OCT4, SOX2, KLF4, AGR2, and NOTCH1), methylation status, copy number variation, and regulatory miRNAs, miR-145, miR-335, miR-150, miR-34a, miR-128, and miR-27a.  Results of previous studies suggest that NANOG may be an important prognostic biomarker in oral squamous cell carcinoma (OSCC), but there are contradictory results regarding NANOG expression patterns on mRNA and protein levels, and the mechanisms of its regulation are poorly understood. Our aim was to analyze the expression and diagnostic significance of NANOG in OSCC, and the possible mechanisms of its regulation, i.e., protein regulators on mRNA level (NANOG promoter was determined using PCR and Sanger sequencing.  Our study included 120 patients with OSCC. Expression of NANOG protein and mRNA was analyzed using immunohistochemistry and qPCR. Expression of regulatory factors, miRNAs, and copy number variation was performed using qPCR. Methylation status of NANOG and OCT4 and downregulation of NOTCH1 and AGR2 mRNA in OSCC with lymph node metastases compared to OSCC without lymph node metastases. We observed a strong positive correlation between mRNAs of NANOG and those of its protein regulators OCT4, SOX2, NOTCH1, AGR2, and KLF4. The expression of NANOG was in positive correlation with the expression of miR-34a. There was also a correlation between T status of OSCC and the expression of miR-335 and miR-150 and a correlation of miR-150 with the N status of T2 OSCC. NANOG promoter methylation and copy number variation were only observed in a small proportion of samples.  We detected upregulation of  Our findings confirm the diagnostic significance of NANOG in OSCC and provide information on NANOG expression patterns on both mRNA and protein levels. They also suggest that protein regulators and microRNAs might play a crucial role, whereas methylation of its promoter and copy number variation probably have a minor role in the regulation of NANOG expression in OSCC. Despite intensive research, head and neck squamous cell carcinomas still have a very high mortality rate 50%) and an overall 5-year survival of only about 40–50%. The main reasons are late diagnosis, high recurrence rate, and lymph node and distant metastases –4. For S0% and anNANOG is an important stem cell transcription factor, with a complex regulation role in human development, determining cell fate, proliferation, and death. After birth, it is expressed at very low levels or is silenced and remains in that state throughout the lifespan. However, NANOG expression is detectable in a proportion of cancer cells that exhibit stem cell-like properties. Cancer stem cells (CSCs) can be the source of malignant transformation, progression, and development of metastases. Elevated NANOG expression has thus been reported in various cancer types , including in OSCC, and it is believed that it can be used as a prognostic biomarker –10.NANOG expression on an mRNA level using quantitative real-time PCR (qPCR) [Data available for NANOG expression in head and neck SCC are mostly based on immunohistochemistry or immunofluorescence , and theR (qPCR) , 13–16.NANOG expression in head and neck SCC [NANOG expression in OSCC (i) in correlation with expression of miRNAs whose target is NANOG, (ii) in correlation with its known protein regulators, (iii) in correlation with the methylation status of its promoter, and (iv) in correlation with the copy number variation (CNV) of the NANOG gene. To understand the regulation of the NANOG in the development and progression of OSCC better, we aimed to characterize the expression of NANOG in correlation with its regulators OCT4, SOX2, KLF4, AGR2, and NOTCH1 and with methylation status [miR-145, miR-335, miR-150, miR-34a, miR-128, and miR-27a [Some NANOG regulators have been previously described as being associated with neck SCC , 17–20. neck SCC , 21–23. n status , CNV, an miR-27a –30. miRNμm, and stained with hematoxylin and eosin (HE). p16-positive OSCC presumably related to infection with human papillomavirus infection was not included in the study.Our study included 120 patients with OSCC who had been treated surgically, including the resection of the primary tumor and regional lymph nodes, and examination of surgical margins. The study was approved by the National Medical Ethics Committee, Ministry of Health, Republic of Slovenia (No 0120-106/2018/6), and was performed in accordance with the ethical guidelines for human research. The need for informed patient consent was waived, and there is no publication of identifying information or images in the manuscript. Resection specimens were dealt with according to standard procedures and, after 24-hour fixation in formalin, representative samples of the tumor, all lymph nodes, and resection margins were taken, cut at 3-4 Group 1 included 15 patients with early OSCC without lymph node metastases (pT1 or T2 N0 M0); 9 were males and 6 females, aged 32 to 87 years (61.60 ± 14.10). Tumors were located in the tongue (7 patients), floor of the mouth (5 patients), buccal mucosa (2 patients), and alveolar ridge (one patient)Group 2 included 15 patients with early OSCC with lymph node metastases (pT1 or T2 N+ M0); 11 were males and 4 females, aged 41 to 76 years (60.53 ± 11.87). Tumors were located in the tongue (9 patients), retromolar trigonum (4 patients), and floor of the mouth (2 patients)In all cases, corresponding tumor-adjacent normal tissues (TANT) were available. For 90 patients, RNAlater-stored tumor and TANT samples were available, while for 30 patients, only formalin-fixed paraffin-embedded (FFPE) tumor and TANT samples were available. After HE examination, 30 patients with OSCC were divided into 2 groups according to TNM classification. Group 1 included 29 patients with early OSCC without lymph node metastases (pT1 or T2 N0 M0); 27 were males and 2 females, aged 37 to 84 years (60.14 ± 10.43). Tumors were located in the tongue (23 patients), floor of the mouth (5 patients) and oropharynx (one patient)Group 2 included 35 patients with early OSCC with lymph node metastases (pT1 or T2 N+ M0); 30 were males and 5 females, aged 41 to 86 years (57.26 ± 10.25). Tumors were located in the tongue (19 patients), floor of the mouth (7 patients), oropharynx (5 patients), buccal mucosa (two patients), uvula (one patient), and alveolar ridge (one patient)Group 3 included 5 patients with advanced OSCC without lymph node metastases (pT3 or T4 N0 M0); all patients were males, aged 42 to 74 years (64.20 ± 12.85). Tumors were located in the tongue (3 patients) and floor of the mouth (2 patients)Group 4 included 21 patients with advanced OSCC with lymph node metastases (pT3 or T4 4N+ M0); 20 were males and one female, aged 44 to 77 years (62.43 ± 8.79). Tumors were located in the tongue (11 patients), floor of the mouth (8 patients), oropharynx (one patient), and retromolar trigone (one patient)RNAlater  stored samples were collected constitutively from 2008 to 2018 and divided into 4 groups:μm thick slides were cut from FFPE tissue blocks, followed by automated antigen retrieval  and staining with commercially available anti-NANOG antibodies  . Reactions were visualized by incubation with peroxidase and 3,3′-diaminobenzidine  and then counterstained with hematoxylin. Negative controls omitting the primary antibody binding were included in every run of samples. Testicular seminoma served as a positive control.For immunohistochemistry, 3-4 We evaluated the extent of positive reaction semiquantitatively , intensity , and staining pattern .μm thick sections were cut and AllPrep DNA/RNA FFPE Kit  was used for total RNA extraction, according to the manufacturer's instructions. All the reagents were from Qiagen, except from ethanol  and deparaffinization solution . NanoDrop-1000  was used for measuring the quantity of RNA by measuring A260.For the isolation of total RNA, four 10 260.For each sample, approximately 10 mg of tissue was collected, separately for the isolation of total RNA (including miRNAs), and for DNA. Total RNA extraction was performed using Maxwell® RSC miRNA Tissue Kit , and extraction of DNA was performed using Maxwell® RSC Tissue DNA Kit . Automatic isolation was performed using Maxwell® RSC Instrument , according to the manufacturer's instructions. All the reagents were from Promega except for ethanol . Quantity of the RNA and DNA was assessed on NanoDrop-1000  by measuring Aμl 6 μM Random primers mix, 5 μl 2x reaction mix and 1 μl 10x enzyme mix. Random primer mix and RNA were mixed and incubated for 5 min at 70°C; then, reaction and enzyme mixes were added and incubated for 5 min at 25°C, 60 min at 42°C, and 4 min at 80°C.OneTaq-PCR kit  was used to reverse transcribe RNA according to the manufacturer's instructions. Each reverse transcription (RT) reaction contained 60 ng of RNA, 1 μl RT reaction contained 150 ng of extracted total RNA, 2 μl HiFlex buffer, 1 μl 10x nucleic mix, 1 μl of miScript RT enzyme, and 0.33 μl of RNaze Inhibitor. The reaction was incubated for 60 min at 37°C and 5 min at 95°C.RT of total RNA (mRNAs and miRNAs) was performed using miScript II RT , according to the manufacturer's instructions. Each 10 All qPCR reactions for miRNA and mRNA expressions after RT using miScript were performed on ViiA 7 Real-Time PCR System . All qPCR reactions for mRNA expression after RT using the OneTaq-PCR kit were performed on Rotor Gene Q . Each sample was done in duplicate.μl of resulting cDNA, 5 μl 2x TaqMan PreAmp Mastermix  and 2.5 μl of pooled 0.2x TaqMan Gene Expression Assays. Pooled TaqMan Gene Expression Assays, followed by dilution to 0.2x using Tris-EDTA buffer solution, pH 8.0 , are summarized in Prior to qPCR, preamplification was performed to measure the expression level of genes, using the TaqMan PreAmp Mastermix . Preamplification was performed using 2.5 Efficiency of qPCR reactions was calculated prior to qPCR as follows: (i) pools of N0 and N+ groups for tumors and TANT samples were created by mixing RNA samples isolated from FFPE tissue and (ii) two separate pools of RNA were created, one from healthy mucosa and one from tumor samples, isolated from RNA-stored tissue samples. Resulting preamplified cDNA was diluted 5-, 25-, 125-, and 625–fold for mRNA efficiency analysis using qPCR (described below). All qPCR reactions for efficiency testing were performed in triplicate. For all mRNA expression analyses, TaqMan technology was used.μl of diluted preamplified cDNA mixture, 5 μl 2x FastStart Essential DNA Probe Master , and 0.5 μl TaqMan Gene Expression Assay   listed in IPO8 was used as the reference gene. Thermal conditions were applied as follows: 2 min at 50°C, 10 min at 95°C and 40 cycles of 15 s at 95°C, and 1 min at 60°C.For FFPE tissue samples, preamplified cDNA was diluted 5-fold. qPCR reaction contained 4.5 μl of 2x TaqMan Gene Expression Master Mix, 0.5 μl 20x TaqMan Gene Expression Assay , and 4.5 μl of diluted preamplified cDNA. Used TaqMan Gene Expression assays are listed in GAPDH, IPO8, and HPRT1 were used as reference genes. Cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C and 45 cycles of 15 s at 95°C, and 1 min at 60°C.For RNAlater-stored tissue samples, preamplified cDNA was diluted 15-fold. qPCR reaction contained 5 RT and qPCR based on SYBR Green technology were performed to measure the expression level of miRNAs. Prior to qPCR, for the purpose of determination of the efficiency, two separate pools of RNA were created, one from TANT and one from tumor samples. RNA pools were processed in terms of RT and preamplification as described above. Resulting cDNA was diluted 5-, 25-, 100-, 125-, 625-, 1000-, and 3125–fold for miRNA efficiency analysis using qPCR (described below). All qPCR reactions for efficiency testing were performed in triplicate.μl of 2x miScript SYBR Green PCR Mix, 1 μl 10x miScript Universal primer, 1 μl 10x miScript primer, 0.05 μl of ROX dye, 0.95 μl ddH2O, and 2 μl of diluted cDNA. All used 10x miScript primer assays  are listed in SNORA73A, SNORD72, SNORD61, and SNORD95 were used as reference genes. Thermal conditions were as follows: 15 min at 90°C and 45 cycles of 15 s at 94°C, 30 s at 55°C, 30 s at 70°C. Afterwards, melting curves were acquired on the SYBR channel using a ramping rate of 0.7°C/60 s for 60–95°C.For miRNA expression analysis using miScript SYBR Green PCR Kit , cDNA was diluted 150-fold. qPCR reaction contained 5 NANOG was analyzed using Sanger sequencing after bisulfite conversion. Bisulfite conversion was performed using the innuCONVERT Bisulfite Basic Kit  according to the manufacturer's instructions using 500 ng of DNA.Methylation status of the promoter region of μl 5 μM primers (forward and reverse) , 0.05 μl (5 units/μl) HotStarTaq Plus DNA Polymerase, 1 μl 10x PCR Buffer, 0.7 μl (3.33 mM) dNTPs, 6.35 μl ddH2O , and 15 ng bisulfite-converted DNA. Thermal conditions for PCR were as follows: 10 min at 95°C, 40 cycles of 1 min at 95°C, 1 min at 61°C, 1 min at 72°C, and final elongation for 10 min at 72°C. Primers used are listed in Additional file 1: Table PCR reaction contained 0.2 Sanger sequencing was performed on SeqStudio Genetic Analyzer . PCR products were directly used for a sequencing reaction after being purified with ExoSAP-IT™ PCR Product Cleanup Reagent , according to the manufacturer's instructions. The sequencing reaction was performed using Big-Dye terminator chemistry version 1.1 . Electropherograms were analyzed using the Sequence Scanner Software 2, Version 2.0 .NANOG was performed using qPCR. Prior to qPCR, for the purpose of determination of the efficiency, two separate pools of DNA were created, one from healthy mucosa and one from tumor samples. As the calibrator sample, the TaqMan Control Genomic DNA  was used. Pooled DNA was diluted 2-, 4-, 8-, 16-, and 32–fold starting with 25 ng/μl of DNA. Each qPCR reaction from diluted samples was performed in triplicate (as described below).CNV analysis of NANOG CN analysis, multiplex reaction was additionally performed in duplicate for each sample, including NANOG-FAM and TERT-VIC. Multiplex qPCR was performed using TaqMan Universal Master Mix II, no UGN  using 5 μl of 2x TaqMan Universal Master Mix II, no UGN, 0.5 μl 20x NANOG-FAM TaqMan CN Assays (Hs04405955_cn), 0.5 μl 20x TaqMan CN Reference Assay , 2 μl ddH2O, and 2 μl of DNA (6.25 ng/μl). Thermal conditions were as follows: 10 min at 95°C and 40 cycles of 15 s at 95°C, 60 s at 60°C. All reagents were from Applied Biosystems, Foster City, CA, USA. All qPCR reactions were performed on ViiA 7 Real-Time PCR System , and each sample was done in duplicate.For the purpose of ∆Cq for statistical analysis.For mRNA and miRNA expression analyses, data were analyzed according to Latham . Geometr∆Cq and Wilcoxon signed rank tests. Further, ∆Cq of the tumor sample was first subtracted from ∆Cq of corresponding TANT to calculate ∆∆Cq (∆∆Cq = ∆TANT − ∆Cq tumor tissue). ∆∆Cq of OSCC N0 and OSCC N+ was compared using the Mann–Whitney test. ΔΔCq was used for calculating fold change using 2ΔΔCq- for graphical presentation.For FFPE tissue samples, for which TANT was available, statistically significant difference was calculated using the ΔCq) [U test. For comparison of methylation status of the promoter region of NANOG and CNV of NANOG to NANOG expression, T status and N status, the Spearman rank-order correlation was performed. The Spearman rank-order correlation was also used for all other nonparametric correlations/associations.For RNAlater-stored tissue samples, comparison of relative quantification of mRNAs and miRNAs (ΔCq)  between p < 0.05 (2-tailed).Statistical analysis of data was performed using IBM SPSS Statistics 24 software . Differences were considered as significant using cut-off NANOG, SOX2, OCT4, AGR2, KLF4, and NOTCH1 in only 11 tumor samples and corresponding TANT (22 FFPE tissue samples).Although a previous study  on a coh∆Cq and Wilcoxon signed rank tests. Expression of genes in OSCC N0 compared to TANT showed downregulation of NANOG, OCT4, and KLF4  and upregulation of SOX2, AGR2, and NOTCH1 . On the other hand, in OSCC N+, expression of NANOG, OCT4, and AGR2 showed upregulation  and SOX2, KLF4, and NOTCH1 showed downregulation , not significant. The p value of the difference of NANOG, OCT4, and NOTCH1 expression levels between OSCC N+ and corresponding TANT groups was just above the cut-off (p = 0.068).Paired FFPE tissue samples (tumor sample and corresponding TANT) were compared using the ∆∆Cq of OSCC N0 (n = 7) and OSCC N+ (n = 4) revealed that expression of NANOG and OCT4 was significantly upregulated in OSCC N+ compared to OSCC N0 (p = 0.008). In contrast, NOTCH1 was significantly downregulated (p = 0.011). For SOX2, AGR2, and KLF4, there were no statistically significant differences, although AGR2 and SOX2 showed similar expression pattern. Results are presented as fold change in Using the Mann–Whitney test, comparison of NANOG was in positive correlation with the expression of OCT4 in TANT and tumor samples .Expression of Thirty cases of OSCC were stained immunohistochemically for NANOG; a positive reaction was found in 27 cases. Immunohistochemical staining was present in the cytoplasm of tumor cells; no nuclear staining was observed . The extNANOG mRNA was present in all tumor samples tested; however, it was undetectable in 10 TANT samples. We observed a stable expression of reference genes in 82 of 90 RNA samples isolated from RNAlater-stored tissue samples of OSCC. These 82 samples were used for further analysis to obtain reliable results.Due to extremely high variability in Cq values among mRNAs, miRNAs, and reference genes isolated from TANT in comparison to tumor samples stored in RNAlater, TANT samples were not appropriate for further analysis.NANOG. It is expected that mRNA of NANOG would not be detected in TANT, since there is no protein expression. However, we observed NANOG expression in TANT using qPCR, but the technical variability was similar to that of reference genes.Another technically challenging question in the case of TANT was the expression of Due to both of the above-mentioned reasons, we excluded samples of normal mucosa from further statistical analyses and comparisons.NANOG and all the tested regulators was observed in tumor samples as follows: OCT4 , KLF4 , NOTCH1 , SOX2 , and AGR2 .Using the Spearman rank-order correlation (2-tailed), strong positive correlation between NANOG. Positive correlation was observed between expressions of SOX2 and AGR2  and between expressions of SOX2 and KLF4 . Expression of OCT4 was positively correlated with expression of KLF4  and NOTCH1 . Expression of KLF4 was in positive correlation with expression of NOTCH1  and expression of AGR2 . All correlations are summarized in We observed correlation also among expressions of other mRNAs beside NANOG and miR-34a   in tumor samples.We observed statistically significant correlation between expression of = 0.018) . On the NANOG expression. Expression of OCT4 was correlated significantly with expression of miR-34a  and miR-27a . Expression of NOTCH1 was correlated with expression of all investigated miRNAs, except miR-128, with miR-150 , miR-34a , miR-145 , miR-335 , and miR-27a .However, we observed correlation between expressions of investigated miRNAs with other genes that have been recognized as regulators of miR-145 was correlated with expression of miR-335 , miR-34a , miR-128 , miR-27a , and miR-150 . Expression of miR-335 was correlated significantly with expression of miR-150 , miR-34a , miR-128 , and miR-27a . Expression of miR-150 was significantly correlated with expression of miR-34a , miR-128 , and miR-27a . There was also correlation between expression of miR-34a and expression of miR-128  and miR-27a . Expression of miR-128 was also correlated with expression of miR-27a . Some results are summarized in Expression of all the tested miRNAs was in correlation to each other in tumor samples. Expression of miR-335  and miR-150  (There was a correlation between the T status of tumor samples and expression of  = 0.05) . No corrn = 45), statistically significant change in expression was observed for miR-150 between OSCC with and without lymph node metastases (p = 0.006) (When considering only tumor stage T2 (= 0.006) .Promoter region of NANOG-201 transcript  was used as reference sequence.NANOG promoter region of 10 tumors and TANT samples . We noticed two additional CpG sites in our samples, numbered by 22 (A->G) and 29 (T->G)  compared to samples where CN was greater in TANT compared to tumor samples . We also calculated average CN for tumor samples (3.45 ± 0.96) and for TANT (3.63 ± 0.47), regardless of N status. There is a difference in CN greater than one copy comparing tumor samples and TANT in 17 samples (19.54%); in contrast, there is a difference in CN lower than one copy in 70 pairs (80.46%).CN of NANOG and NANOG CNV of tumor samples using the Spearman rank-order correlation.No correlation was observed between expression of NANOG in OSCC and confirmed its important role in the development and progression of OSCC. As expected, NANOG was not expressed in normal tissue but it was expressed in OSCC, as already reported in many types of human cancers, including head and neck cancer [NANOG expression was significantly higher in OSCC patients with nodal metastases than in those without nodal metastases, suggesting its role as a prognostic biomarker. Immunohistochemical analysis of NANOG expression showed positive staining in a large proportion of OSCC cases, with a cytoplasmic pattern, in contrast to the nuclear pattern observed in germ cell tumors. The significance of the two different staining patterns remains unclear. The three negative cases, interestingly, were more than 10 years old, which might had influenced the immunohistochemical staining. In areas of the dysplastic epithelium adjacent to OSCC, positive staining was found, being very weak in mild dysplasia and intense in severe dysplasia. Occasionally, staining was more intense in the dysplastic mucosa adjacent to OSCC than in OSCC itself.We analyzed the expression and regulation of k cancer . FurtherDespite an important role of NANOG in the development and progression of OSCC, little is known about the regulation of its expression. We therefore studied possible mechanisms and factors of NANOG expression regulation, such as protein regulators on the mRNA level, microRNAs, CNV, and methylation status.NANOG and mRNA levels of all the tested protein regulators in tumor tissue samples, indicating that these regulators affect NANOG expression in OSCC, too. We preliminarily unraveled the correlation in the expression between NANOG and other stemness factors.On the basis of previous studies of NANOG regulators in various cancers, we chose the following protein regulators for further analysis: AGR2, KLF4, NOTCH1, OCT4, and SOX2. We found a strong positive correlation between POU5F1) and SOX2, which regulate NANOG expression through binding to its promoter. NANOG, OCT4, and SOX2 mRNA expressions have previously been shown to be significantly higher in samples of moderately and poorly differentiated compared to well-differentiated squamous cell carcinoma [OCT4 and SOX2 are correlated with NANOG in OSCC. Another analyzed transcription factor, KLF4, has been reported to be associated with both oncogenesis and tumor suppression [OCT4 and SOX2 in cooperation with KLF4 can activate NANOG promoter synergistically [KLF4 expression can be used as a poor prognostic factor in head and neck cancer [Previous studies were mainly focused on the expression of two transcription factors, OCT4 . Copies can be identical, occurring in 97% of cases, but their transcripts are often differentially spliced [NANOG in OSCC samples, there were on average four copies of NANOG in most of the tested samples. We found that there is a difference in NANOG CNV in tumor samples compared to TANT; however, it seems that NANOG CNV has a certain role in its regulation but is not crucial for tumorigenesis.The  spliced , 8, 46. In addition to NANOG, which was the main focus in this study, there are also other important known stemness regulators in head and neck SCC including OSCC, such as stem cell markers . Among known stemness transcription regulators, transcription factors that are inducers of epithelial-mesenchymal transition  might also lead tumor cells to achieve stem cell-like properties , 47. HowNANOG in TANT showed highly variable values, from undetectable expression to high expression similar to invasive carcinoma. We therefore excluded samples of normal mucosa from further statistical analyses and comparisons. Another limitation was the subjective cut-off when stratifying our samples and also for immunescoring results. We were only able to analyze NANOG in relation to TNM status of our samples as we described. However, in view of the new General Data Protection Regulation, we were unable to obtain additional patient information for prognostic value evaluation. We focused on mRNA expression levels of NANOG protein regulators and that is a limitation, since mRNA correlation does not necessarily represent protein correlation.The present study has several strengths and limitations. The main strength is related to our approach, which enabled insight into NANOG expression on both mRNA and protein levels and various possible mechanisms and factors of its regulation. Another strength is related to the fact that our study included both FFPE tissue and tissue stored in RNAlater. In FFPE tissue, nucleic acids are fragmented and therefore difficult to analyze, but a great advantage of FFPE tissue is that all samples are first evaluated by pathologists, enabling appropriate diagnosis. In our study, only samples that had successfully passed initial quality control with stable expression of reference genes were further analyzed, thus limiting the number of included samples. In contrast, another limitation was in tissue stored in RNAlater, where nucleic acids are well-preserved, but pathohistological analysis is not possible and samples taken from macroscopically looking normal mucosa may contain foci of dysplasia or even carcinoma, as shown in Our findings confirm the diagnostic significance of NANOG in OSCC and provide information on NANOG expression patterns on both mRNA and protein levels. They also suggest that NANOG protein regulators and miRNAs play a crucial role, whereas methylation of its promoter and CNV probably have a minor role in its regulation. Further studies should focus on the diagnostic and prognostic significance of NANOG and its regulators in the whole process of cancerogenesis, including preneoplastic stages.


EKO), MR (MREKO), or both  in response to dexamethasone (Dex). The overall transcriptional response was abolished in GREKO and DKO skin suggesting dependence of the underlying dermis on the presence of epidermal GR. Indeed, the observed dermal GC resistance correlated with a constitutive decrease in GR activity and up-regulation of p38 activity in this skin compartment. Upon Dex treatment, more than 90% of differentially expressed genes (DEGs) in CO overlapped with MREKO. However, the number of DEGs was fourfold increased and the magnitude of response was higher in MREKO vs CO, affecting both gene induction and repression. Taken together our data reveal that, in the cutaneous transcriptional response to GCs mediated through endogenous receptors, epidermal GR is mandatory while epidermal MR acts as a chief modulator of gene expression.Glucocorticoid (GC) actions are mediated through two closely related ligand-dependent transcription factors, the GC receptor (GR) and the mineralocorticoid receptor (MR). Given the wide and effective use of GCs to combat skin inflammatory diseases, it is important to understand the relative contribution of these receptors to the transcriptional response to topical GCs. We evaluated the gene expression profiles in the skin of mice with epidermal-specific loss of GR (GR However, the outcome of GC treatments is highly variable among patients, with differences in sensitivity occurring due to a variety of factors, many of which are still to be defined. GC response can be influenced by heritable mutations or polymorphisms in GR and resistance that develops as a consequence of pathological processes4. Some patients show adverse effects following short treatments while others tolerate GCs well for longer time periods4. Inadequate GC therapy, due to generalized rather than patient-specific treatment strategies, results in reduced efficacy and also constitutes a socio-economic burden with considerable impact on health care costs. Moreover, detrimental side effects ranging from skin thinning to impaired wound healing preclude long term treatments5. Understanding differential responses among patients holds promise to optimize current GC based therapies minimizing unwanted effects and maximizing therapeutic responses.Glucocorticoid (GC) derivatives are widely used as topical treatments to combat skin inflammatory diseases due to their anti-proliferative and anti-inflammatory actions3. We previously assessed the relative roles of these receptors in the skin by generating mice with epidermal-specific inactivation of GR, MR, or both 8. The characterization of these mouse models demonstrated that: i) during development, epidermal GR and MR play non-overlapping functions and act cooperatively to regulate skin morphogenesis; and ii) in adulthood, neither GR nor MR are required in the epidermis under basal circumstances; however, both act as anti-inflammatory mediators in pathological conditions.GC effects can be mediated through the closely related GC receptor (GR) and the mineralocorticoid receptor (MR), which are ligand-dependent transcription factorsEKO, MREKO, and DKO mice8. However, while GC targets have been previously reported in human cultured keratinocytes and skin10, the relative contribution of GR and MR to GC actions remains unknown. This study aims to understand the global contribution of the endogenous GC receptors, individual or combined, to the GC transcriptional response in the whole tissue, by using an in vivo approach.We have also demonstrated that both epidermal GR and MR are required for the anti-proliferative and protective actions of GCs in inflamed skin as the effects of Dex were drastically reduced in GREKO, MREKO, and DKO mice8 with dexamethasone (Dex) or vehicle (V) for 24 h. RNA-sequencing was performed using whole skin and differentially expressed genes (DEGs) within each genotype were identified by calculating the fold-change (FC) of Dex vs V  or Nr3c2 (MR) in vehicle-treated CO, GREKO, and MREKO mice, by RT-qPCR , including E2f1, Aurka, Ccna2, Ccnd1, Uhrf1, Mki67, Cenpf, Birc5, and Plk1  clustering of DEGs identified in CO and MR MR Fig. . For the Dataset . Other elk1 Fig. . Other oEKO list identified several categories that were coincident to those overrepresented in gene sets common to CO and MREKO, including regulation of apoptosis  and cell cycle   Fig. . These fEKO also clustered into novel GO categories , cytoskeleton organization (3%), response to wounding (3%), fatty acid metabolism (2.6%), epithelium development (2.6%), inflammatory response (1.9%), regulation of T cell activation (1.7%), and steroid metabolic process (1.5%)  Fig. . These cCcna2, Cdk1, and Foxm1, and the chemokine Cxcl10 were repressed by Dex while Ddit4, Igfl3, Mt1, Tfcp2l1, and Tsc22d3/Gilz1, were induced , the global or epidermal-specific phenotypes of the knock-out mouse models for these transcription factors clearly indicate that they do not play overlapping functions10, further validating the use of mice to model skin responses to GCs. The fact that proliferation and apoptosis, two crucial processes modulated by GCs, were overrepresented in both CO and MREKO suggest that epidermal MR may not be strictly necessary for these GC actions. However, as proliferation and apoptosis were also overrepresented in MREKO, it is feasible that the absence of epidermal MR modulates the sensitivity to GCs of specific gene subsets.The DEGs identified in in this study as well as the overrepresented processes are highly coincident with those recently reported in human skin treated with clobetasol7. It is known that the induction or repression of GC targets above or below proper threshold can contribute to detrimental effects of GC excess in several tissues. For example, up-regulation of Zbtb16 and Txnip/Vdup has been reported in bone tissue in Cushing patients37. It is also worth noting the high overlap between the DEGs identified in this study and the gene expression changes in human skin of aged vs young subjects10.The idea that endogenous epidermal MR may modulate the skin sensitivity to GCs would support the topical use of MR antagonists in combination with GCs as an advantageous therapeutic approach. However, it is crucial to address first the question of whether an increased response to GCs is beneficial per se, or could rather mediate some of the GC-associated adverse effects; these options are not mutually exclusiveEKO vs CO skin are difficult to interpret. On one hand, the repression of Foxm1, a transcription factor contributing to the proliferation/differentiation balance in keratinocytes, as well as its target genes Cenpf, Top2a, Ccna2, Birc5, and Plk1, was twofold higher in MREKO vs CO . Groups of 3 to 6 of mice were housed in conventional cages with ad libitum access to food and water, controlled temperature (20–22 °C), humidity (50–60%) and a 12-h light:dark cycle.Nr3c140 and/or Nr3c241 flanked by loxP sites were crossed with those expressing Cre under the control of the keratin 5 promoter (K5-Cre)42 to generate mice lacking epidermal GR (GREKO)6, MR (MREKO)7, or both (DKO)8. Mice were kept on the B6D2 background and littermates with loxP flanked Nr3c1 and/or Nr3c2 were used as controls. Genotyping was performed using primers in Table Mice with the third exon of Dorsal flanks of 2-month old female mice in the telogen phase of the hair cycle were shaved 48 h prior to topical treatment with Vehicle  or 8 µg Dex , which was carried out in the morning. Upon sacrifice at indicated timepoints, skin was collected and snap frozen in liquid nitrogen.Hprt1. Technical triplicates were used; and 3–6 biological replicates per experimental group were assessed to calculate the mean value ± SD. Primer sequences are in Table 43.RNA from mouse cells or tissues was isolated using Trizol . Tissues were homogenized in Trizol with a polytron . The RevertAid H Minus Reverse Transcriptase kit (ThermoFisher) was used to generate cDNA. The Quant Studio 5 Real-Time PCR System  was used for QPCR with gene specific oligonucleotides (0.3 μM each) and the FastStart Universal SYBR Green Master mix ROX (Merck). Cts were normalized to those of the housekeeping gene EKO, MREKO or DKO mice (n = 3/genotype) were treated with vehicle or 8 µg Dex for 24 h and then sacrificed and RNA was prepared from dorsal skin. Integrity of total RNA in the samples was assessed with the Agilent 2100 Bioanalyzer using the RNA 6000 nano Kit . Purified RNA from each sample was prepared for sequencing using the TruSeq Stranded mRNA Sample Preparation Kit with PolyA selection for ribo depletion . Libraries were sequenced on Illumina NextSeq 500 instrument. Depth of 2237 million single 75 pb reads were generated for each sample.CO, GR44 and FastQC v0.11.845. Trimmed sequences were mapped to the Mus musculus genome  using TopHat2 (v2.1.0)46. Seqmonk version 1.4147 was used for quality control, visualization, and quantification. Raw read counts were generated by counting uniquely mapped reads over protein-coding genes using the RNA-seq quantification pipeline assuming opposing strand specificity. Downstream expression analysis was conducted with the EdgeR R/Bioconductor package (v3.20.9) using R (v3.4.4)48. Genes that not satisfy the condition of having at least 1 count-per-million in at least 3 samples were removed. Differential gene expression analyses between experimental defined groups were performed using generalized linear model approach and quasi-likelihood F-test 40. Benjamini–Hochberg false discovery rate (FDR) q values ≤ 0.05 was considered to be significant. The heat map was generated from a cluster analysis of the normalized read counts /σ), euclidean distance, complete-linkage) using the 'pheatmap' package (v 1.0.12) in the R statistical language . Gene ontology was determined using DAVID functional clustering with an EASE score less than 0.05 and medium stringency49.Raw sequence reads were checked for quality, adapter trimmed and filtered using Cutadapt v1.8.32.Following excision of dorsal skin, the hypodermis was removed by scraping the underside of the skin with a scalpel. Next, the remaining skin was floated dermal side down in 3.8% ammonium thiocyanate in DPBS for 10 min at room temperature. The epidermis was then separated from the dermis; both tissues were washed extensively in DPBS, minced and snap frozen in liquid N7 using buffers supplemented with Complete protease and phosphatase inhibitor cocktails (Merck). Protein concentrations were measured using Bradford reagent  and 20–30 µg of protein/sample was boiled in Laemmli buffer, separated on SDS-PAGE, and transferred to Hybond ECL nitrocellulose . Nitrocellulose membranes were stained with Ponceau S (Merck) to verify equal protein loading and transfer prior to blocking and antibody incubations. Primary antibodies used were from Cell Signalling Technology : p-GR Ser211, #4161S, 1/2000; p-ERK Thr202/Tyr204, #4376, 1/1000; p-p38 Thr180/Tyr182 #4631, 1/1000; p-JNK Thr183/Tyr185 #9251, 1/1000, and JNK #9252, 1/1000; Santa Cruz Biotechnology : GR, sc1004, 1/2000; Sigma: actin, A2066, 1/4000; and tubulin, T6199, 1/4000. Peroxidase-conjugated secondary antibodies were from GE Healthcare: anti-rabbit, NA934 and anti-mouse NXA931. Immunoreactive bands were detected using Pierce ECL Plus Western Blotting Substrate (ThermoFisher) and the ImageQuant 4000 Biomolecular Imager . Band intensities were quantitated using Image J software and were normalized to the loading controls, actin, or tubulin.Lysates from whole skin, epidermis, dermis or cultured cells were prepared as previously describedUnless otherwise mentioned cell culture reagents were from Biowest .J2-3T3 fibroblasts were maintained in DMEM supplemented with 7.5% bovine serum , 100 U/ml penicillin/100 μg/ml streptomycin, 2 mM glutamine and 0.25 μg/ml amphotericin B.11. For that purpose, mitotically inactivated J2-3T3 fibroblasts  were plated in flasks coated with type I collagen from rat tail . Next, keratinocytes were added to flasks and cells were cultured in keratinocyte medium: calcium-free DMEM  and Ham’s F12 mixed at a 3:1 ratio and supplemented with 1.8 × 10 − 4 mol/l adenine (Sigma), 0.35 mM calcium (Sigma), 7.5% FBS Gold, 100 U/ml penicillin/100 μg/ml streptomycin, 2 mM glutamine, 0.25 μg/ml amphotericin B, 5 μg/ml insulin (Sigma), 10−10 M cholera toxin (Sigma), and 10 ng/ml EGF .Fibroblasts were used only as feeders for culture and maintenance of the spontaneously immortalized keratinocyte cell lines, which was previously describedFor experimentation, fibroblasts were removed from flasks with Versene and keratinocytes were plated and cultured alone in collagen I-coated tissue culture dishes in keratinocyte medium until around 80% confluent. At this point, cells were washed with DPBS and cells were cultured for 24 h in keratinocyte medium containing charcoal-stripped FBS. Then, keratinocytes were treated with the indicated concentrations of Dex for 3 h.Experimental data were analyzed with Microsoft Excel and IBM SPSS Statistics 25 software. In all graphs mean values ± SD are shown. When statistical analysis was performed with relative values, data were first subjected to logarithmic transformation. The Levene’s test was used to determine whether samples within groups had equal variance before parametric testing. The Student’s unpaired two-tailed t-test was used for comparisons between two experimental groups. For comparisons among higher numbers of experimental groups, the one-way ANOVA and post hoc Tukey multiple comparison test was used.Supplementary Information 1.Supplementary Dataset 1.Supplementary Dataset 2.


JT), Abalakov jump height (ABKH), half-squat test 1 repetition maximum (HST1RM) between T1 and T2 showed significant differences by Wilcoxon signed rank test only in LOW and MED (p < 0.05), but not in the HIGH group (p > 0.05). Internal load was significantly lower in the HIGH group (p = 0.017) regarding LOW and MED by Mann Whitney u test. A significantly lower change during the study in ABKJT (p = 0.038), ABKH (p = 0.038) HST1RM (p = 0.041) and in terms of fatigue (p = 0.018) and lactate (p = 0.012) within the aerobic power-capacity test was presented in HIGH relative to LOW and MED. Conclusions: 120 g/h CHO intake during a mountain marathon might limit neuromuscular fatigue and improve recovery of high intensity run capacity 24 h after a physiologically challenging event when compared to 90 g/h and 60 g/h.Background: Current carbohydrate (CHO) intake recommendations for ultra-trail activities lasting more than 2.5 h is 90 g/h. However, the benefits of ingesting 120 g/h during a mountain marathon in terms of post-exercise muscle damage have been recently demonstrated. Therefore, the aim of this study was to analyze and compare the effects of 120 g/h CHO intake with the recommendations (90 g/h) and the usual intake for ultra-endurance athletes (60 g/h) during a mountain marathon on internal exercise load, and post-exercise neuromuscular function and recovery of high intensity run capacity. Methods: Twenty-six elite trail-runners were randomly distributed into three groups: LOW (60 g/h), MED (90 g/h) and HIGH (120 g/h), according to CHO intake during a 4000-m cumulative slope mountain marathon. Runners were measured using the Abalakov Jump test, a maximum a half-squat test and an aerobic power-capacity test at baseline (T1) and 24 h after completing the race (T2). Results: Changes in Abalakov jump time (ABK Participation in ultra-endurance mountain race events >4 h) has increased in recent years . The dif h has inCarbohydrate (CHO) intake during endurance exercise has been shown to delay neuromuscular fatigue and improve exercise capacity and work rate significantly in a dose–response relationship ,10. In t2+ release from the sarcoplasmic reticulum (SR), has been established in the literature [2+ after completing 1 h of maximum effort [2+ release after 4 h post-exercise between the group that consumed CHO (1 g/kg body weight/h) and the placebo one (water), suggesting that CHO intake during exercise might take on a major role in improving short term glycogen replenishment and muscle function [Peripheral fatigue is understood as the failure of local mechanisms in the muscle and, therefore, the decrease in the contraction and relaxation function that are related with the energetic status of the muscle cell ,19. Amonterature ,22. A st2+ was still reduced at this point and muscle damage biomarkers returned to the baseline on day 5 [2+ release from the SR, Ortenblad et al. [2+ release rate returned to baseline levels.Additionally, both short- and long-term recovery periods following exercise play an important role in ensuring a suitable return to physiological and metabolic homeostasis in athletes. When the neuromuscular function has been affected, replenishment of glycogen stores is required to restore muscle function ,24. Afteon day 5 . In geneon day 5 , althougon day 5 . Furtherd et al.  found thOptimal nutritional protocols to ensure recovery following exercise have been extensively documented in the literature regarding glycogen resynthesis, protein synthesis and rehydration ,28,29,30The main hypothesis of this research was to ascertain whether 120 g /h CHO intake could reduce neuromuscular fatigue by internal exercise load and improve long term recovery compared to current recommendations for ultra-endurance events (90 g/h) ,35, and The current research was put together as a randomized trial with the aim of examining the effects of 120 g/h of CHO intake during a trail marathon race on internal exercise load during exercise and muscle recovery 24 h following exercise. The 120 g/h of CHO supplementation was compared to international references for ultra-endurance events (90 g/h) ,35, and Thirty-one elite male athletes were assessed for eligibility in this study  and the International Association of Athletics Federations (IAAF)). After removing 5 athletes because they failed to meet the inclusion criteria , (II) group that was supplemented with 90 g/h of CHO  and (III) group that was supplemented with 120 g/h of CHO . The runners were instructed that as soon as they felt any damage and/or gastrointestinal distress which might compromise their athletic performance, they should withdraw from the marathon to avoid interference in the results. As a result, 6 athletes withdrew during the race . The remaining athletes finished the marathon without experiencing any injuries and/or gastrointestinal difficulties. Consequently, the sample included in the current research comprised 20 athletes, including 2 world champions  [The runners were randomized into three different groups by an independent statistician via a randomization sequence using SPSS software as follows: (I) group that was supplemented with 60 g/h of CHO  .ad libitum during the race, the participants ingested no food other than the gels. The athletes were instructed to extract the maximum content of the gels so that the residual amount was minimal. Likewise, all the athletes who finished the race confirmed through a questionnaire that all the gels had been taken at the time and in the manner previously indicated.All participants ingested CHO during the race using the same gels made for this research at the University of Valladolid Physiology Laboratory (Soria) by an experienced pharmacist using a gel packaging machine. The gels contained 30 g of maltodextrin (glucose) and fructose (ratio 2:1) to increase exogenous CHO oxidation during exercise . The athThe “marathon of Oiartzun” is a mountain marathon race (42.195 km) which began at 9:00 am in Oiartzun (Guipúzcoa-Spain)  and was controlled by official chronometers. The race consisted of an entry and exit to a circuit that the runners had to complete 3 times. The height accumulated gradient of the mountain marathon race was 3980.80 m  , while tmean) and maximum HR (HRmax) during the event were also recorded.During the race, the heat rate (HR) was documented using a HR monitor, and the mean HR .By way of an inclusion criterion, it was stated that all athletes should have undertaken gut training which involved ensuring >90 g/h CHO intakes > 2 days/week over the 4 weeks prior to the race ,40.Regarding diets and menus during the research period, all were put together individually for each athlete in accordance with international recommendations for ultra-endurance sports ,35 by thThe athletes obtained a personalized diet for a 48-h period prior to T1 and marathon race day with 9 g CHO/kg BM/day, 1.5 g protein/kg BM/day and 0.5 g fat/kg BM/day to adjust their glycogen content. This diet comprised, among other foods, vegetables, olive oil and fish, although it did not contain any fatty meat or butter . MoreoveAt the end of the marathon, each athlete ingested 1.2 g of CHO/kg BM with 0.3 g of whey isolate protein/kg BM in the recovery shakes.Within the next 24 h until T2, each runner consumed a suitable diet  in order to replenish glycogen levels to almost 90–93% of previous muscle glycogen ,42.Runners attended the laboratory for an athletic performance test the week prior to the trail marathon race (T1) and 24 h after it (T2). The internal exercise load was estimated by training impulse (TRIMP) and athletic recovery was assessed using the Abalakov jump test (ABK), Drop jump test (DJ), Half Squat test (HST) and aerobic power-capacity test.The two test sessions were carried out at the ElikaEsport Health Center under standard conditions (temperature: 20 °C and humidity: 55%) for both test sessions, and the tests were completed following a standardized 15-min warm-up. The warm-up involved 10 min of running with two 1-min speeding up (at 3 min and 5 min) and 5 min of injury prevention drills consisting of general movements, dynamic/static stretching and core stability.Abalakov jump test  and halfJT), and Abalakov jump height (ABKH) was calculated [Abalakov jump test: Participants made 3 countermovement with 30-s breaks between jumps . All runlculated . The bes1-RM) using a Multipower machine . After 5 min rest from HST1-RM the runners performed three repetitions at maximal speed for a load of 70% of their 1-RM in this half-squad exercise, with 1-min rest between repetitions to determinate the concentric movement speed of half-squad test (HSTSpeed). During half squat test the athletes performed a knee flexion until the thigh was parallel to the ground (90° knee angle) and, following a command, moved the bar up as fast as possible, without their shoulder losing contact with the bar [Half Squat test: 5-min following Abalakov jump test, runners performed a 1-repetition maximum test (1RM) of half squat test . The protocol used involved maintaining a constant speed of 20 km/h, with a slope of 1%—this being the maximum possible time until exhaustion . During mean and HRmax were recorded, and internal exercise load was also calculated using individualized training impulse (TRIMP) [mean − resting HR)/(HRmax − resting HR).During the mountain marathon racing, the heat rate (HR) of all runners was monitored continuously using the same HR monitors as used in the aerobic power-capacity test. Likewise, during the race the HR (TRIMP) ,50. TRIMAll anthropometric and measurements were administered by an internationally certified level 3 anthropometrist. The anthropometrist administered measurements at T1 in accordance with the International Society for the Advancement of Kineanthrometry (ISAK). All measurements were taken in duplicate, with the exception of those that exceeded 5% difference between each other, which required a third measurement.2 (kg/m2), while triceps, subscapular, suprailiac, abdominal, front thigh and medial calf skinfolds were examined using a skinfold caliber , with 0.2 mm precision. The sum of these 6 skinfolds (mm) was then calculated. A Lufkin model W606PM measuring tape with 1 mm precision was used to measure the perimeters  in cm. These girths were all corrected for the skinfold at the site using the following formula: (corrected girth = girth − (π × skinfold thickness at the site)). Relaxed arm girth was corrected for triceps skinfold (CAG), mid-thigh girth corrected for front thigh skinfold (CTG), and calf girth was corrected for medial calf skinfold (CCG). Muscle mass (MM) was predicted using the Lee [BM (kg) and height (cm) were measured using a scale , with 0.1 kg and 1 mm precision respectively. Body Mass Index (BMI) was considered in accordance with the equation: BM/height the Lee  equationp < 0.05.Statistical analysis was completed by Statistical Package for the Social Sciences 24.0 , with results being shown as mean and standard deviation. The significance level for all analyses was set at n < 50), non-parametric tests were performed because the sample in each of the study group was very small. The percentage changes of the physical test between T1 and T2 were considered as Δ (%): ((T2 − T1)/T1) × 100. Δ (%) of athletic recovery  was compared among 3 CHO intake groups using Kruskal–Wallis test with the CHO intake groups as the fixed factor. A Mann Whitney u tests test was completed for pairwise comparisons between groups.Although the data obtained presented a parametric distribution after using the Shapiro–Wilk test .JT and ABKH in LOW and MED (p < 0.05), while the HIGH did not show any significant differences in these parameters between T1 and T2 (p > 0.05).1-RM in HSTSpeed in the HIGH  in terms of LOW  and MED , no significant differences were observed in the group-by-time for these parameters (p > 0.05). However, there were significant declines in HSTSpeed in MED between T1 and T2 (p < 0.05).On the other hand, although there was a tendency towards a smaller loss in HSTp < 0.05). Moreover, although there was a tendency for a decreased lactate in MED ), only LOW evidenced a significant reduction in lactate between T1 (7.45 ± 1.42 mmol/l) and T2 (5.65 ± 1.27 mmol/l) (p < 0.05). However, HR max showed significant declines both in LOW and MED between T1 and T2 (p < 0.05). On the other hand, Borg showed a significant decrease in HIGH throughout the study , and the Borg value in T2 was significantly less compared to LOW (18.83 ± 0.98) and MED (18.71 ± 1.38) (p < 0.05).p = 0.017). Specifically, a significant higher TRIMP was observed in LOW (399.8 ± 17.5) and MED (371.2 ± 16.2) compared to HIGH (314.8 ± 16.2).JT (p = 0.038) and ABKH (p = 0.038). In this sense, there were significant improvements (p < 0.05) in ABKJT in the HIGH (0.53 ± 5.12%) compared to LOW (−5.90 ± 4.18%) and MED (−5.02 ± 4.22%). Likewise, there was a significant improvement (p < 0.05) in ABKH in the HIGH (1.19 ± 8.05%) regarding LOW (−11.33 ± 8.00%) and MED (−9.66 ± 8.19%). 1-RM among groups between T1 and T2 (p = 0.041). Specifically, a significant smaller decline was noted (p < 0.05) in HST1-RM in HIGH (−2.35 ± 7.23%) compared to LOW (15.30 ± 7.54%) and MED (−13.84 ± 9.74%) between T1 and T2, while HIGH (−4.19 ± 11.31%) evidenced a significant smaller decline (p < 0.05) in HSTSpeed compared to MED (−20.06 ± 14.75%) during the study.p = 0.018) between T1 and T2. Specifically, HIGH (1.28 ± 8.55%) showed a significantly better change in time in aerobic power-capacity test compared to LOW (−14.09 ± 14.98%) and MED (−14.87 ± 11.86%) between T1 and T2 (p < 0.05). Likewise, HIGH (1.69 ± 11.78%) displayed a significantly higher increase in lactate (p = 0.012) compared to LOW (−22.69 ± 9.38%) and MED (−13.94 ± 12.41%). Furthermore, although the percentage change in HIGH showed a tendency towards better values in Borg compared to LOW and MED, these differences were not significant (p = 0.066).The main purpose of this study was to analyze the effects of 120 g/h CHO intake during a mountain marathon on long-term recovery and compare it to current international recommendations for endurance exercise (90 g/h) ,35 and rMountain running events such as marathons and ultra-marathons represent extreme physiological and metabolic challenges as they involve long distances and high exercise intensities, combined with a wide variety of up and down terrain . The run2+ release from the sarcoplasmic reticulum (SR)) and (III) the interaction between actin-myosin proteins [Although fatigue is multifactorial, neuromuscular fatigue represents a determining factor in mountain endurance events ,20, and proteins ,56,57. Aproteins , could e2+ concentration in the cytosol mediated by the reduced Ca2+ release from the SR [2+ reduction could be explained by three main factors: (I) failure in translation of the neural command in the plasma membrane [2+ release [Moreover, it has been shown that mountain running endurance events have a high impact on peripheral fatigue, reducing muscle function and performance ,20,58. Sm the SR . This Camembrane , (II) unmembrane . The resmembrane ,63. In tmembrane , suggestmembrane . As lactmembrane . Thus, i release .As the athletes in the HIGH group had significantly higher blood lactate concentrations and it was correlated, at the same point, with a significantly greater high intensity running capacity 24 h post-marathon, it is possible to state that they showed a higher glycolytic capacity. In endurance events, the metabolic flexibility, which is described as referring to the capacity to use different substrates according to acute metabolic demands of the exercise, constitutes a determining factor in exercise performance and may be indirectly assessed by blood lactate concentration, determining the capacity to use glucose as a fuel during high intensity efforts . In thisAs glycogen represents a major regulator of glycolysis and, therefore, performance capacity ,67, its This study presents some methodological limitations that could have improved the quality of the conclusions drawn. First of all, the activity was not carried out in a laboratory and, therefore, some relevant physiological variables such as oxygen consumption and substrate oxidation were not measured. As these could add important information about the athlete’s physiological and metabolic intensity during exercise, the authors assume this to be a major limitation. Moreover, the absence of glycogen measurements might represent a major limitation that could have enabled us to understand the mechanisms of and reasons for the findings . A direcAdditionally, the study shows some strengths that justify its practical importance in the current literature. It was fully completed under real conditions with a mountain race organized by the authors. This involved a realistic effort from athletes and a unique opportunity for researchers to look into the real effects of CHO intake in a mountain marathon. In this sense, the fact that this trial was carried out in a real scenario helps us to understand the real effects of different CHO intakes in performance and recovery. As demonstrated in our previous research  this triThese findings might open up some relevant future lines of research about the potential benefits of ingesting CHO during exercise to delay neuromuscular fatigue and high intensity run capacity, thus improving post-exercise recovery. Thus, more research is needed in order to understand the best recovery strategies in the field of sports and, specifically, in those multi-stage events in which recovery time is limited and plays a major role in performance. Moreover, this study, besides the previous one published by our group  showed tThis study underscores the importance of CHO ingestion during highly demanding endurance events in order to delay fatigue and improve recovery. Athletes competing in endurance and ultra-endurance events should train in nutritional planning and carry out gut training protocols to optimize performance and recovery from competitions. Moreover, coaches and nutritionists could use high CHO intake during exercise to improve high intensity run capacity and post-exercise recovery in multi-stage competitions or during extreme load training programs. It is our understanding that every athlete should focus on working on the individual maximum CHO intake possible (up to 120 g/h) to ingest without experiencing any gastrointestinal problems. Lastly, 2:1 glucose–fructose ratio could be considered a suitable composition for attaining high amounts of CHO ingestion, although more research is needed in this area.An intake of 120 g/h CHO during a mountain marathon could limit neuromuscular fatigue. In addition, 120 g/h CHO during a mountain marathon would seem to improve long-term muscle recovery by limiting the decrease in neuromuscular function and high intensity run capacity 24 h following a challenging race compared to current recommendations for ultra-endurance events (90 g/h CHO) and regular intake by these athletes (60 g/h CHO). Therefore, high CHO intakes during highly demanding exercises of up to 120 g/h could represent a new, more suitable strategy for optimizing post-exercise recovery during consecutive efforts. Moreover, it has been demonstrated that 120 g/h CHO might be well tolerated in physiologically extreme conditions following a gut training plan.


Twelve men, median 55 years, ECOG 0, median PSA 14.7 ng/dL, clinical stages M0—1/12 (8%), M1a—3/12 (25%) and M1b—8/12 (67%), were treated. 18F-DCFPyL scan was utilized in 58% of cases. Therapies included prostatectomy 12/12 (100%), neoadjuvant , adjuvant radiation , and LHRH agonist 12/12 (100%)]. 2/5 (40%) initial patients developed neutropenic fever (NF), while 0/6 (0%) subsequent patients given modified docetaxel dosing developed NF. Otherwise, TET resulted in no additive toxicities. Median follow-up was 48.8 months. Overall survival was 12/12 (100%). 1-, 2-, and 3-year undetectable PSA’s were 12/12 (100%), 10/12 (83%) and 8/12 (67%), respectively. Median time to biochemical recurrence was not reached. The outcomes suggest TET in men with newly diagnosed OMPCa is safe, does not appear to cause additive toxicities, and may result in an extended interval of undetectable PSA.To evaluate the outcomes of total eradication therapy (TET), designed to eradicate all sites of visible cancer and micrometastases, in men with newly diagnosed oligometastatic prostate cancer (OMPCa). Men with ≤ 5 sites of metastases were enrolled in a prospective registry study, underwent neoadjuvant chemohormonal therapy, followed by radical prostatectomy, adjuvant radiation (RT) to prostate bed/pelvis, stereotactic body radiation therapy (SBRT) to oligometastases, and adjuvant hormonal therapy (HT). When possible, the prostate-specific membrane antigen targeted  In 1995, the term oligometastases (OM) was coined to describe a disease state existing between localized and widespread systemic disease [Men newly diagnosed with PCa and distant metastases have an overall 5 year survival rate of 31% . HoweverIn OM, or low-burden metastatic PCa, randomized studies support radiation to the primary site , metasta18F-DCFPyL PET/CT scan (18F-DCFPyL scan) imaging findings. In men where all PSMA avid lesions were treated, there was decreased risk of later metastases, which supports consolidative MDT. However, the investigators also pointed out that most men do not achieve an undetectable PSA after SABR, which suggests micrometastatic disease is still present.The ORIOLE trial  found thO’Shaughnessy et al. published a pilot study of multimodality treatment to definitively treat known sites of disease in early metastatic prostate cancer . 20% of 18F-DCFPyL scan [In 2015, we utilized a prospective patient registry to follow men under treatment for newly diagnosed metastatic PCa, including oligometastatic prostate cancer (OMPCa). A series of patients underwent treatment to eradicate all clinically evident disease, including neoadjuvant and/or adjuvant chemohormonal therapy, definitive therapy to the primary lesion through radical prostatectomy or external beam radiation therapy, and metastasis directed therapy (MDT) using radiation to the metastatic lesions. The registry allowed for inclusion of real-world clinical practice. The introduction of the PyL scan , with thPyL scan , changedPyL scan , therefoHere we discuss and report outcomes in men who presented with newly diagnosed, treatment naïve OMPCa, and chose to be treated with total eradication therapy (TET)—defined as definitive therapy to the primary lesion (prostatectomy), adjuvant radiation therapy (aRT) to the prostate bed and pelvis as needed, MDT for oligometastases (radiation), and systemic therapy for micrometastases .Men with newly diagnosed, untreated OMPCa were considered for TET, with the intent to eradicate all clinically evident and microscopic disease. In addition to the TET approach, patients were offered hormone therapy alone (discussed as a standard of care), hormone therapy plus radiation therapy, and any available clinical trial where entry criteria were met. Patients who elected TET, and had at least 2 years of follow-up, were included in this report.Pretreatment criteria for TET included histologic confirmation of prostate cancer and ECOG performance status ≤ 1. None of the patients had prior systemic therapy for PCa, prior local therapy to a metastatic site, prior primary therapy (prostatectomy or radiation), abnormal end organ function, active cardiac disease, or a prior history of malignancy (unless in remission > 5 years). Hormone therapy started < 90 days before initiating TET was allowed. All patients provided written informed consent to be followed in the Registry trial as approved by The Johns Hopkins University School of Medicine Institutional Review Board .99mTc-bone scan and a contrast-enhanced CT of the abdomen/pelvis. During the study period, the novel PSMA targeted 18F-DCFPyL scan [OM was defined as ≤ 5 metastatic lesions, including bone lesions and lymph nodes, as demonstrated on a PyL scan , was intADT with a luteinizing hormone-releasing hormone (LHRH) agonist was initiated along with chemotherapy, for 1- or 2- years duration based on physician discretion. Bicalutamide (50 mg daily) was allowed prior to or at the start of LHRH therapy, for ≤ 1 month. During the study period, the addition of abiraterone and prednisone to ADT was found to increase overall survival in men with newly diagnosed, metastatic castrate-sensitive PCa . Hence, 2 dose) and treatment delays  were allowed to mitigate and manage toxicities. Over time, a higher than expected incidence of chemotherapy-related NF was noted, and determined to be due to competition of metabolic pathways when initiating docetaxel and hormonal therapy simultaneously in castrate-sensitive patients [2 for cycles 1 and 2, and of 60 mg/m2 for cycles 3 and 4. In one patient, prostatic adenocarcinoma was mixed with small cell carcinoma (SCC) and cisplatin and etoposide (CIS-ETOP) at 80 mg/m2 and 100 mg/m2, respectively, were given for six, 21-day cycles.Docetaxel was given for up to six, 21-day cycles. Premedication included dexamethasone (8 mg) twice daily, beginning the day before chemotherapy, for 3 days. Clinical and laboratory assessments were performed at baseline, with each cycle, and 3 weeks post-chemotherapy. Dose reductions  and the post-operative PSA.At 12–16 weeks following prostatectomy, aRT to the prostate bed/pelvis was given, as elected following surgery. In the same time frame, or immediately adjacent to aRT, SBRT was used to treat metastatic sites located outside of the adjuvant radiation field.Beyond the 8 weeks post-radiation visit, patients were followed and PSA levels were measured every 3 months. Testosterone levels were measured post-chemotherapy, post-radiation, and then every 3 months after completion of ADT. Imaging was performed at baseline and as clinically indicated.Response to therapy was defined as PSA < 0.1 ng/mL post-radiation. If PSA was detectable, it was monitored monthly to confirm recurrence.Adverse events (AEs) were graded using the National Cancer Institute Common Terminology Criteria for AEs (CTCAE) version 4.0. Toxicities recorded were ADT associated side effects, abiraterone-specific toxicities , chemotherapy-associated neutropenia and NF (worst grade experienced), neuropathy and renal insufficiency, and radiation-associated toxicities. Post-surgical complications were graded using the Clavien-Dindo classification .Patients were observed from initiation of chemotherapy until treatment failure (defined as detectable PSA at the completion of consolidative therapy) or the data closure date (3/5/2020).Tracked outcomes included follow-up time (defined as the time from start of chemotherapy until the data closure date), 1-, 2- and 3-year undetectable PSA, defined as a PSA < 0.1 ng/mL at 1-, 2-, and 3-years from the start of ADT; normalization of testosterone (defined as ≥ 200 ng/dL after completion of ADT); biochemical recurrence (BCR) ; time to androgen independence (defined as failure of androgen deprivation therapy), and overall survival defined as the time from the start of ADT until death. As an observational report, all results are descriptive; continuous variables are expressed as medians with interquartile ranges (IQRs). Kaplan–Meier estimates were used for event-time distributions. All statistical analyses were done with STATA version 15.1 .Twelve patients with newly diagnosed, untreated OMPCa initiated treatment between 10/17/2014 and 1/9/2017   or osseous (M1b)  metastases. The 18F-DCFPyL scan was intermittently available through several imaging protocols [18F-DCFPyL scan modified patient staging: upstaged to OM (n = 1), down-staged to OM (n = 1)  and excluded (n = 4) metastatic sites.100% 12/12) of patients had a rotocols –24 and o/12 of paTreatment details are shown in Fig. 2, in 4 cycles, over a median 9 weeks (IQR 9–10). The initial docetaxel dosing regimen was used in 45% (5/11) of patients, whereas the revised regimen was used in 55% (6/11). The dosing regimens differed in total docetaxel given  within a similar time period (4 cycles within 9.5 versus 9 weeks). Neoadjuvant CIS-ETOP, total dose 480/600 mg/m2, was given in 6 cycles over 15 weeks to the patient with mixed adenocarcinoma and SCC. Median post-chemotherapy PSA was 0.2 (IQR 0–0.45). Four patients achieved an undetectable PSA prior to surgery.Median time to start chemotherapy was 0 days (IQR 0–21.5) after initiating ADT. Neoadjuvant docetaxel was given to 11 patients, total median dose 190 mg/mAll patients underwent a radical prostatectomy, a median 2.3 months (IQR 1.7–2.7) after the final chemotherapy dose. Pathologic findings revealed residual disease in all patients with a histologically apparent treatment effect in 67% (8/12), including significant treatment effects , partial treatment effects 17%, 2/12), and hormonal therapy effects ; T2, T3a, and T3b disease was observed in 33% (4/12), 25% (3/12), and 42% (5/12), respectively, N1 disease was present in 67% (8/12), and positive surgical margins in 33% (4/12). Post-operative PSA was undetectable in 83% (10/12), and was not assessed in 17% (2/12) patients.Following prostatectomy, all patients underwent radiation therapy: adjuvant radiation to the prostate bed/pelvis only , adjuvant radiation to the prostate bed/pelvis and SBRT to metastatic sites outside of the adjuvant radiation field , and SBRT only . Adjuvant radiation to the prostate bed and pelvis was given at a median interval of 3.7 months (IQR 3.2–5.0) following prostatectomy. Post-radiation PSA was undetectable in all patients.Chemotherapy-related NF, grades 3/4, occurred in 18% (2/11) of patients; both were treated on the initial docetaxel dosing regimen. Neutropenia, grade 3, occurred in 45% (5/11) of patients: 3/6 treated in the initial dosing regimen (including the 2 patients with NF) and 2/5 treated in the revised dosing regimen. Chemotherapy-related renal insufficiency and peripheral neuropathy (both grade 1), occurred in the patient treated with CIS-ETOP.LHRH therapy-related grade 2 vasomotor symptoms, requiring medical therapy, occurred in 17% (2/12) of patients. Of the 6 patients given abiraterone, related toxicities occurred in 3/6, and included elevated alanine aminotransferase (grade 3), cough (grade 1) and fatigue (grade 3). In all cases, the toxicity improved or resolved with dose reduction or discontinuation of abiraterone.n = 1 and resolved, n = 1). No other complications were reported.Post-prostatectomy urethral strictures  occurred in 17% (2/12) of patients , grade 1 constipation (n = 2), nausea, vomiting, proctitis, urinary frequency/urgency ; no long-term toxicities were reported.Radiation-related toxicities during treatment occurred in 3/6 patients who underwent adjuvant radiation to the prostate bed/pelvis: grades 1–2 hemorrhoids (n = 1) and grade 1 diarrhea (n = 1); no long-term toxicities were reported.Of the 6 patients who underwent SBRT only, 2/6 reported toxicities during treatment: grade 2 dysuria, diarrhea, hemorrhoids  (Table .6–60.6 (n = 1), SBRT and ADT (n = 1), SBRT (n = 1), salvage RT and ADT (n = 1), and lymph node dissection and ADT (n = 1)  ( 1) Fig. .Men newly diagnosed with PCa and distant metastases have an overall 5 year survival rate of 31% . There iTET is an intensive treatment protocol given over 1 year, after which therapy is complete and testosterone is expected to recover. 67% (8/12) of the men treated with TET had a 3YR undetectable PSA, along with a recovered testosterone and freedom of ADT side effects. While patients treated with hormone therapy alone may experience an undetectable PSA at 3YR, it is expected that at a mean of 2–3 years on hormone therapy, androgen independence will occur , leaving18F-DCFPyL PET scan was recently shown to identify putative sites of disease in a majority of men in a prospective study of men with BCR. In that study, 68% (21/31) had at least one site of PSMA uptake consistent with a site of PCa, despite having negative conventional imaging [18F-DCFPyL scan changed management by altering the eligibility for the diagnosis of OM, as well as by altering the number or location of metastases treated with SBRT.OM remains a diagnosis of imaging, by virtue of the lone biomarker—the number of lesions – which traditionally has been assessed by bone scan and CT. The  imaging . In our Large, controlled trials , 28, 29,Although multiple studies are underway to optimize the treatment of men with newly diagnosed metastatic and OMPCa, they do not typically attempt to definitively treat all sites of clinically evident disease, as well as micrometastases. The addition of radiation to the primary tumor to ADT has been shown to increase survival . SWOG 18Our registry study of patients with newly diagnosed, untreated OMPCa, treated with TET, served as a model to inform the optimal study design for prospective TET trials. We then initiated two prospective phase II clinical trials (protocols available in eSupplements) to assess the efficacy of TET in men with newly diagnosed OMPCa (< 5 sites of metastases). The first trial, TED-1 , is for treatment naïve men with OMPCa. The second trial, TED-2, is for men diagnosed with OMPCa within 6 months of prostatectomy. The TED-1 study design includes neoadjuvant treatment encompassing up to 6 months of androgen deprivation and up to 6 cycles of docetaxel with concurrent abiraterone and prednisone. Following docetaxel therapy, patients with a PSA response of at least a 50% decrease from baseline, proceed to maximum consolidative therapy. Consolidative therapy includes definitive local therapy with radical prostatectomy (RP), ± adjuvant radiation therapy (RT) (given in the setting of adverse pathologic features), ± consolidative stereotactic body radiation therapy (SBRT) to the oligometastatic sites. Androgen deprivation is continued through consolidative therapy; it is given for 1 year in total. The TED-2 study design, for men post-prostatectomy (< 6 months), includes adjuvant treatment encompassing up to 6 months of androgen deprivation and up to 6 cycles of docetaxel with concurrent abiraterone and prednisone. Following docetaxel therapy, patients proceed to maximum consolidative therapy. Consolidative therapy includes ± adjuvant radiation therapy (RT) (given in the setting of adverse pathologic features) ± consolidative stereotactic body radiation therapy (SBRT) to the oligometastatic sites. Androgen deprivation therapy is continued through consolidative therapy; it is given for two years in total. Follow-up for TED-1 and TED-2 is for 2 years after completion of ADT treatment and includes serial labwork and imaging. The primary endpoint is 2- and 3-year progression-free PSA (< 0.2 ng/mL), respectively.Potential toxicities associated with the individual modalities of TET include post-chemotherapy neutropenia and febrile neutropenia, and post-prostatectomy or radiotherapy non-neutropenic toxicities including urinary incontinence, urinary complications requiring instrumentation, rectal or anal complications requiring instrumentation, and secondary malignancy. To mitigate potential additive toxicity from TET multimodal therapies, the TED 1 and 2 protocols include early stopping boundaries. Based on prior probabilities of the average risks of toxicities, the early stopping boundaries incorporate safety monitoring after each patient, and outline when to stop the studies should the threshold of acceptable toxicity be crossed.Tracking the treatment and outcomes of a registry study of patients with newly diagnosed, untreated OMPCa allowed us to assess the impact of TET. Our outcomes suggest that multi-modality therapies can be combined safely resulting in durable responses. Therefore, we have since initiated clinical trials utilizing TET in men with newly diagnosed OM disease, both prior to prostatectomy (NCT02716974 ) and pos


Most acute promyelocytic leukemia (APL) patients respond to all-trans-retinoic acid (ATRA)and have a good prognosis. However, variants APL who carry PLZF/RARа, STAT5B/RARа, and STAT3/RARа are insensitive to ATRA and have poor prognoses. The standard treatment for variants APL is still unclear due to the small sample size.9/L), anemia (hemoglobin 73 g/L), and thrombocytopenia (54 × 109/L). Prothrombin time and activated partial thromboplastin time were normal.Here we reported a Chinese male who was admitted to our hospital with the complaint of rib pain, dyspnea, and fever (37.5°C). Blood tests showed leukopenia .The patient relapsed soon after his first molecular complete remission (CRm), fortunately, he got a second CRm with DCAG. He has survived for more than 9 months and remains CRm, now he is looking for a suitable donor to prepare for hematopoietic stem cell transplantation (HSCT).APL patients with STAT5B-RARa is not only resistant to ATRA, but also to conventional combination chemotherapy such as daunorubicin and cytarabine/idarubicin and cytarabine or other regimens. Relapse and extramedullary infiltration is common, HSCT is a effective treatment, and the best time for HSCT is after the first CR. It should be noted that this patient got CRm with DCAG after relapse, so the role of decitabine in APL with STAT5B-RARa needs to be considered. The STAT5B/RARа fusion gene is rare in APL and was first reported in 1996. To date, 16 cases have been reported. The standard treatment for variants APL is still unclear due to the small sample size. Here, we report a 62-year-old man diagnosed as APL with STAT5B/RARа and summarize the clinical features and effective treatment, hoping to improve the prognosis of these patients.More than 98% of acute promyelocytic leukemia (APL) patients have classic t and promyelocytic leukemia-retinoic acid receptorА (PML-RARА) fusion genes; they respond to all-trans retinoic acid (ATRA) and have a good prognosis. However, RARа fuses with other partner genes in patients with variants APL (1%–2%), and some new fusion proteins have been formed, such as PLZF/RARа, NPM/RARа, NuMA/RARа, STAT5B/RARа, FIP1L1/RARa, PRKAR1A/RARa, and BCOR/RARa. APL patients with PLZF/RARа, STAT5B/RARа, and STAT3/RARа are insensitive to ATRA.29/L, hemoglobin 73 g/L, platelet count 54 × 109/L, prothrombin time 13.6 seconds, activated partial thromboplastin time 38.8 seconds, fibrinogen 5.8 g/L, D-dimer 2.4 μg/mL, fibrinogen degradation products 8.15 μg/mL. Abnormal promyelocyte in bone marrow aspiration showed 80.4% of blast, and flow cytometry indicated mainly positivity for CD33 and myeloperoxidase, partially positivity for CD117, CD9, CD11b, CD13, CD15 and CD64; negativity for CD7, CD19, CD10, cCD3, cCD79a, CD16, CD123, HLA-DR, CD14, CD56, CD3, CD4, CD8, CD2, and CD11c. Cytogenetics showed 43,−46,XY,+2,−5,+8,14p+,−16,17q−,17q+,+18,−19,−20,−21,+mar1,+mar2[CP5]/46,XY[15] . This patient is now looking for a suitable donor to prepare for hematopoietic stem cell transplantation (HSCT).A 62-year-old man was admitted to the hospital with the complaint of rib pain, dyspnea, and fever (37.5°C). Blood tests showed the following results: white blood cell count 1.83 × 103STAT5B is located on chromosome (Chr)17q21.1-21.2 and mainly distributed in the cytoplasm and nucleus. It regulates cell proliferation and induces leukemia through JAK-STAT signaling pathways. STAT5B plays a role in preventing further differentiation of hematopoietic cells together with the corepressor complex of deacetylase, this complex cannot be released by ATRA. This may explain the resistance of APL with STAT5B/RARa to ATRA.The current case showed a complex karyotype and an unsatisfactory treatment process. He was responsive to ATRA and ATO at the beginning and achieved CRm after the fourth course; however, he relapsed quickly (molecular biology relapse). To date, a total of 16 cases of APL with STAT5B/RARа have been reported worldwide; this disease is extremely rare (Table We can conclude that APL patients with STAT5B-RARa is not only resistant to ATRA, but also to conventional combination chemotherapy such as DA/IA or other regimens. Relapse and extramedullary infiltration is common, HSCT is a effective treatment, and the best time for HSCT is after the first CR.The authors would like to thank American Journal Experts who provided language editing.Data curation: Lei Wang.Funding acquisition: Xiaojing Yan.Software: Lei Wang.Writing – original draft: Lei Wang.Writing – review & editing: Lei Wang, Juan He.


Arabidopsis thaliana plants under normal and NaCl-stressed conditions. Methods: For this study, ex vitro and in vitro growth experiments were conducted to assess the effect of both NaCl and BC204 on basic physiological parameters such as biomass, chlorophyll, proline, malondialdehyde, stomatal conductivity, Fv/Fm and the expression of four NaCl-responsive genes. Results: This study provides preliminary evidence that BC204 mitigates salt stress in Arabidopsis thaliana. BC204 treatment increased chlorophyll content, fresh and dry weights, whilst reducing proline, anthocyanin and malondialdehyde content in the presence of 10 dS·m−1 electroconductivity (EC) salt stress. Stomatal conductivity was also reduced by BC204 and NaCl in source leaves. In addition, BC204 had a significant effect on the expression of salinity-related genes, stimulating the expression of salinity-related genes RD29A and SOS1 independently of NaCl-stress. Conclusions: BC204 stimulated plant growth under normal growth conditions by increasing above-ground shoot tissue and root and shoot growth in vitro. BC204 also increased chlorophyll content while reducing stomatal conductivity. BC204 furthermore mitigated moderate to severe salt stress (10–20 dS·m−1) in A. thaliana. Under salt stress conditions, BC204 reduced the levels of proline, anthocyanin and malondialdehyde. The exact mechanism by which this occurs is unknown, but the results in this study suggest that BC204 may act as a priming agent, stimulating the expression of genes such as SOS1 and RD29A.Aims: The aim of this study was to assess the effect of BC204 as a plant biostimulant on  Although all of these ions can cause soil salinisation, high concentrations of Na+ as a result of excess NaCl is the most prevalent and the subject of most salt-stress related research  BC204, 50 mM NaCl, 50 mM NaCl+ 0.01% [v/v] BC204) were selected for measurement of stomatal conductance, using a SC-1 leaf porometer , and Fv/Fm, using an 0S30p+ chlorophyll fluorometer .Ninety minutes after the final BC204 treatment, 12 plants from each treatment group  + (0.00802 × A645).Chlorophyll was extracted from plants grown on peat disks using an adapted protocol . Nine plTM no.9, South Africa) using an adapted protocol [2O (9:1:10) buffer. Absorbance was measured at 530 nm and 637 nm, then anthocyanin content was calculated as A530 − (0.25 − A637) and expressed as Abs530/g·FW.Anthocyanins were extracted from plants grown on Jiffy peat disks (Jiffyprotocol . Nine plw/v) sulfosalicylic acid (SAS). Samples were vortexed and centrifuged at 10,000× g for 5 min. A 100 µL aliquot of the supernatant was added to 500 µL of a reaction mixture of 3% (w/v) SAS/glacial acetic acid/acidic ninhydrin (1:2:2). This mixture was heated at 95 °C for 60 min before being centrifuged at 10,000× g for 5 min. Absorbance was measured at 520 nm.Proline content was determined using an adapted protocol . Nine plw/v) trichloroacetic acid (TCA), centrifuged at 10,000× g for 5 min and the supernatant mixed with 20% (w/v) TCA and 0.5% (w/v) thiobarbituric acid (TBA). The mixture was boiled for 15 min before being transferred to ice and centrifuged at 10,000× g for 2 min. The absorbance of the supernatant was measured at 532 nm. SOD activity was determined according to manufacturer’s protocol using the InvitrogenTM K335-100 Superoxide Dismutase (SOD) Activity Colorimetric Assay kit .Malondialdehyde was extracted and quantified according to Rao and Sresty . Leaf ti® 16 AS2000 Instrument with the Maxwell® 16 Total RNA Purification Kit, as per the manufacturer’s protocol . One microgram of total RNA was used to obtain complementary DNA (cDNA) via reverse transcription using an oligo(dT)18 primer and RevertAid reverse transcriptase , according to manufacturer’s protocol. The PowerUpTM SYBRTM Green Master Mix kit and the QuantStudio 3 Real-Time PCR System was used for reverse transcriptase quantitative PCR (RT-qPCR) analysis and the relative expression calculated using the 2−ΔΔCT method [Ef1α (At1g18070) was used for as an internal control as it has previously been shown to be a suitable reference gene for A. thaliana under abiotic stress conditions, including salt stress [Nine plants from each treatment were randomly selected and pooled into three representative samples (3 plants per sample), flash frozen, ground in liquid nitrogen using a mortar and pestle and total RNA extracted in a MaxwellT method . Expresst stress . Primer All experiments were independently replicated at least three times to ensure reproducibility. Statistical significance between control, treated and NaCl-stressed groups was determined by the one-way ANOVA function in Excel followed by the Fischer’s least significant difference (LSD) test at the 0.05 probability level.−1) in A. thaliana. Under salt stress conditions, BC204 reduces the levels of proline, anthocyanin and malondialdehyde. The exact mechanism is unknown, but the results in this study indicate that BC204 acts as a priming agent in some sense, stimulating the expression of genes such as SOS1 and RD29A.BC204 stimulates plant growth under normal growth conditions by increasing above-ground shoot tissue and root and shoot growth in vitro. BC204 also increases chlorophyll content while reducing stomatal conductivity. BC204 furthermore mitigates moderate salt stress (10–20 dS·m+ and K+) will also be valuable to shed light on the effect of BC204. For gene expression, several samples should be harvested at different time points within the experiment to account for the circadian control of specifically genes like RD29A [This study focused on salt stress at a single growth stage, at the end of vegetative growth. However, investigating how the plant reacts to BC204 during oxidative stress/early salt stress will be valuable as it will give insight into a possible priming effect of BC204 during earlier stages of plant growth. Ion measurements (Nake RD29A .There is an overlap in how plants perceive and respond to salt, drought and, to a lesser extent cold stress as many genes that are regulated by salt stress also respond to cold or drought stress . Therefo