prescribed.


The association of free testosterone (FT) with sarcopenia and its components is well known in men but incompletely understood in women. We examined the association of baseline FT with the prevalence and incidence of sarcopenia and its components in community-dwelling older adults.Cross-sectional and longitudinal analysis from the prospective population-based Korean Frailty and Aging Cohort Study.A total of 1,879 community-dwelling older adults aged 70–84 years were enrolled for cross-sectional analysis and 1,583 subjects who participated in the 2-year follow-up survey were included for longitudinal analysis. Baseline FT levels was measured by radioimmunoassay. Skeletal muscle mass, handgrip strength, and physical performance tests were measured at baseline and after 2-year follow-up. Sarcopenia was defined by the diagnostic criteria of the Asian Working Group for Sarcopenia (AWGS).Continuous FT levels was positively associated with the prevalence of sarcopenia in men ] and women  after adjusting for multiple confounders. In prospective analysis, low FT levels was associated with a decrease in handgrip strength in women  and a reduction in Timed “Up and Go” (TUG) test  in men after 2 years. No significant correlations were found between FT levels and the incidence of sarcopenia.Low levels of FT may be a significant determinant of decreases in muscle strength in women and declines in physical performance in men after 2 years. Low FT do not predict loss of muscle mass in both men and women. Sarcopenia, or the loss of skeletal muscle mass and strength with age, accelerates the risk of adverse health outcomes, such as functional impairment, falls, disability, and mortality in older adults , 2. As tTestosterone, which is an anabolic steroid hormone, helps maintain muscle mass through influencing satellite cell proliferation and myonuclear number , 11. Appvia aromatization to estradiol in women  is an ongoing longitudinal cohort study with a baseline survey whose purpose is to identify frailty status and frailty transitions over time in populations of older adults. Baseline survey was conducted from May 2016 to November 2017 with longitudinal visits planned for every 2 years. A total of 3,013 participants were recruited in the baseline round from sex- and age-stratified community-dwelling older adults aged 70–84 years in ten study centers, which included urban, suburban, and rural areas throughout South Korea . The ratBlood samples were collected from the antecubital vein between 0730 h and 0800 h after an overnight fasting. Participants were required to not take medications prior to blood collection. The blood samples collected in serum separator tubes were clotted at room temperature for 30–60 min. After separation at 3,000 rpm for 10 min, blood samples were immediately stored at 2–8°C and transferred to the central clinical laboratories  within 24 h. All serum samples were stored at -80°C prior to analysis. Serum FT levels were measured at baseline. FT levels were measured using radioimmunoassay (AMP 10-R4100), with a detection limit of 0.40 pg/mL. The inter-assay coefficients of variation (CVs) were 9.3%, 5.7%, and 11,7%, and the intra-assay CVs were 19.5%, 7.3%, and 9.1% in the low, medium, and high pools, respectively. Conventional units of FT concentrations (pg/mL) were multiplied by 3.47 to convert to SI units (pmol/L). The other hematological, biochemical, metabolic, and inflammatory parameters were as follows: serum albumin , creatinine , hemoglobin , glycosylated hemoglobin , total cholesterol , triglyceride , high-density lipoprotein cholesterol , 25-hydroxy vitamin D , and high-sensitivity C-reactive protein . Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: [fasting insulin (µU/mL) × fasting glucose (mmol/L)]/22.5.The Asian Working Group for Sarcopenia (AWGS) defines sarcopenia as low muscle mass, low muscle strength, and/or low physical performance . The appThe Short Physical Performance Battery (SPPB) test is a validated test that assesses lower extremity function by measuring balance, gait speed, and time to rise from a chair five times . ParticiFor the health examinations, all participants underwent face-to-face interviews based on standardized surveys. Sociodemographic and lifestyle information on education level, smoking status, alcohol consumption, physical activity level, and living conditions were obtained by trained investigators. Comorbidities were self-reported status diagnosed by physicians and defined as two or more of the following diseases: hypertension, diabetes mellitus, dyslipidemia, myocardial infarction, congestive heart failure, angina pectoris, cerebrovascular disease, peripheral vascular disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, or chronic obstructive pulmonary disease. Low physical activity level was defined as total energy consumption of <494.65 kcal for men and <283.50 kcal for women, which corresponds to the lowest 20% of the total energy consumed in the general population of Korean older adults . The parBaseline descriptive statistical analyses were carried out to compare the participants’ characteristics according to FT quartiles. One-way analysis of variance with Bonferroni post-hoc test for continuous variables and the chi-squared test or Fisher’s exact test for categorical variables were conducted. Cross-sectional associations between serum FT levels and the prevalence of sarcopenia measured during the baseline assessment were assessed using multivariate logistic regression. FT levels are expressed as continuous (per standard deviation [SD]) and as quartiles, with the first quartile as the reference group. We analyzed the associations of continuous FT levels and FT quartiles with sarcopenia to identify the linear and threshold effects of FT levels on sarcopenia, respectively.The associations between serum FT levels and the incidence of sarcopenia was investigated using multivariate logistic regression. In additional analyses, generalized estimating equation (GEE) models were employed to examine the associations of low baseline serum FT as a binary variable with changes in muscle mass, muscle strength, and physical performance over 2 years. GEE models, which is an extension of the generalized linear model, consider the dependency of repeated measures within participants. For this analysis, baseline FT (continuous and dichotomous) levels, time and time interactions with baseline FT was included, and the correlation structure was set to autoregressive. In GEE models, we also presented False Discovery Rate (FDR) adjusted p-value to reduce the risk of false positive due to multiple comparisons. A low FT level was defined as the lowest quartile group.To account for potential confounders, all models were adjusted as follows: model 1, unadjusted; model 2, adjusted for age and waist circumference ; model 3Baseline characteristics according to the FT quartiles are presented in In men, the cross-sectional association between circulating levels of FT and sarcopenia are presented in The results of the multivariate logistic regression analysis of the cross-sectional association between circulating levels of FT and sarcopenia in women are shown in The associations of baseline FT levels with changes in muscle mass, muscle strength, and physical performance over 2 years are demonstrated in Of the 1,237 participants classified as non-sarcopenia at baseline, the incidence of sarcopenia was 11.1% (n=70) in men and 9.7% (n=59) in women after 2 years. In this prospective cohort study of community-dwelling older adults aged over 70 years, low baseline FT levels were associated with a decrease in handgrip strength in women and a decline in the TUG test in men after a follow-up period of 2 years. To the best of our knowledge, this is the first prospective study that identified sex-specific differences in the association of baseline FT levels with changes in sarcopenia components in older adults using longitudinal analyses.In women, we found a relationship between low FT levels and a decrease in muscle strength despite a short follow-up period. A previous study reported that low FT levels accelerated the risk of sarcopenia in Japanese women aged 40–79 years on follow-up after an 8.3-year period; however, the only criterion for sarcopenia was low muscle mass, and the study did not evaluate muscle strength and physical performance . In our vs 76.3). Another differences in results may be partially attributable to assay methodology. FT level was calculated by Vermeulen method in LASA, Health ABC, and EMAS study; however, we directly estimated FT level through radioimmunoassay method, which is more accurate. Meanwhile, the lack of association of low FT levels with muscle mass loss is consistent with previous prospective study. Gielen, E. et al. , which was funded by the Ministry of Health and Welfare, Republic of Korea .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.