Brassinosteroid (BR) signaling regulates plant growth and development in concert with other signaling pathways. Although many genes have been identified that play a role in BR signaling, the biological and functional consequences of disrupting those key BR genes still require detailed investigation.A. thaliana lines carrying a loss-of-function mutation in BRI1 gene, bri1–5, that exhibits a dwarf phenotype and its three activation-tag suppressor lines that were able to partially revert the bri1–5 mutant phenotype to a WS2 phenotype, namely bri1–5/bri1–1D, bri1–5/brs1–1D, and bri1–5/bak1–1D. From the three investigated bri1–5 suppressors, bri1–5/bak1–1D was the most effective suppressor at the transcriptional level. All three bri1–5 suppressors showed altered expression of the genes in the abscisic acid  pathway, indicating that ABA likely contributes to the partial recovery of the wild-type phenotype in these bri1–5 suppressors. Network analysis revealed crosstalk between BR and other phytohormone signaling pathways, suggesting that interference with one hormone signaling pathway affects other hormone signaling pathways. In addition, differential expression analysis suggested the existence of a strong negative feedback from BR signaling on BR biosynthesis and also predicted that BRS1, rather than being directly involved in signaling, might be responsible for providing an optimal environment for the interaction between BRI1 and its ligand.Here we performed phenotypic and transcriptomic comparisons of BRS1.Our study provides insights into the molecular mechanisms and functions of key brassinosteroid (BR) signaling genes, especially The online version contains supplementary material available at 10.1186/s12864-021-07778-w. Because BRI1 is the core receptor of BRs, mutants of BRI1 have been used as genetic background to identify suppressors, i.e. other genes that when mutated, suppress the bri1 phenotype and thus may play a role in BR signaling. For example, the function of BZR1 has been unveiled by using the null allele of BRI, bri1–116 [bri1, bri1–5, can be rescued by overexpression of BAK1 and BRI Suppressor 1 (BRS1) [BRS1 can suppress two weak BRI1 extracellular domain mutants, bri1–5 and bri1–9, but not the strong cytoplasmic domain mutant bri1–1, implies that BRS1, unlike the downstream genes, BZR1 or BES1, may function upstream of the BR signaling pathway or in a close regulatory relationship with BRI1 [BRS1’s homologs amongst which ECS1 (Extra Carpels and Seeds 1) can also partially suppress the phenotype of the bri1–5 mutant observed in leaves. Overexpression of BRS1’s homologs also increases the number of carpels and seeds, confirming the role of BRS1 and its homologs in the BR signaling [BRS1 potentially interacts with other BR genes in order to maintain balance in BR signaling is still unknown.Brassinosteroids (BRs) are essential plant hormones, regulating multiple processes amongst which plant growth, flowering, senescence, and seed germination . BR bios mutants , 3. In t mutants , 7. Afte mutants , 5. Upon mutants –10. In t1 (BRS1) . BRS1 isignaling . Yet, thbes1–1D and bzr1–1D backgrounds are not responsive to exogenous BRs, suggesting that BES1 and BZRI have also other functions than BR signaling [bri1 mutants show in addition to reduced growth, an increased stress-tolerance, further confirming the complexity and dosage sensitivity of BR signaling and regulation [BRI1 and BES1 [BRI1, BAK1, and BRS1 in regulating/restoring the response to BRs and/or in other functions independent of BR signaling.Some genes involved in BR signaling are also involved in other processes, such as stress response, and can act independently of the presence of BRs. Several studies found that ignaling , 15. In ignaling , 16–18. ignaling . Some brgulation , 21. Traand BES1 . Therefobri1–5 and its three activation-tag suppressors along with their corresponding wild-type, WS2. Two suppressor strains bri1–5/bak1–1D and bri1–5/brs-1D were obtained from [bri1–5/bri1–1D mutant was generated in the framework of the current study (see Methods). Sequencing the BRI1 flanking region from the suppressor bri1–5/bri1–1D showed that the activation tag was inserted 534 bp downstream of the BRI1 gene  lines displayed larger seedlings than the bri1–5 mutant, but still significantly smaller than the WS2 . The contribution of the epidermal cell length in recovering the bri1–5 is marginal in the bri1–5/bak1–1D (line with the largest seeding) but seems much more pronounced in the bri1–5/brs1–1D  and the bri1–5 background were sampled at a 7-day seedling stage. To assess the reproducibility of the expression analysis, we measured the extent to which the expression profiles of replicate samples were similar using Principal Component Analysis (PCA): PCA indeed showed that the largest fraction of the variation in gene expression between the samples could be assigned to differences in genetic background and not to differences between replicates of the same genetic background, confirming the reproducibility . This is in line with its observed phenotypic behavior as indeed bri1–5/bak1–1D seems to also phenotypically best compensate for the bri1–5 mutation.We determined for each mutant line its differential expression versus the same common reference i.e. the expression state in WS2, resulting in a total of 1413 differentially expressed genes . This suggests a similar role for BRI1 and BRS1 in the BR signaling pathway. Note that in Fig. bri1–5). In this way, the correlation analysis is driven by the expression of the genes that change their expression relative to bri1–5. Although this results in lower correlation values than when directly comparing the expression values of the mutant lines, it better reflects the consistency between mutant lines in restoring genes affected in the bri1–5 mutant.The Venn diagram in Fig. bri1–5 mutation, we compiled a list of marker genes representative of downstream pathways affected by BR signaling . Fig. bri1–5 mutant and how some of those genes got restored in the suppressor mutants. These results confirm what we observed based on the global expression analysis, i.e. that the bri1–5/bak1–1D restored the bri1–5 affected marker genes to the largest extent, and that molecularly the bri1–5/brs1–1D and bri1–5/bri1–1D mutant tend to behave more similarly in restoring the same marker genes.To confirm the extent to which the different suppressor strains molecularly restore the defects in the bri1–5 and all of the suppressor lines. These represent the pathways that are responsible for the aberrant growth phenotype in the bri1–5 mutant and that could not entirely be restored or compensated for in the suppressor lines. Among others, pathways related to cell wall synthesis , protein and lipid metabolism can explain the residual discrepancy between the WS2 growth phenotype and the suppressors.Pathway analysis (see Methods) unveiled the pathways overrepresented amongst the differentially expressed gene sets in each of the mutant lines. Fig. bri1–5 mutant, they could do so because they either restore the pathways disrupted in the bri1–5 mutant to wild-type levels or they induce genes that compensate for the bri1–5 affected pathways. Both mechanisms are reflected in the expression data. Processes that are aberrantly expressed in the bri1–5 mutant, but not in any of the suppressor lines, represent pathways that are restored to WS2 levels in all of the suppressors. This seems to be the case for some genes related to cytochrome P450 oxidase , ii) genes that were compensatory in most of the suppressors  and iii) genes altered in the bri1–5 mutant that most likely were not restored in the suppressors . This combined set of genes (789 genes) is referred to as the set of seed genes or the genes we want to maximally connect on the interaction network.To study the interaction between non-restored, restored, and compensatory pathways in more depth, we combined the following gene sets for network analysis see Fig. : i) genebri1–5 signaling deficiency in the suppressor strains.Network analysis  identified 8 sub-networks Fig.  containiHAI1, HAB1 ABI1, ABI2, and PP2CA acted as compensatory genes: these were are up-regulated in at least two bri1–5 suppressor lines compared to wild-type, but were not affected in the bri1–5 mutant , and HAB2 was identified as a connector node.In-depth analysis shows that the subnetwork enriched in ABA signaling  were identified as differentially expressed in all three suppressors . This indicates that ABA signaling has indeed been affected in the suppressor strains to compensate for the bri1–5 signaling deficiency. Of these, DTX50, HVA22D, PUB19, COR15B could not be connected by PheNetic on the interaction network, implying they are either not annotated in the interaction network (COR15B) or quite distantly located from each other in the network.Interestingly, several targets of the ABA signaling pathway . PP2C is known to indirectly repress ABI5, the main activator of ABA signaling [PYR/PYL/RCAR family as connector genes . The PYR/PYL/RCAR family constitutes the receptor of ABA signaling and promotes the activation of SnRKs by repressing PP2C [The aforementioned negative regulators of ABA signaling in subnetwork 1 belong to the protein phosphatase 2C (PP2C) gene family which has nine members in total  and the PP2C gene family. By partially recovering BR signaling in the suppressor lines, one would expect that PP2C gene expression levels restore to WS2 expression level. However, they appear to become up-regulated in suppressors, indicating that further compensatory repression of the ABA signaling is required in order to restore the bri1–5 phenotype.We could not find any link in the literature to explain how ABA signaling through BR signaling might affect other phytohormone pathways as well. Based on the pathway analysis using MapMan and the network analysis we can conclude that ABA signaling is mostly a compensatory pathway , ROS signaling (subnetwork 6), and ethylene signaling (subnetwork 8) were also detected. It is well known that crosstalk between these phytohormone signaling pathways exists , 29. HenCYP90C1, CYP90B1/DWF4, CYP85A2, CYP90A1/DWF3) that are differentially expressed in the bri1–5 lines and all of the suppressors. Those genes, belonging to the cytochrome P450 superfamily play a role in BR biosynthesis by converting the sterol “campesterol” to BRs [CYP708A3, another BR biosynthesis gene was found to be differentially expressed in all suppressors and the bri1–5 mutant  treatment [.The network result shows that the BR biosynthesis (subnetwork 7) is not well recovered in any of the suppressors. This subnetwork contains 4 genes , the less we expect the expression of the BR biosynthesis to be aberrant. We indeed found that the expression of the BR-biosynthesis genes  are less affected in the strains that better mimic the wild-type phenotype . This further supports the existence of negative feedback from BR regulation on BR biosynthesis: a more sustained BR signaling results in decreased BR biosynthesis, whereas suboptimal BR signaling is compensated for by higher transcriptional activity of BR biosynthetic genes.The fact that the expression of BR biosynthetic genes is affected by mutations in BR signaling genes points towards the existence of negative feedback of BR signaling on BR biosynthesis. If indeed negative feedback exists between BR signaling and biosynthesis, this feedback should be reflected in quantitative differences in overexpression of the BR signaling and biosynthesis genes in the BZR1 and BES1 [bri1–5 signaling deficiency by suppressors mutants, the transcript level of some stress-response genes is restored to normal, but other stress-response genes become induced . Based on GO analysis we mainly found stress related processes to be overrepresented in each of the groups. Genes involved in these stress related processes seem to be scattered over the interaction network as they could not be recovered as well delineated subnetworks, indicating that different stress-related genes are induced in the different lines. According to the literature, there is crosstalk between BR signaling and signaling by other hormones in response to stress, especially via ABA and auxin signaling . In the and BES1 , 15  are not only overrepresented in stress related processes (see above) but also in glutathione transferase (up-regulated),  were uniquely up-regulated in bri1–1D/brs1–1D  [+-ATPase transporters, CBC1, was significantly down-regulated in bri1–5/brs1–1D , but not in the other suppressors. This implies that H+-ATPase transporters are more active in bri1–5/brs1–1D to export H+ from cytosol into apoplast, making the apoplast more acidic , Fig. . This ovels Fig. . We also–1D Fig. . Membersgroup D, 3 genes aporters) . Accordiant Fig.  might beIn this study, we explored the alteration of gene expression in BR signaling mutants to better understand BR signaling and the functions of key BR genes.bri1–5 expression phenotype to WS2 levels. It is also obvious that restoring pathways can depend on the presence of compensatory genes in the same pathway. The crosstalk between BR signaling and signaling by ABA observed in our study is in line with the literature [SLAC1, KAT1 [bri1–5), SnRK2.3 can mimic the presence of ABA in triggering ABA signaling, once it is phosphorylated by BIN2 [ABI1 or ABI2, which encodes the negative regulator of ABA signaling could promote BR signaling [bri1–5 suppressor strains suppressed the ABA signaling (SnRKs) through up-regulating its negative regulator, PP2C  are transcription factors that affect root and shoot elongation [Links between BR signaling and auxin we observed are also supported by literature and in line with the observed phenotype of the ongation . In the ongation , 40. Thiongation , 41. Henongation .Pisum sativum [bri1–5 suppressor lines, supporting the impact of BR signaling on the development, and the composition of fatty acids.BR signaling controls development and growth by regulating metabolic processes such as fatty acid metabolism. For example, exogenous BR treatment was shown to promote leaf senescence, likely via the alteration of fatty acid composition in  sativum . Exogeno sativum . Similar sativum  and drou sativum  stress. bri1–5 mutant and is partially restored by suppressor lines.Transcription factors, regulators, oligopeptides, and proteins are an essential part of BR- and other signaling pathways. Previous studies showed that BR treatment increases the protein levels in the nuclei of hypocotyl cells , 45. Trabri1 mutants by the presence of a negative effect of the BR signaling proteins BZR1/BES1 on the BR-biosynthesis pathway. Although it cannot be excluded that the previously reported accumulation of BR precursors in bri1–5 mutants results from the inability of the mutant lines to use available BRs, our observation in BR signaling mutants suggests that aberrant regulation of BR biosynthesis can also contribute to the accumulation of BRs precursors: it seems that non-aberrant BR signaling is required for homeostasis of appropriate levels of endogenous BRs. Our results also show that the level of negative feedback depends on the degree to which the suppressor could compensate for the phenotypic difference between the bri1–5 and WS2. The better the defects in BR signaling were alleviated (as reflected by the phenotype), the less pronounced the observed effects on the BR biosynthetic genes. This observation also supports the hypothesis made by Gruszka et al. [In addition, our results provide evidence for negative feedback between BR signaling and BR biosynthesis. This hypothesis was already made by Noguchi et al.  who expla et al.  that BINbri1–5) the activated SnRK2 by BIN2 mimics the presence of ABA, activates ABI5, and finally regulates stress response genes  described in the literature or why treatment with exogenous BR give rises to a phenotypic response that is worse than the one observed in the bri1–5 mutant (shorter root) [Because BR signaling regulates response to a wide spectrum of stresses , it is ner root) . This fuIn addition, there is evidence that BAK1 plays a role in regulating stress-response pathways independently from BR signaling . Since Bbri1–5/brs1–1D suppressor line to compensate for the bri1–5 mutant showed that BRS1 seems involved in the acidification of the apoplast environment. We hypothesize that this acidification could contribute to an improved BRI1-BRs binding or BRI1-BAK1 dimerization and hence restoration of the bri1–5 mutant phenotype. In vitro studies have indeed shown that BRs preferentially bind to BRI1 in an acidic cell wall environment (pH < 5) [brs1–1D [bri1–5/brs1–1D line at least partially restores the epidermal cell length  , 48. It (pH < 5) . The sam(pH < 5) . In addi[brs1–1D . This isgth Fig. . These obri1–5/brs1–1D suppressor line also induces glutathione transferase activity which is necessary for redox homeostasis. This link between BR and redox signaling is in line with the literature [bri1–5 signal deficiency, it can be suggested that BRS1 does not have a regulatory role and only provides a better condition for triggering the BR signaling by making the apoplast environment more acidic.The terature . An oxidterature . Since aterature  but its bri1–5 dwarf phenotype. The fact that also other phytohormone signaling pathways are restored to the wild-type expression level in all bri1–5 suppressors confirms the crosstalk between BR and other phytohormone signalings. The negative feedback from BR signaling on BR biosynthesis was also confirmed by quantitative evidence. In addition, a new indirect role for BRS1 in BR signaling was suggested. However, our study is limited to transcriptome analysis and BR signaling is likely regulated to a large extent at the post-transcriptional level (i.e. phosphorylation). By using network analysis we can partially deal with this missing information, but this approach is limited by the incompleteness of the interaction network. Hence, more extensive validation studies are required to confirm our hypotheses. Besides, this study mainly focuses on the suppressor lines of bri1–5. Future work by adding enhancer lines such as bri1–5/bri1–1, bri1–5/brs1–1, and bri1–5/bak1–1 or by studying the effect of suppressors not only in the BRI1 as a loss-of-function background but also using double or triple mutants of multiple genes of the BRI1 gene family and/or BRI1-like genes [BRI signaling. In addition, future research by scRNA-seq of bri1–5 suppressor mutants across different time points of hypocotyl growth would be key to elucidating the detailed mechanisms of BR signaling at the level of cell types [In this study, we performed expression, pathway, and network analysis to provide more insight into the BR signaling by taking advantage of the availability of mutants for keys genes in BR signaling. Our results suggest that ABA signaling plays a significant role in alleviating the ke genes  could bell types , 54.bri1–5, bri1–5/bak1–1D, and bri1–5/brs1–1D were obtained from our previous study [bri1–5/bri1–1D was generated in this study as previously described [bri1–5), and its three suppressor mutants  were grown at 22 °C in a long-day condition (16 h of light and 8 h of dark) in a greenhouse for 7 days. All mutants were generated from the WS2 ecotype background. Microarray analysis of all genotypes was performed with three biological replicates. Total RNA of 7-day whole seedlings was isolated, labeled, and hybridized with an Arabidopsis ATH1 genome array according to the Affymetrix instructions. Scanning of the array was performed using the Agilent GeneArray Scanner. The data are available in GEO (GSE70843). The CEL files were preprocessed using the AFFY package ) [The two activation tagging suppressors of us study . An addiescribed . Wild-tyzation)) . The arrzation)) . All samzation)) . To defization)) . The prization))  and GO ezation)) .The phenotypic impact of suppressor lines was evaluated through the measurement of the root, hypocotyl, and epidermal cell length on the 7-day old seedlings. The root and hypocotyl lengths were measured on 40 plants and hypocotyl epidermal cells were scanned using the electron microscope (SEM) on samples taken from 3 random plants for each line. The mean of epidermal cell length per image was determined using Fiji software . Statistbri1–5 mutant line with the list of “high-confidence” BR-responsive genes was used to evaluate the consistency between the expression data of our study and literature and to determine which suppressor best restored the expression of those genes to WS2 level.We performed a literature study to compile a list of “high-confidence” BR-responsive genes, i.e. genes regulated by exogenous BR treatment or in a line containing a gain-of-function mutation in a BR signaling gene with consistent transcriptional response from at least five references , 63–71. A high confidence Arabidopsis interaction network was compiled from the following sources: 64185 FunTFBS regulatory interactions were obtained from PlantRegMap , 96,827 p-value to this logFC, we empirically estimated the distribution of the observed max logFC for all genes. As we expected that most of the genes would not change their expression this is an estimate of the null distribution. The mean (μ) and standard deviation (σ) of this distribution was estimated empirically using maximum likelihood implemented in the MASS package [To perform network analysis with PheNetic  network  package . Using t package : max(logFC)).gene _ score = abs(1 − 2 ∗ ϕThe edge weight between a source (S) and target (T) nodes was derived by the product of the S gene-score and T gene-score.expression subcommand was run in the downstream mode with the following parameters: min cost: 0.1; max cost: 5; step size: log scale between max and min cost with 28 steps; path-length = 4; k-best paths: 50; for all other parameters the default values were used. For each edge cost, the highest-scoring subnetwork was selected. Furthermore, the Jaccard index of all the subnetworks with the same edge cost was computed. For each cost, the subnetwork is rejected if it has a low stability score  or if it is too large (more than 500 interactions). The final subnetworks are then the union of all the “best subnetworks” for each edge penalty that passed the stability and size requirements.This weighted interaction network was used together with the seed gene list  in PheNetic , 24. Phebri1–5 mutant, but restored to WS2 level in at least 2 suppressors), compensatory genes , non-restored genes  and genes that are uniquely affected in respectively the bri1–5/brs1–1D (group D), the bri1–5/bak1–1D (group F), and the bri1–5/bri-1D (group E)  consists of 149 genes that are affected in bri1–5 mutant but not restored by any suppressors. However, as the size of the gene sets is dependent on the choice of an arbitrary threshold, we assumed that some of the genes belonging to the processes represented by these core gene sets were found to be significantly differentially expressed in two of the three lines only (and slightly below the threshold in the third). That is why we extended the core gene sets with the genes that were differentially expressed in at least two lines, rather than in all three of them. Extended gene sets A, B and C were subsequently combined to study the interaction between the restored, compensatory, and non-restored genes.To study the interaction between restored, compensatory, and non-restored genes, we combined the genes of groups A, B, and C (789 genes) to perform network analysis. Each group consists of its core genes. The core of group A (restored genes) consists of 118 genes that are restored to the wild-type state by all suppressors. The core of group B (compensatory genes) consists of 23 genes, i.e. genes that were significantly differentially expressed in all three Additional file 1. List of differentially expressed genes as compared to WS2 in each line and group membership. (XLSM 104 kb)Additional file 2. List of marker genes being differentially expressed upon addition of external BRs or in line with a gain of mutation in BR signaling genes at least in 5 studies.Additional file 3. GO enrichment for genes exclusively differentially expressed in each suppressor.Additional file 4: Fig. S1. The T-DNA insertion site for bri1–1D (A), and microscopic images of 7-day old hypocotyl cells for WS2 (B), bri1–5 (C), bri1–5/bak1–1D (D), bri1–5/bri1–1D (E), bri1–5/brs1–1D (F). Fig. S2. PCA plot for assessing the reproducibility of the gene expression dataset. Samples taken from the same genotype are represented in the same color. The plot indicates high consistency between replicate samples as they are located close to each other when plotted on the first and second principal components. Fig. S3. RT-qPCR results for relative expression of selected genes and their corresponding values from microarray analysis. The values represent the log2 of relative expression (sample1/sample2). Rows indicate gene names and columns show the comparison between the indicated lines. Columns with pink header represent the RT-qPCR values, and columns with yellow header are microarray measurements. The red color on the heatmap indicates that the gene has been up-regulated in sample1 as compared to sample2, while blue indicates down-regulation. Fig. S4. Comparing genome-wide expression impact between bri1–5 suppressor lines. Fig. S5. Heatmap of expression of the marker genes that up/down regulation of their expression was confirmed by at least 5 independent references and also affected in the bri1–5 line of our study. For each line, the row-scaled normalized expression data of the 3 biological replicates are shown as adjacent columns. In each row the gradient red color indicates the higher expression for the gene compared to other samples while blue indicates the lower expression. Fig. S6. Pathway analysis (MapMan metabolism) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S7. Pathway analysis (MapMan: large enzyme families) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S8. Pathway analysis (MapMan: gene regulation) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S9. Expression pattern in each mutant line of genes related to ABA signaling, Glutathione metabolism, and ion related hemostasis as discussed in the main text. Mutant lines are represented in the x-axis. The y-axis indicates the log2 normalized expression value of the gene. Table S1. RT-qPCR test of log-fold change (log-FC) of the genes that are overexpressed by activation-tagging in the suppressors at the 7 days seedling stage. Table S2. Summary of the most significant results obtained by MapMan pathway analysis . Left column: enriched pathways; entries provide for each line the degree to which the pathway is enriched. P-values are FDR corrected using Benjamini-Hochberg). Table S3. Designed primers for RT-qPCR.


This was supported by comments in the semi-structured interviews. Parent-reported PedsQL scores were lower than their child's post treatment (p = 0.007). Testosterone therapy for pubertal induction was associated with an improvement in QoL and the observed physical changes during puberty played an important role. Low self-esteem was also a prevailing theme. This data supports the inclusion of testosterone therapy for pubertal induction as a Standard of Care.Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy in childhood. It is associated with progressive muscle function decline and premature death. Long-term oral glucocorticoid use slows muscle weakness but is associated with several side effects including delayed puberty. This study assessed the impact of a 2-year incremental intramuscular testosterone regimen on quality of life (QoL) in a cohort of 15 adolescents with DMD. The Pediatric Quality of Life Inventory (PedsQL) Neuromuscular module was used to assess QoL and was completed by parent-child dyads. Semi-structured interviews were carried out to understand patient views on testosterone therapy. QoL scores increased in 10 of the 15 participants during treatment, with a mean total PedsQL score of 74.6 pre-treatment v 80.2 post treatment ( The quantitative outcome measures of this study have been reported elsewhere (1)The Pediatric Quality of Life questionnaire (PedsQL) to assess quality of life and(2)Semi-structured interviews to gain a deeper understanding of the challenges faced in the adolescent with DMD and during pubertal induction.The objectives were to use:22.1Participants were part of a single center study (NCT02571205) following the progress of 15 adolescents with DMD and delayed puberty as they were treated with testosterone to induce puberty. Males aged between 12 and 17 years old who were receiving GC were recruited between December 2015 and December 2016. Full details of the protocol have been published 2.2The 25-item PedsQL 3.0 Neuromuscular Module is validated for the assessment of QoL in DMD ,9. The P2.3Participants were given the option of consenting to a semi-structured interview to gain insight into the boys’ views on the challenges they face and the use of testosterone therapy. Interviews with the adolescents were carried out by the same member of the study team (CW). Some of the participants chose to have a parent present during the interview. A sample question list was used as a guide and drawn up after consultation with a qualitative data analysis expert with individual interviews guided by responses. Coding and thematic analysis was used and themes explored until saturation 2.4Paired t-tests were used to compare QoL before and after testosterone treatment and unpaired t-tests to compare parents estimations of their son's perceived QoL compared with the self-reported scores.33.1p = 0.04) (p = 0.44)), although post-treatment mean scores were greater across all domains  out of a maximum of 100 at baseline. Following the 2-year incremental testosterone regimen, there was an increase to 80.9  , Fig. 1. domains , Fig. 1. domains .Table 1Pp = 0.12) and post-treatment  (p = 0.24) and post-treatment patient 75.5, parent 61.1 (p = 0.22)).Patient-reported PedsQL scores were higher than parent-reported scores pre-  . This su3.2‘not fitting in’ was an overarching theme throughout the interviews I'm hoping my voice will change a bit”Feeling self-conscious about the pitch of voice- “(b)“Everyone when they look at me thinks that I am younger. They ask what year I am in, not in a nasty way, but am always surprised when I say….”Looking younger-(c)“Looking forward to being hairier.”Lack of hair growth-(d)“I want to take part, to make going to the toilet easier, you know.” (Mum then explained that toileting would be easier if his penis was bigger.)Penis size- Concerns regarding pubertal development were also expressed in a variety of ways including:If these individual concerns were grouped together to represent concern over ‘lack of pubertal development’ it may be considered as an equally common concern to short stature.“The best thing to come from the testosterone injections is that I have grown taller.”“I've grown in height and my voice has broken.”I feel more confident with young people of the same age group as myself.”““I feel much better and stronger.”After testosterone treatment, several of the boys commented on feeling and looking older and having grown: I would like a bit more confidence.” Another boy described having to get changed separately from the other children at school for PE because he takes longer as “a relief so people don't notice me” and another said “I don't like to use my wheelchair at school.” Some parents (who sat in with their children at the request of the child) expressed concerns about confidence levels:“He definitely has low self-esteem.”“He doesn't perceive that any girl would find him attractive. If he gets any attention, he's chuffed to bits.”The theme of low self-esteem featured throughout the interviews . Some bodifficulties negotiating transparency and openness, referring to the young person's struggles to communicate with their peers about their condition  are making me short and look young but I am doing ok with them so don't want to stop.”It was widely recognised among the boys that short stature was largely a result of their steroid medication but many acknowledged the beneficial effects of steroids in terms of preserving muscle function, 4Higher PedsQL scores were reported by most participants at the end of the study, suggesting that the testosterone regimen was well-tolerated and that pubertal induction was associated with improved QoL. This is consistent with the TSQM data, which showed a mean total score of 48.5  out of a possible 59 points, with similar scores in each domain. To the nearest integer, the mean score for all but one treatment domain was 6 .None.


Domestication of wild animals results in a reduction in the effective population size, and this could affect the deleterious mutation load of domesticated breeds. Furthermore, artificial selection will also contribute to the accumulation of deleterious mutations due to the increased rate of inbreeding among these animals. The process of domestication, founder population size, and artificial selection differ between cattle breeds, which could lead to a variation in their deleterious mutation loads. We investigated this using mitochondrial genome data from 364 animals belonging to 18 cattle breeds of the world.Our analysis revealed more than a fivefold difference in the deleterious mutation load among cattle breeds. We also observed a negative correlation between the breed age and the proportion of deleterious amino acid-changing polymorphisms. This suggests a proportionally higher deleterious SNPs in young breeds compared to older breeds. Our results highlight the magnitude of difference in the deleterious mutations present in the mitochondrial genomes of various breeds. The results of this study could be useful in predicting the rate of incidence of genetic diseases in different breeds.The online version contains supplementary material available at 10.1186/s13104-021-05664-y. Earlier studies revealed a much higher ω for domesticated pig, dog, rabbit, silkworm, rice and sunflower compared to their wild relatives [ω estimated for different breeds of dogs were found to correlate with their silent diversity [Domestication of wild animals results in a drastic reduction of effective population size as only a subsample of the wild population is used. Artificial selection further reduces the population size due to inbreeding within domesticated animals, which occurs during breed formation . The rednal load –6. Sinceelatives –5, 7, 8.iversity . Since tiversity . Young hThe present study is focused on investigating this by estimating the deleterious mutational load of the mitochondrial genomes of 18 cattle breeds and comparing them with the age of the breeds.Bos taurus were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) by searching using the keywords Bos taurus, mitochondrion, and complete genome. The records without specific breed information were excluded. Furthermore, only the breeds that have mitochondrial genomes from more than four individuals were included. To reduce estimation errors, breeds having at least one synonymous and nonsynonymous variant or SNP were included. This resulted in 364 mitogenomes belonging to 18 breeds. The accession number and locations are given in Additional file mafft [https://mafft.cbrc.jp/alignment/server/). After alignment, all 13 protein-coding genes (CDS) were to form a super alignment containing 11,229 base pairs from 364 animals.A total of 578 mitochondrial genomes of le mafft —online vπN) and synonymous (πS) diversities using the Pamilo-Bianchi-Li method [ωP = πN/πS) for each breed was estimated. Furthermore, the ratio (ωS) of interspecies synonymous (dS) and nonsynonymous (dN) divergence between cow and bison mitochondrial protein-coding genes was also computed (ωS = dN/dS). Using the population and species level estimates of the ratios, it is possible to calculate the proportion of deleterious mutations (δ) segregating in a population using the following formula [The CDS alignment was then used to estimate nonsynonymous  and synonymous (πS) diversities were estimated for the concatenated alignment of 13 mitochondrial protein-coding genes, and their ratio (ωP = πN/πS) was calculated for each breed population. As shown in Fig. ωP varies more than fivefold (5.1) among cattle breeds, with Creole having the highest ωP of 0.59 and the lowest was observed for Iraqi (0.116). This suggests much higher harmful mutations are segregating in the breed Creole compared to their Iraqi counterparts.The complete mitochondrial genome sequences of 364 individuals belonging to 18 cattle breeds were obtained from GenBank  segregating in each breed using the equation (see “ωS = dN/dS) by comparing the mitochondrial protein-coding genes of cow and bison. While ωS indicates the proportion of nonsynonymous mutations fixed in the cow lineage, ωP denotes the proportion of nonsynonymous mutations or SNPs segregating in the cattle populations. Population genetic theories predict that neutral (mutations with no harmful effects) mutations are fixed in a species. Therefore, the value of ωS suggests the proportion of neutral nonsynonymous mutations fixed in cattle. If ωP = ωS, then the nonsynonymous SNPs segregating in cow breeds are effectively neutral. However, ωP > ωS suggests an excess of nonsynonymous SNPs segregating in cattle breed populations. This excess SNPs are potentially deleterious, and they will be removed from the population over time. The deleterious excess fraction could be calculated using the equation in “To accurately quantify the harmful mutations, we calculated the proportion of harmful mutations (ion see “” developδ) and a highly significant negative correlation was observed   Fig. . This suA deeper look at the haplogroups of the mitogenomes revealed that young breeds belong to only a single haplogroup, whereas older breeds belong to broader haplogroups. For instance, the age of Creole and Japanese Black are around 8.3 Kyrs, and they exclusively belong to T1 and T3 haplogroups, respectively (Additional file It is well known that deleterious mutations segregate in young populations, and over time they are removed by natural selection. This has been well demonstrated in human populations, and the young European haplogroups such as H and T have more deleterious mutations than the older African haplogroups such as L0 and L1 . Our resThe present study revealed that the deleterious mutation load significantly varies between different cattle breeds. Hence the results of this study could be useful in predicting the rate of incidence of mitochondrial genetic diseases in various breeds.This study is based on mitochondrial genomes that are uniparentally inherited. Furthermore, recombination is not well documented in mitogenomes. Therefore, it is unclear whether a similar pattern of mutation load is expected in nuclear genomes. Hence, further studies based on large number of nuclear genomes is required to investigate this.Additional file 1: Table S1. List of Genbank accession numbers and breed information. Table S2. Haplogroups and age of breeds


Microfluidics offers promising methods for aligning cells in physiologically relevant configurations to recapitulate human organ functionality. Specifically, microstructures within microfluidic devices facilitate 3D cell culture by guiding hydrogel precursors containing cells. Conventional approaches utilize capillary forces of hydrogel precursors to guide fluid flow into desired areas of high wettability. These methods, however, require complicated fabrication processes and subtle loading protocols, thus limiting device throughput and experimental yield. Here, we present a swift and robust hydrogel patterning technique for 3D cell culture, where preloaded hydrogel solution in a microfluidic device is aspirated while only leaving a portion of the solution in desired channels. The device is designed such that differing critical capillary pressure conditions are established over the interfaces of the loaded hydrogel solution, which leads to controlled removal of the solution during aspiration. A proposed theoretical model of capillary pressure conditions provides physical insights to inform generalized design rules for device structures. We demonstrate formation of multiple, discontinuous hollow channels with a single aspiration. Then we test vasculogenic capacity of various cell types using a microfluidic device obtained by our technique to illustrate its capabilities as a viable micro-manufacturing scheme for high-throughput cellular co-culture. Microfluidic co-culture platforms embedding multiple cell types suspended in hydrogels can recapitulate specific organ functions and thus provide insights into complex physiological systems4. Microstructures within these platforms facilitate the arrangement of cells into physiologically relevant layouts5. For example, micropillars6 or bumps (named as "phageguide")7 constructed on hydrophobic surfaces form narrow gaps between microchannels, resulting in a high bursting pressure for a liquid to infiltrate into adjacent microchannels. When a hydrogel precursor is loaded into a microchannel with lower pressure than the bursting pressure of the gap, the liquid will completely fill the microchannel without disturbing adjacent channels. Different types of cell suspensions or hydrogels containing cells can later fill the remaining adjacent channels after cross-linking of the pre-loaded hydrogel precursor. These microstructure-mediated hydrogel patterning methods can mediate meniscus-pinning for precisely segregating co-cultures in hydrogels to model vasculature10, tumor extravasation dynamics11, glomerular filtration barrier12, and intestinal epithelium tubes13. The hydrogel loading guided by surrounding microstructures, however, fails when the hydrogel pressure exceeds a limit, or capillary-bursting pressure, causing the gel to invade the undesired region15. Thus, a great care should be taken to limit the injection pressure, resulting in yield variation depending on the operator’s pipetting skill and low throughput associated with short available time of hydrogel precursor16. Furthermore, the complex microscale features of such devices, achieved typically through soft lithography, preclude application of scalable manufacturing processes like injection molding.Proper understanding of interactions between various cell types is essential in tissue engineering and design of in vitro organ models22. Here, rails denote long hydrophilic plates between which liquids are confined owing to capillary forces, while the side areas are exposed to the air23. In those studies, spontaneous capillary flow along corner or rail structures enhanced yield by reducing the effect of user’s dispensing pressure. Furthermore, their structures are simple to produce with injection molding so that the open microfluidic design also enhanced productivity. However, the air–liquid interfaces in rail-based microfluidics are highly sensitive to the volume of liquid dispensed, thus requiring a careful loading process with accurate volume of cell-laden hydrogel precursors24 or requiring additional design for rail structures to reduce the reliance on a precise volume25.Open microfluidic devices that utilize spontaneous capillary wicking along rail structures present an alternative avenue for gel patterning without relying on the capillary-burst valve26. Even though some aspiration-mediated patterning within microfluidic systems have been introduced29, they were limited to provide full understanding of the patterning mechanism under rail structures. Here, we further characterize the aspiration-mediated patterning method under unique rail structures which yields agile, easy and accurate positioning of hydrogels with only a simple pipetting activity. The scheme relies on the capillary holding capacity of rail-based microstructures against aspiration upon their geometry, which can be tailored to leave hydrogel precisely in desired location. Although delineated below in detail, the working principle is based on the dynamic characteristics of a solid–liquid-gas contact line that it starts to recede when the local contact angle gets smaller than the critical receding angle. By defining where the recession starts and stop with geometric parameters like aspiration port diameter and rail height, we can selectively remove hydrogel from undesired regions. We characterized design rules for successful patterning based on theoretical analysis and experimental verification using simplified structures. Proper design provides control over the sequence of microchannel formation, which enables the formation of multiple discrete channels with a single aspiration. A proof-of-concept device featuring four microchannels separated by hydrogel structures is shown to be able to co-culture five distinct cell types. Our rail-based open microfluidic devices are amenable to fabrication by injection molding26, thereby suggesting their great potential as an easy-to-use co-culture platform with low cost and high manufacturing scalablity.In our previous study, we have introduced an aspiration-mediated hydrogel micropatterning technique within a rail-based open microfluidic device and utilized it for 3D immune cell cytotoxicity assayl, is 5 mm, and the width of low and high rail, h = 100 μm and H = 300 μm. Two through-holes at the ends of a high rail function as fluid ports for liquid injection and aspiration. Air plasma treatment activates the surface of the device by increasing hydrophilicity. We first inject a hydrogel precursor solution through one of the fluid ports, which fills the area under all rails  film. 3D-printed rail structures consisted of high rails (HRs) surrounded by low rail (LRs) and reservoir walls which supports rail structures and are bonded to the PSA film. HRs and LRs have different heights from the PSA film to retain liquid with different strengths Fig. a. Figurementclass2pt{minimmentclass2pt{minimmentclass2pt{minima) and the liquid (pl): ∆p = pa − pl = γ(1/R1 + 1/R2), where γ is the gas–liquid interfacial tension and R1 and R2 are two radii of curvature in orthogonal planes intersecting the interface30. The contact line where the gas–liquid interface meets the solid surface is initially immobile while resisting the pulling force, because it can recede only when the contact angle is reduced below the critical receding contact angle, θR,c. The patterning of hydrogel that originally fills the gaps under both HRs and LRs critically depends on which interface starts to recede first by reaching θR,c.As the pressure at a port is lowered by aspiration with a micropipette, the interior pressure of the hydrogel precursor solution is also lowered. The Young–Laplace equation states that the liquid–gas interface of the hydrogel precursor solution originally filling the gaps caves inward , where θ is the instantaneous contact angle of liquid, h is the height of the rail, and η is the radius of curvature on a plane parallel to the substrate. During aspiration, θ decreases until reaching θR,c when the capillary pressure is maximized. We experimentally found that θR,c is nearly zero for hydrogel precursor solution on the hydrophilized 3D printed part of resin and PSA film surfaces. Hence, we estimate cosθR,c to be 1 due to the small receding contact angle, θR,c. Furthermore, η scales with l, the length of rails, and h <  < l for most rail designs, thus the 1/η term is negligible. Hence, we estimate the maximum pressure difference for the side area below LRs as ∆pL = 2γ/h.For the initial hydrogel solution-loaded configuration, liquid–gas interfaces arise at the remaining port opposite to the port of aspiration, and lateral faces of the fluid filling the gaps below the LRs. We will refer to the opposing port as an “open port” in the following analysis. For the open port of diameter O < ∆pL, the critical liquid pressure for interface recession is reached at the open port before the side area. Once burst at the open port, the interface spontaneously bulges accompanying the decrease of interface curvature to meet the bottom substrate. Then the interface curvature is newly defined by the height (H) and width (wH) of HR . If ∆pL > ∆pO > ∆pH, the liquid pressure is already low enough to ensure continuous recession of the liquid beneath HR. If ∆pL > ∆pH > ∆pO, further decrease of the liquid pressure following bursting of the open port can cause the liquid to recede along a lane beneath the HR. In short, both cases of ∆pL > ∆pO > ∆pH and ∆pL > ∆pH > ∆pO result in successful patterning. However, if ∆pH > ∆pL, the liquid will stop upon bursting at the open port and recede beneath LRs, rather than HRs, resulting in patterning failure.If ∆pL is greater than both ∆pO and ∆pH, and thus the interface recession occurs from the open port, continues beneath HR, to result in a straight empty lane flanked by the hydrogel beneath LR. In the middle column, the side areas beneath LRs burst before the open port because ∆pL < ∆pO. In the right column, the open port bursts first because ∆pL > ∆pO, but further decrease of the liquid pressure bursts the side areas beneath LRs before the interface from the open port starts to recede beneath HR.Figure L = ∆pO, or D = 2 h. For D > 2 h, we get ∆pL > ∆pO, and thus the port bursts first, leading to successful channel formation in the beginning. Otherwise, the interface moves at the side area of LR before at the port, preventing the formation of the microchannel under HR. Figure L = ∆pH, or H = h/(1 − h). For H > h/(1 − h), we get ∆pL > ∆pH, leading to continuous recession of the interface under the HR from the open port. Otherwise, the interface under LR starts to recede, eventually merging with the interface originated from the open port. We see in Fig. Figure or H = h/ − h. ForL = 1439 Pa, ∆pO = 479.8 Pa, and ∆pH = 311.9 Pa. To apply different aspiration pressures, we applied maximum aspiration pressures of four different types of micropipettes whose maximum handling volumes are 10, 20, 100, and 200 μl, respectively. Maximum aspiration pressures of the four micropipettes were measured by tracking the velocity of receding fluid interfaces in straight microfluidic channels as detailed in the supplementary information and Table PL under the LR also allow for successful patterning. We expect that further studies of meniscus kinetics using pressure-controlled pumps may explain how successful patterning can still result from aspiration pressures greater than the capillary pressure at interfaces under LRs.To determine the effect of aspiration pressure on patterning, we conducted patterning tests by aspirating deionized water that filled the region under a 3D-printed basic rail structure whose D, h, and H are 600 μm, 100 μm, and 300 μm, respectively, with various aspirating pressures. For the designed parameters, maximum capillary pressures at each interface are ∆p26. The reduced uptake procedure can enhance experimental throughput when loading hydrogel precursors in microfluidic devices. This is especially beneficial when handling gels with a short pot life, such as fibrin gels whose cross-linking time is approximately 1 min16. Using aspiration-mediated patterning, it took around 20 s to fill a dozen devices, while the conventional method that injects the exact amount of hydrogel solution for each device took approximately two-fold longer.The unique nature of this hydrogel patterning method is advantageous in filling multiple devices. To fill a series of devices using conventional pipetting methods, the operator must repeatedly uptake the exact volume of a single device from the stock container before loading the next device. In contrast, the present technique can uptake the combined volume of all desired devices at the onset of loading. Each device is initially overfilled by arbitrarily depressing the pipette prior to aspiration of the excess fluid by releasing the pipette piston. Video In this manner, the aspiration-mediated patterning method achieves improvements in throughput using a manual micropipette that typically necessitate costlier electronic pipette equipment with automated dispensing capabilities. Furthermore, our experiments show that such electronic pipettes become less reliable with certain chip designs that require small fluid volumes (~ 1 µL) and produce high capillary action. Video D, h, and H are 600 μm, 100 μl, and 300 μm, respectively. We measured the area of dyed water that remained after aspiration and compared it with the area of 1.2 μl of dyed water injected through one of two ports in the HR. Even though water is injected through a port, the water fills the area under LRs instead of the HR due to large capillary force. Consequently, both methods result in similar positioning of dyed water. The uniformities of the remained dyed water were 92.5% and 89.0% for the aspiration method and manual injection through a port, respectively, and they showed no statistical difference as shown in Figure To further characterize patterning uniformity, we conducted aspiration-mediated patterning with dyed water using a basic 3D-printed device, whose Our fluid dynamic studies using basic rail structures in the previous sections verify that the air–liquid interface with the smallest critical capillary pressure tends to move first along the rail. Leveraging this phenomenon can provide control over the sequential formation of discrete microchannels with a single aspiration. Figure 30. In the video, a continuous microchannel forms in the top right HR because it contains the port of aspiration, and thus only a single port for air infiltration. In contrast, a similar device employing asymmetrical port sizes in each HR exhibits unidirectional movement of air–liquid interfaces beneath HRs as shown in Video During multiple channel formation, differences in port diameter within the same channel lead to orderly movement of the air–liquid interface. If ports of the same diameters are employed, air can infiltrate into the channel through the identical ports simultaneously, to form a thin film of liquid between the infiltrating air from the two ports, as shown in Video This unique liquid patterning method enables versatile rendering of microchannel in hydrogels. Figure 31, it is not always observed in in vitro models. Previous studies also showed very diverse vasculogenic capacities of cancer cell lines from different organs within an injection-molded device33, and required additional co-culturing of endothelial cells or fibroblasts to mimic in tumor vasculature34. These results guided us to develop a microfluidic model to screen vasculogenic capacities of cell lines for better recapitulation of tumor angiogenesis. Modification of the device in Fig. 6 cells/ml) and primary human normal lung fibroblasts  under the LR, and subsequent seeding of fibrin gels containing different cell types into the rendered microchannels establishes the basic experimental setup  3D Printing  produced the bodies of open microfluidic devices with a photo curable resin . Rinsing with isopropyl alcohol in an ultrasonic cleaner for 15 min followed by curing ultraviolet curing  thoroughly solidified the printed pieces. Vapor deposition of parylene  onto the 3D printed parts ensured device biocompatibility. Attaching the bottom side of the printed part to a PSA film completed the open microfluidic devices. Air plasma treatment of the device  prior to fluid injection imparted hydrophilicity to the device surface for facilitating patterning of fluids under rail structures.Three unbiased students performed aspiration-mediated patterning and injection through a hole three times respectively for each method using 3D printed basic rail structures. A digital microscope  captured images after patterning green dyed water under low rails. ImageJ splited the images based on R, G and B values, and wand tool in ImageJ with tolerance value of 60 selected the area of green dyed water in splited images based on G values. We measured the area of green dyed water and calculated mean deviation uniformity following the equation, U = [1 − (Max − Min)/(2Avg)] × 100 (%).Cell culture experiments utilized human umbilical vein endothelial cells  cultured to passages between 4 and 7 in endothelial growth medium , and normal human lung fibroblasts  cultured up to passage 7 in fibroblast growth medium . CCD-18Co, HepG2, U87MG and H1299 cells were cultured in RPMI 1640 (Thermo Fisher) supplemented with 10% of FBS, penicillin (100 U/ml) and streptomycin (100 U/ml). 0.25% trypsin–EDTA (HyClone) facilitated detachment of cultured cells from the culture dishes. Re-suspension with appropriate amounts of culture medium provided target cell densities before mixing the cell suspension with bovine fibrinogen solutions in ratios of 3:1.3D printed rail structures consisted of high rails surrounded by low rails. HRs housed holes that served as ports for injection of fluids and air infiltration. Injection of fluid via a micropipette through a port in the HR and immediate aspiration generated micropatterned fluid under LRs. Subsequent cross-linking of the solution resulted in patterned hydrogels and adjacent microchannels. Visualization of the patterns employed a solution of bovine fibrinogen  dissolved in water and mixed with green food dye. Mixing with 50 U/ml of thrombin (Sigma) solution in a 30:1 ratio (v/v) at room temperature began the polymerization reaction of fibrinogen to fibrin. Exposing the loaded device to ambient conditions for 5 min resulted in fully crosslinked green dyed fibrin gel.2, 3 min) resulted in fully crosslinked fibrin gel encapsulating cells. Culture of the cellular gels involved adding EGM-2 into each well of the device, followed by incubation for five days with media changes every two days.For microfluidic co-cultures, fibrinogen solution (10 mg/ml) mixed with cell suspension in ratios of 1:3 (v/v) yielded cellular gels with a final fibrinogen concentration of 2.5 mg/ml. Mixing with thrombin solution (25 U/ml) in a 50:1 ratio immediately before injection into the fluid ports began the polymerization reaction. Immediate patterning and incubation in a cell incubator  was used for visualizing blood vessel networks. Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 15 min, and stained with the fluorescence labed antibody diluted to 1:200 in 3% bovine serum albumin for overnight at 4 °C.AlexaFluorA digital microscope  captured time-lapse images of microchannel formation and images of various microchannel designs. Image analysis using ImageJ of 3D printed bodies provided measured diameters of fluid ports. Confocal microscopy  imaged labeled blood vessel networks through optical z-sectioning . Image analysis for vascularized area measurement utilized Z-projection and conversion to binary images using auto threshold in Fiji following the “Li” method.One-way ANOVA with pair-wise comparisons by the Tukey post hoc test was used to determine whether six data-sets were statistically significant. At least five samples for each condition within two independent experiments were used for the imaging and data analysis. *** denotes p < 0.001, ** denotes p < 0.01, and * denotes p < 0.05.Supplementary Information 1.Supplementary Video S1.Supplementary Video S2.Supplementary Video S3.Supplementary Video S4.Supplementary Video S5.Supplementary Video S6.


The effect sizes for the 2 main independent variables in the 2 columns in Table 3 are comparable . Thus, the study’s conclusions remain unchanged. Also, from “Age group” on down to “Metropolitan area,” in the 2 columns representing model B and model C in Table 3, the aRRs and 95% CIs were inadvertently switched. Similarly, eTable 3 had columns that were inadvertently switched but with comparable effect sizes, again leaving the study’s conclusions unchanged. Finally, the title of eTable 2 has been corrected to better reflect that death counts rather than mortality rates were used as a dependent variable in statistical modeling. This article has been corrected.1In the Original Investigation titled “Association of Pharmaceutical Industry Marketing of Opioid Products With Mortality From Opioid-Related Overdoses,”


However, the PETM onset coincides with a mercury low, suggesting at least one other carbon reservoir released significant greenhouse gases in response to initial warming. Our results support the existence of ‘tipping points’ in the Earth system, which can trigger release of additional carbon reservoirs and drive Earth’s climate into a hotter state.The Paleocene–Eocene Thermal Maximum (PETM) was a period of geologically-rapid carbon release and global warming ~56 million years ago. Although modelling, outcrop and proxy records suggest volcanic carbon release occurred, it has not yet been possible to identify the PETM trigger, or if multiple reservoirs of carbon were involved. Here we report elevated levels of mercury relative to organic carbon—a proxy for volcanism—directly preceding and within the early PETM from two North Sea sedimentary cores, signifying pulsed volcanism from the North Atlantic Igneous Province likely provided the trigger and subsequently sustained elevated CO The Paleocene–Eocene boundary coincided with runaway global warming possibly analogous to future climate change, but the sources of greenhouse gasses have remained unresolved. Here, the authors reveal volcanism triggered initial warming, and subsequent carbon was released after crossing a tipping point. However, the major sources of carbon and the causal mechanisms triggering its release have remained under debate7, stymying our ability to draw firm inferences relevant to the future. Plausible carbon sources include peatlands and permafrost3, methane hydrates8, sedimentary marine organic matter10, and the mantle5, while proposed hypotheses for the PETM trigger include changes in orbital insolation11, volcanic activity of the North Atlantic Igneous Province  values observed in the sedimentary record marking the PETM onset and importantly, separating triggers from carbon-climate feedbacks. For example, a recent study5 concluded that volcanically produced carbon was the main source to explain proxy records of ocean pH across the PETM, but was unable to resolve the relative timing and contribution of different kinds of volcanic and non-volcanic carbon. Other studies have shown that NAIP volcanic rocks  were formed approximately around the time of the PETM12, but limited dating of individual local geological features makes it difficult to conclude confidently whether volcanism provided the trigger. Here, we investigate new sediment cores from the North Sea for both high resolution δ13C stratigraphy and sedimentary mercury (Hg) as a proxy for volcanic emissions.The relative geological rapidity of warming and CO16 as well as earlier large igneous provinces17. Today, approximately 20–40% of natural global Hg emissions come from the Earth’s crust via volcanism18. Released to the atmosphere, the ~0.5–1 year residence time of Hg allows it to be mixed globally, and when subsequently deposited to the ocean or as peatlands its preservation is predominantly via association with organic matter23. As such, normalised to total organic carbon (TOC), Hg has previously been used as a proxy for global volcanism17. Mercury can also be released hydrothermally, with modern hydrothermal vents being associated with locally-elevated Hg concentrations in water, sediments, and biota24. In the case of the PETM, Hg released via hydrothermal vents12 associated with the NAIP may not necessarily have entered the atmosphere but instead be detectible in NAIP-proximal ocean sediments7, where the modern residence time of Hg in the open ocean is decades to centuries, and on shelves is ~4 months which is considerably less than the residence time of shelf water25.Variation in the concentration of Hg in organic carbon-rich marine sediments has shown promise as a direct means of elucidating regional-scale volcanic activity such as associated with the PETM26) and E−8X 27 compared to atmospheric deposition (19.5 ± 9.5 Mmol year−1)28, isotopic studies have shown terrestrial Hg to be constrained to coastal and nearshore shelf environments29 whilst our core sites are >200 km distal in the central North Sea. Well sites 22/10a−4 and E−8X are located ~400 km and ~600 km proximal to NAIP volcanic centres, respectively , although these datasets are skewed towards studies of Large Igneous Province volcanism and may therefore be above overall average shale background. In contrast, our records show Hg spikes up to values comparable to modern contaminated sediments of >1000 ppb (ref. 35), and Hg/TOC spikes of 200–500 ppb/wt%. Looking further afield to Svalbard , but also shows numerous Hg/TOC pulses  and sedimentary transport/preservation effects might also come into play, submarine eruptions would likely produce predominantly local Hg enrichments36. Any occurrence of marine anoxia cannot account for the Hg, as increased sedimentary drawdown would have rapidly depleted the water column if dissolved Hg were not continuously replenished17. Neither can Hg released from permafrost melting account for the observed (up to a factor of 4) Hg increases, as releasing even a modern permafrost inventory of 330–1300 Gg of Hg (ref. 23) over 2 kyear (our estimated approximate Hg pulse duration) equates to a release rate no more than the modern volcanic background rate of 700 Mg Hg/year (ref. 18), with this doubling unlikely to register clearly in the composition of sediments given other causes of background fluctuations. Finally, our records are characterised by multiple peaks of Hg/TOC, inconsistent with a single bolide impact or permafrost melting event16. Therefore, our results do suggest pulsed volcanic Hg releases occurred sporadically throughout the study interval37, and in particular around the PETM onset.Both cores E−8X and 22/10a−4 show numerous high frequency and high amplitude fluctuations in Hg and Hg/TOC above background levels of ~50 ppb and ~40 ppb/wt%, respectively , and a shift towards lower TOC values at E−8X (~2024.5 m). Further Hg pulses occur at E−8X and BH9/05 and then decline further as δ13C continues to fully recover.Core 22/10a−4 has the longer pre-PETM section Fig.  and reco26 Fig. , althoug13C is consistent with volcanism triggering PETM carbon release and also contributing to sustaining the CIE during its main phase12. This is evidenced by the Hg and Hg/TOC peaks immediately before and during the initial negative CIE onset, and later within the main phase of the PETM before δ13C begins to increase again  are essentially synchronous at our (continuous at E−8X) sampling resolution (>100 years per sample). This is because the oceanic residence time of shelf Hg, and the time it would take volcanic CO2 to reach the atmosphere, mix, and be sequestered into ocean sediment via phytoplankton, is months to years. These Hg/TOC pulses are not likely to have been caused by changing sedimentation rates, as reporting Hg as a ratio to TOC aims to remove the influence of changing background sedimentation. However, changing delivery and source of sedimentary TOC (transportation and reworking) can modify Hg/TOC trends. Well sites E−8X and 22/10a−4 were within the deepest part of the North Sea Basin ~200 km from land, at palaeo-water depths of up to ~500 m, well below storm wave base26. Well site E−8X is exceptionally useful for Hg analysis as we see no evidence for highly variable sedimentation rates in the high TOC fine-grained claystone. The location and sedimentation suggest the carbon was at least partly of marine origin, as the δ13C record shows a similar CIE shift to that measured on dinoflagellate cysts at New Jersey38, and shows no smoothing that might typify a transported terrestrial and/or reworked carbon setting39.The timing of Hg/TOC pulses relative to the evolution of δ7 are somewhat different , or Bass River (New Jersey)  which is argued to have exposed that site to significant reworked terrestrial carbon that could have residence times of thousands of years39, possibly muting some Hg signals. However, records from Svalbard and Denmark are useful for constraining the extent of Hg signals, and broadly show a baseline decrease in Hg/TOC and Hg with distance from the NAIP , although we acknowledge our records cannot discount potential additional sources of CO2. The latter part of CIE step 1 coincides with increased Hg  that could reduce Hg/TOC signals, but E−8X shows no sign of reworking with a clear and rapid CIE onset and central deep North Sea Basin location. In Svalbard, there is a general increase in the Hg/TOC baseline over the CIE onset, and at Fur sedimentation rates significantly increase during the PETM41 such that Hg deposition rates likely increase even though concentrations do not. However, there is no Hg/TOC evidence for any substantial increase in volcanism during CIE step 2 (unlike step 1). We note that all other E-8X instances of reduced Hg/TOC in this interval , and increases , although the changes outside of the CIE steps are subtle.The high resolution and clear carbon isotope signals from core E-8X allow examination of the structure of the CIE onset, where the negative δear Fig. . Althougear Fig. , CIE ste Hg Fig. , before 2, annual release rates would have been an order of magnitude above modern volcanic CO2 emissions, and therefore possibly raised annual Hg deposition to at least an order of magnitude above modern18 and be detected in our samples. Alternatively, if intruded into organic-rich mudrocks such as those that underlay much of the NAIP12 we might expect even higher Hg deposition in the North Sea sediments. Therefore, this short-lived reduction in Hg/TOC during CIE step 2 is important as it points to a secondary phase of carbon release from a reservoir not directly linked to Hg emissions and hence likely not to volcanism. The main possibilities for such a feedback reservoir are suggested to include methane hydrates8 and permafrost carbon3.Although we do not rule out background volcanic activity occurring during CIE step 2, if volcanism was voluminous enough to produce the required CO13C as CIE step 1, they do consistently co-occur with modest decreases in δ13C both before and after the CIE onset  increase42. We correlate E−8X δ13C with Bass River dinoflagellate cyst δ13CDINO, which also shows the two-step CIE onset  index values and changing sedimentation rates making it harder to correlate, but do show a possible fall stratigraphically below the CIE that has been suggested to reflect local cooling from volcanism44.While other pulses of Hg/TOC at E−8X do not correlate with such a large decrease in δset Fig. , and finset Fig.  corresposet Fig. , which a13C  are presented in Supplementary Fig. 46, the preservation of which also indicates the mud was never buried to temperatures where smectite recrystallizes (>90 °C). The dominant facies of the Ve Member is homogeneous red claystone with small  white concretions of unknown mineralogy. Small green patches are locally present in the dark red clay. Two 15–20 cm thick beds with graded bedding, and a small slump fold near the base, occur below 2030 m. The muddy matrix contains quartz sand and white spheres (<1 mm in diameter), tentatively interpreted as redeposited concretions. The graded beds are tentatively interpreted as gravity flows. The boundary towards the Bue Member is transitional, and the uppermost 20 cm of the Ve Member are green or variegated, and weakly laminated. The Ve Member is correlated with the Holmehus Formation, onshore Denmark, and the latter has been interpreted as fully marine clay, deposited very slowly at water depths of >500 m (ref. 47). The slow sedimentation rates may partly explain the oxidation of the marine clay.For the Ve Member of the Lista Formation, only the uppermost 1.5 m of the Ve Member (2030.03–2029.55 m) is shown on the sedimentological log and is dominated by red, homogeneous mudstone. The lithology is consolidated mud, compacted by the overlying 2 km of sediments. The Ve Member is rich in smectiteZoophycos sp. and Chondrites sp.). The Zoophycos burrows are ~2 mm in diameter, and the Chondrites burrows are ~1 mm. The ichnogenera Chondrites and Zoophycos are common in depositional environments with dysoxic conditions48. These trace fossils are known to form a deep tier below the sea floor51, and it is possible that other, shallow-burrowing organisms produced the nearly homogeneous clayey mudstone. Pyrite is observed in the bioturbated sediment, either as small concretions or as pyritic laminae. In the upper part of the Bue Member (>2026.85 m), the lithology changes gradually to a greenish-black mudstone where neither laminations nor burrows are observed. Upwards, the mudstone becomes more greyish and locally fissile. The mudstone has no visible pyrite and no visible trace fossils. Shallow burrows in a ‘soup ground’ sediment may not be preserved as distinct trace fossils51. The mudstone contains few layers of volcanic ash. Towards the Sele Formation, the Bue Member is a weakly laminated, greyish black claystone or clayey mudstone, locally slightly greenish with jarosite. The TOC content increases in these upper facies   was deposited from the onset of PETM, which demonstrates that anoxic conditions were regional in the North Sea Basin during the PETM53.The laminated, anoxic mudstones of the Sele Formation are also known from other wells in the North Sea26; Heilmann-Clausen et al. 53). Core 22/10a−4 was found to include average ~0.08 mm-thick couplets of pale and dark laminae, and the Stolle Klint Clay ~0.25 mm-thick couplets. If annual, the ~3.5 m-thick laminated part of 22/10a−4 would have been deposited in ~40 kyear, and the 24.4 m-thick Stolle Klint Clay7 deposited in ~100 kyear.Sedimentation rates may be estimated from laminated sediments if the stacked laminae are nearly identical and deposited annually (as varves). Laminated sediment from 22/10a-4 (North Sea) and the Stolle Klint Clay (Denmark) was studied in a thin section and suggested to have been annually deposited . One is taken from the petrophysical logs (not shown). The depth to the top of Core 3, Box 1, was measured as 6630′ (2020.82 m) at the time of drilling, and the base of Box 11 had a depth of 6661′6″ (2030.43 m). Each Box was subsequently given a depth in feet based on the length of the measured core  augustum, a planktonic dinoflagellate cyst species used as the diagnostic marker for the PETM in the North Sea and Arctic54, is present within the CIE of sediment core E−8X (Extended Data Table 55) or ~135 kyear (ref. 56). The age models we use for Fig. 40. We correlate the E−8X record . To correlate 22/10a−4 with E−8X—a site some 350 km distant—we use a tie point at the onset of the CIE, and another at the beginning of the CIE main phase   or 2.1 cm  kyear−1 (BH9/05 option B).The age model for E−8X is constructed with biostratigraphy and carbon isotope stratigraphy. ar ref. 5. The agear ref. 5. The age26) Fig. . The ave13C occurred over 6 cm or 2–3 kyear. Although the CIE onset has been previously estimated as lasting ~20 kyear from modelling Svalbard core BH9/05, the assumption that BH9/05 records the true onset length has been challenged39 as that location was a marginal marine and proximal to land57, and possibly impacted by terrestrial organic carbon lag times.Our records from E−8X indicate that the CIE onset took place over about 10 cm, which could be within 3–5 kyears. The largest shift in the δ13CTOC at high resolution (~1 cm) from sediment core E−8X  at Bass River  were measured into a glass measuring boat and its precise mass determined. Samples were heated in the Pyrolyzer to ~700 °C to volatilise the Hg within the sample. Following this, gaseous Hg was transported into the Analyzer and abundance was measured, providing the abundance mass of Hg as parts per billion (Supplementary Data n = 82), and the standard deviation of the offset from repeated samples from 22/10a−4 and E−8X (n = 72) was 26.7 ppb. For both records, sedimentary Hg shows highly fluctuating values from 6 to 1500 ppb  and hydrofluoric (HF) acids, and zinc bromide was used as a heavy liquid to separate and remove acid-resistant mineral grains. Slides were mounted using Elvacite. All palynomorphs were analyzed with a Nikon transmitted light microscope, counting the total number of palynomorphs on a strew slide (Extended Data Table Palynology samples were prepared using standard preparation proceduresSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Nitrate needs are most likely meet by ingesting approximately 250–500 g of leafy and root vegetables per day; however, dietary supplements might represent a more convenient and accurate way of covering an athlete’s nitrate needs. Athletes should refrain from mouthwash usage when nitrate supplementation benefits are desired. Future research should focus on the potential beneficial effects of nitrate supplementation on brain function, possible negative impacts of chronic nitrate supplementation through different nitrate sources, and the effectiveness of nitrate supplementation on strength and high-intensity intermittent exercise.Nitrates have become increasingly popular for their potential role as an ergogenic aid. The purpose of this article was to review the current scientific evidence of nitrate supplementation on human performance. The current recommendation of nitrate supplementation is discussed, as well as possible health complications associated with nitrate intake for athletes, and dietary strategies of covering nitrate needs through sufficient intake of nitrate-rich foods alone are presented. Pubmed, Scopus, and Web of Science were searched for articles on the effects of nitrate supplementation in humans. Nitrates are an effective ergogenic aid when taken acutely or chronically in the range of ~5–16.8 mmol (~300–1041 mg) 2–3 h before exercise and primarily in the case of exercise duration of ~10–17 min in less trained individuals (VO NO3− is an active compound found in certain NO3− rich vegetables and elicits potentially ergogenic as well as health-promoting effects. As such, NO3− presents as an interesting compound from both a sports perspective as an ergogenic aid as well as a potentially cost-effective tool for reducing the likelihood of cardiovascular events [Nitric oxide (NO) is a signaling molecule involved in numerous vascular and cellular functions, such as cellular respiration, vasodilation, and angiogenesis. NO is produced through both endogenous and exogenous pathways by dietary nitrate  were included. Studies completed in animal models or individuals with certain medical conditions were excluded from the review.NO3− may improve sports performance primarily through its effects on skeletal muscle, blood vessels, and the brain [3− on blood vessels is manifested through blood pressure reduction, an observation also seen in otherwise healthy populations with blood pressure values in reference ranges. NO3− may also increase heat loss through the skin during physical activity [3− on the brain is currently in its infancy and is not as well understood as are the effects on skeletal muscles and blood vessels. As such, there is currently no direct evidence that the addition of NO3− could, in fact, increase the availability of NO in the brain. Nevertheless, research completed in animal models reports that NO in the brain reduces oxygen consumption during exercise [NOhe brain . The effexercise , accelerexercise , and potexercise ,7. 3−, L-arginine, L-citrulline, or through endogenous synthesis. NO synthesis from L-arginine takes place via its oxidation, catalyzed by the family of enzymes called nitric oxide synthase (NOS), and takes place in the presence of oxygen. The oxidation of L-arginine to NO was once thought to be the only way NO is formed in the body. However, we now know that this is not the case and that NO can be produced through the reduction of NO3− and nitrites (NO2−) in the body [3−–NO2−–NO pathway takes place with a gradual decrease in oxygen concentration (hypoxia), which occurs during intense physical activity. This pathway can be described as an alternative or complementary pathway of NO formation in the absence of oxygen to the aforementioned pathway via the amino acid L-arginine [NO is synthesized either by ingesting foods rich in NOthe body . NO formarginine .3− from diet or dietary supplements, plasma NO3− levels peak after approximately 1–2 h or after 2–3 h for NO2− before concentrations of both compounds gradually decrease and return to baseline levels after approximately 24 h [After ingestion of NOely 24 h .3− and NO2− begins in the mouth, where a certain portion (~25%) of ingested NO3− is digested by saliva via anaerobic bacteria that reside there and reduce NO3− to NO2−. The greater part of NO3− reduction takes place later in the stomach because of the low pH of the environment. Further reduction of NO2− takes place via a non-enzymatic reaction in the gastric lumen. Most of the circulating NO3− is eventually excreted through urine. However, approximately 20–25% of NO3− is taken up by the pancreas from the bloodstream and concentrated in saliva. The cycle of NO3− metabolism is thus completed and is then repeated, where the anaerobic bacteria in human saliva initially reduce NO3− to NO2−. NO2− and remaining non-reduced NO3− is then swallowed again where a smaller percentage of NO2− is further reduced to NO by the low pH environment of the gastric lumen [2−, however, re-enters the systemic circulation and is transported to specific locations throughout the body where they are reduced to NO via various enzymatic or non-enzymatic degradation pathways. The exact mechanism of NO2− re-entry into the circulation is unknown, but the reduction takes place primarily in the vascular system under conditions of hypoxia and reduced pH levels. Through the NO3−–NO2−–NO pathway of NO3− degradation, endogenously ingested nitrates are recycled by oral bacteria and act as a kind of reservoir of NO synthesis precursors [Digestion of ingested NOic lumen . Most ofecursors .3− recycling process is severely impaired when oral mouthwashes are used on a regular basis as they destroy oral bacteria and consequently lower plasma NO3− levels [3− supplementation periods. Interestingly, chlorine-sterilized pool water does not seem to impair the NO3− reduction pathway through oral bacteria breakdown, as previously speculated by some. This has important implications for swimmers, a population where NO3− supplementation might be efficacious [It is important to note that this NO− levels ,13. Thisicacious .3− have been frequently researched over the past decade and a half in terms of their impact on sports performance in a wide variety of training modalities. One of the first of such studies reported positive effects of NO3− supplementation on time to exhaustion [3− supplementation was found to have a positive impact on lowering oxygen consumption and time to exhaustion [NOhaustion . Afterwahaustion . 3− intake, such as lowering blood pressure [3− supplementations on exercise performance is presented in Several other studies report positive benefits of NOpressure ,18, redupressure , reducedpressure ,20,21, apressure ,22,23,24pressure ,29,30,313− supplementation effects are less pronounced in better-trained individuals is also supported in a systematic review and meta-analysis by Campos et al. (2018) [3− on the performance of well-trained individuals, the effect of NO3− should not be neglected. NO3− is currently regarded as one of a handful of dietary supplements with a direct, positive effect on athlete’s performance based on the latest consensus statement by the International Olympic Committee (IOC) [3− supplementation on performance may be particularly desirable on competition day, where the differences between the competitors are marginal.The observation that NOee (IOC) . The eff3− supplementation effects in well-trained individuals. This effect, speculative in nature, relates to the general eating habits of elite athletes. We can assume that most top athletes include decent amounts of foods rich in NO3− as well as L-arginine and L-citrulline, making the effect of the dietary supplementation less pronounced [3−-rich foods, and if the intake of NO3− from the diet is too low, dietary supplement usage will most likely yield greater benefits. A parallel may be drawn with creatine supplementation. Vegetarians and vegans who consume diets poorest in creatine  have the lowest levels of muscle creatine phosphate, and the effect of creatine supplementation on performance is significantly more pronounced in this population versus omnivores [Additionally, diet may also influence NOonounced . Of courmnivores .3− is ingested at once without the presence of other ingredients affecting its digestion. In this context, a major concern might be the interaction between NO and superoxide (O2) leading to peroxynitrite (OONO−) formation—a highly reactive nitrogen species (RNS) affecting mitochondrial function, signal transduction, and stress response [− formation might directly lead to the production of RNS and reactive oxygen species (ROS) in other subcellular compartments, leading to increased oxidative stress [3− supplementation. For example, high doses of vitamin C and E have been shown to blunt aerobic exercise adaptations [3− supplementation, no such guidelines can be given, and to our knowledge, no research has looked directly into differences in RNS formation from NO3− supplementation or via NO3− rich diet but should be taken into account, especially when chronic NO3− supplementation is being considered.Lastly, a difference in performance outcomes observed in studies using NO supplementation might be attributed to NO’s ability to interact with other free radicals. As NO half-life in vivo is in the order of a few seconds, this reaction might be dependent on its initial concentration , thus maresponse ,76. Chroe stress , which mptations ,79. Highptations . This mi3− supplementation on exercise performance. Hoon et al. (2013) reported a statistically significant improvement in performance for constant power/speed tests and smaller, statistically insignificant but positive effects in the case of incremental tests and time trials [3− supplementation protocols between trials as well as the training status of individuals. Pawlak-Chaouch et al. published a meta-analysis including 26 randomized and placebo-controlled studies in 2016 and reported a significant reduction in VO2 during submaximal exercise [3− supplementation can improve tolerance to and efficiency of continuous high-intensity exercise and maximal exercise with increasing intensity [3− supplementation (up to 15 days) when taken in an amount of 5–9 mmol with similar conclusions being found in a meta-analysis by McMahon et al. (2016) on the impact of NO3− supplementation specifically on endurance performance [3− supplementation was efficient on submaximal aerobic capacity, but lower effectiveness for time trial tests, which is in line with past research by Hoon et al. (2013) [There are also several literature reviews and meta-analyses examining the effectiveness of NOe trials . Howeverexercise . Afterwantensity . The autformance . This me. (2013) .3− ingested and the increase in plasma NO3− levels, we can assume that there is an upper limit of NO3− intake that still elicits a positive effect on performance. Wylie et al. (2013) reported that ingestion of 4.2 mmol NO3− did not affect VO2 during moderate-intensity cycling, but VO2 was affected by NO3− supplemented at 8.4 mmol and 16.8 mmol [3− compared to 8.4 mmol NO3− did not provide additional benefits. Thus, we can assume that the effect of NO3− on performance exists within a specific interval. This interval is most likely between 5–9 mmol (310–560 mg) NO3− taken either acutely 2–3 h before exercise [3− is not statistically significant if NO3− is taken less than 2 h before exercise as this does not allow enough time for NO3− to NO conversion.Even though a linear correlation exists between the amount of NO6.8 mmol . Howeverexercise  or chronexercise  and Seneexercise . The aut3− intake is expected to be similar based on the current literature [3− intake of more than 3 consecutive days before the race may potentially reap greater benefits for well-trained athletes [The effect of acute or chronic NOterature ; howeverathletes .3− differs across not only training status but exercise type and trial duration as well [3− supplementation is reported to be in the range of 4–25% for time to exhaustion tests and 1–3 % for sport-specific tests lasting less than 40 min. NO3− is expected to have the greatest effect between the range of approximately 12 and 40 min. Furthermore, within this time frame, the effects of NO3− supplementation are likely most pronounced for exercise lasting between 601 and 999 s (~10–17 min), with the effects of NO3− being effective regardless of normoxic or hypoxic conditions [The IOC reports the effectiveness of NO as well . As suchnditions .3− supplementation have been studied in a wide variety of performance tests. However, the effects are most likely especially pronounced in time to exhaustion tests rather than time trial tests or incremental power tests. This may be due to the fact that time to exhaustion tests are supposedly better at measuring an athlete’s endurance capacity and are highly influenced by psychological factors  [3− on cycling and running, the most commonly studied training modalities in research on NO3− supplementation, but not in knee extension tests or rowing rests. However, the lack of effect is most likely due to the relatively low proportion of studies completed on these two forms of performance tests rather than the exercise type per se [Effects of NOm, etc.) . As for e per se .3− might be particularly effective for team sports athletes because of their potential beneficial effect on cognition. Athletes who participate in team sports are forced to make many quick decisions during training and competition. However, prolonged high-intensity exercise can have a negative impact on reaction time and task performance [3− supplementation in the amount of 6.4 mmol to 12.8 mmol for 7 consecutive days [3− supplementation on cognition may arise from the positive effect of NO on neurovascular coupling [3− thus has a potentially positive effect on reducing the decline in cognitive function, primarily athlete’s reaction time, which is otherwise associated with repetitive high-intensity intermittent exercise.NOformance . Thompsoive days . These pcoupling  and incrcoupling . NO3− th3− supplementation effects on performance have been completed on endurance tests. Some research, however, focuses on investigating these effects on high-intensity exercise and strength, where mixed results are observed. Thompson et al. (2016) reported improvements in sprints in the Yo-Yo test after NO3− supplementation [3− intake of 6.4 mmol in the Wingate test, primarily in the first half of the sprints [3− on the protocol of 8 s sprints with 30 s pauses [A large majority of studies on NOentation . A similentation . Cuenca  sprints . Howevers pauses .3− on weight training in an otherwise limited sample of four studies reported a positive effect of NO3− on upper body strength and the number of repetitions performed in upper body strength test (bench press) as well as lower body strength test (squat) [3− on high-intensity exercise and strength, but preliminary results suggest that NO3− could be beneficial in this sport context as well.A systematic review by San Juan et al. (2020) on the effect of NO (squat) . We cert3− effects on performance is research completed in extreme conditions, such as hypoxic and cold environmental settings . As altitude increases, hypoxic conditions reduce O2 availability and decrease exercise performance. We can somewhat overcome this problem with altitude acclimatization; however, this process may take up to several weeks to fully manifest, which is not always possible in certain sports situations. Additionally, physical fitness otherwise seen at sea level might not ever be fully regained, even with prolonged acclimatization [2 availability to the working muscle and brain tissue during hypoxic conditions [3− supplementation on certain physiological parameters . However, these findings do not seem to be observed in field tests at ’real’ altitudes, making real-world applications limited [3− supplementation might even be detrimental for athletes training at altitude from a perspective of possibly blunting hypoxic adaptations by decreasing arterial and muscle O2 saturation, which may act as a signal for such adaptations [Another avenue of NOtization . It is snditions ,95. Certnditions . Indeed, limited ,96,97. Cptations . As such3− and NO2− is either through NO3− rich foods or through endogenous productions. Of these pathways, nutrition represents the one with greater potential to supply the body with a higher amount of NO3− as the endogenous supply of NO3− is relatively limited, and only a bowl of green leafy vegetables contains a higher amount of NO3− than is formed endogenously throughout the entire day [3− through nutrition, either with NO3− supplementation or through NO3− rich foods—primarily leafy greens and root vegetables.The primary sources of NOtire day . Athlete3− content of these vegetables varies greatly, as it depends on many factors such as the origin of the vegetable, the quality and pH of the soil in which the vegetables are grown, type and frequency of nitrogen fertilizers, type of vegetable cultivation, time of vegetable harvesting, age of the plant at harvest, conditions of vegetable storage and weather conditions in which vegetables are grown, and method of vegetable preparation, etc. [However, the NOon, etc. .3− via the diet due to the large number of variable factors that affect the NO3− content in the diet. Speculations can be made based on current data on the average NO3− of NO3− rich foods  that this figure would be set at 150 g of aforementioned foods at the lowest [3− intake.Given all these factors, it is difficult to make a specific recommendation for athletes to meet the needs of NOe lowest . However3− via supplementation might hold a greater risk of peroxynitrite production relative to covering NO3− needs through diet. This might be another limiting factor of NO3− supplementation, as discussed in the chapter titled ’Nitrates and exercise performance’.Additionally, ingesting a bolus of NO3− in the form of a dietary supplement may represent a more convenient and accurate way to cover the needs for NO3−; however, as with any other dietary supplement, there is always the possibility of supplement contamination [Lastly, NOmination , and an 3− represents an effective ergogenic aid for improving performance through various mechanisms and is useful in a variety of sports situations and exercise modalities. The effect of NO3− is most pronounced in less-trained individuals when taken acutely or chronically in the range of ~5–16.8 mmol (~300–1041 mg NO3−) 2–3 h before exercise and primarily in the case of exercise duration of ~10–17 min. Nitrate supplementation is less pronounced in well-trained individuals (VO2max > 65 mL/kg/min); however, it might still be desirable, especially during competition. Athletes should refrain from mouthwash usage when nitrate supplementation benefits are desired.Based on current literature, NO3− is found in certain vegetables, but due to many variable factors, we cannot make exact recommendations to cover these needs through diet alone. Given that there is a potential for supplement contamination, it would make sense to explore how dietary needs for NO3− can be covered through dietary sources. Currently, speculations can be made that this figure is roughly 250–500 g of leafy and root vegetables per day. Dietary supplements might represent a more convenient and accurate way of covering one’s needs for nitrate; however, potential supplement contamination and an athlete’s budget must be considered.NO3− on the brain, especially in regard to sport-specific situations, and on the effectiveness of NO3− in strength training and high-intensity intermittent training.Future research should focus on the potential beneficial effects of NO