In children with CKD, Protein Energy Wasting (PEW) is common, which affects the outcome of children and is an important cause of poor prognosis. We are aiming to explore the pathogenesis of muscle wasting in CKD-PEW children.Blood samples of 32 children diagnosed with chronic kidney disease (CKD) and protein energy wasting (PEW) in our hospital from January 2016 to June 2021 were collected. RNA sequencing and bioinformatics analysis were performed.P = 2.81 × 10–16), and its expression was up-regulated by 3.92 times . Followed by RETN (adjusted P = 1.63 × 10–7), and its expression was up-regulated by 8.306 times . SELE gene were secondly significant (adjusted P = 5.81 × 10–7), and its expression was down-regulated by 22.05 times .Based on GO (Gene Ontology) functional enrichment analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis and differential gene expression analysis, a total of 25 CKD-PEW related genes were obtained including CRP, IL6, TNF, IL1B, CXCL8, IL12B, IL12A, IL18, IL1A, IL4, IL10, TGFB2, TGFB1, TGFB3, ADIPOQ, NAMPT, RETN, RETNLB, LEP, CD163, ICAM1, VCAM1, SELE, NF-κB1, NF-κB2. The most significantly differentially expressed gene was NF-κB2 (adjusted A variety of inflammatory factors are involved in the pathogenesis of CKD-PEW in children, and chronic inflammation may lead to the development of muscle atrophy in CKD-PEW. It is suggested for the first time that NF-κB is a key gene in the pathogenesis of muscle wasting in CKD-PEW children, and its increased expression may play an important role in the pathogenesis of muscle wasting in children with CKD-PEW. Chronic Kidney Disease (CKD) involves a gradual loss of kidney function. Malnutrition is an important complication of CKD and leads to poor prognosis , but theTranscriptome refers to the sum of all RNAs transcribed in a specific tissue or cell at a certain time or state, mainly including mRNA and non-coding RNA . RNA seqThe pathogenesis of PEW in children with CKD is very complex, and the exact mechanism is currently unclear. Some studies have shown that it is related to factors such as decreased appetite, inflammatory state, insufficient energy intake, increased consumption, and dialysis, etc. . HoweverA total of 32 children diagnosed with CKD PEW in our hospital from January 2016 to June 2021 were enrolled. The study was approved by the Ethics committee of Beijing Children's Hospital and strictly followed the Declaration of Helsinki.2) ≥ 3 months, with or without evidence of kidney injury.(1) Diagnostic criteria for chronic kidney disease : ① Kidne2);Stage 1 eGFR ≥ 90 ml/(min · 1.73 m2);Stage 2 eGFR 60–89 ml/(min · 1.73 m2);Stage 3 eGFR 30–59 ml/(min · 1.73 m2);Stage 4 eGFR 15–29 ml/(min · 1.73 m2).Stage 5 eGFR < 15 ml/The diagnostic criteria for protein-energy wasting were based on the adult diagnostic criteria of the International Society of Renal Nutrition and Metabolism, and the modified diagnostic criteria for children were combined with the characteristics of children and relevant literature, including , 9, 10: (1) complicated with other organ dysfunction; (2) complicated with infection; (3) receiving blood purification treatment.Three to four ml blood samples were drawn from each child and stored in PAXgene Blood RNA storage tubes of BD company. Blood samples were kept with 4° ice bags, placed in an incubator, and delivered immediately for RNA sequencing. RNA-seq experiment and high through-put sequencing and data analysis were conducted by MyGenostics Inc. .https://doi.org/10.1006/abio.1987.9999). DNA digestion was carried out after RNA extraction by DNaseI. RNA quality was determined by examining A260/A280 with NanodropTM OneCspectrophotometer (Thermo Fisher Scientific Inc). RNA Integrity was confirmed by 1.5% agarose gel electrophoresis. Qualified RNAs were finally quantified by Qubit3.0 with QubitTM RNA Broad Range Assay kit .Total RNAs were extracted from peripheral blood cells of 32 children with CKD PEW using TRIzol Reagent  following the methods by Chomczynski et al.  following the manufacturer’s instruction. PCR products corresponding to 200–700 bps were enriched, quantified and finally sequenced on DNBSEQ-T7 sequencer  with PE150 model.In this study, the eukaryotic mRNA was enriched by magnetic beads with Oligo (dT). The mRNA was broken into short fragments by adding lysis buffer. The first cDNA strand was synthesized with random hexamers using mRNA as template, and then the second cDNA strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. After purification by QiaQuick PCR kit, elution with EB buffer and the end repair, polyA and sequencing adapter were connected, and then agarose gel electrophoresis was used for fragment size selection, and finally PCR amplification was performed. The constructed sequencing library was sequenced by high-throughput sequencing instrument. The specific experimental procedure is shown in Fig. ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna/) using STRA software (version 2.5.3a) with default parameters. Reads mapped to the exon regions of each gene were counted by featureCounts  and then RPKMs were calculated. Genes differentially expressed between groups were identified using the edgeR package (version 3.12.1). A p-value cutoff of 0.05 and fold-change cutoff of 2 were used to judge the statistical significance of gene expression differences. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis for differentially expressed genes were both implemented by KOBAS software (version: 2.1.1) with a p value cutoff of 0.05 to judge statistically significant enrichment. Alternative splicing events were detected by using rMATS (version 3.2.5) with a FDR value cutoff of 0.05 and an absolute value of Δψ of 0.05.Raw sequencing data was first filtered by Trimmomatic (version 0.36), low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed.clean data were mapped to the reference genome of human from Ensembl database , and cluster analysis, gene ontology (GO) functional annotation analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for significant DEGs. The analysis process is shown in Fig. P < 0.05 was considered statistically significant.SPSS19.0 software was used to establish a database and analyze the data. A total of 32 children were included in the study, with an average age of 9.27 years, including 21 males and 11 females.P < 0.05), of which 4998 genes were up-regulated, 13,798 genes were down-regulated. There were 1102 genes with log2foldchange > 2 and 10,495 genes with log2foldchange < -2.DESeq2 software was used to analyze 32 cases versus 18 controls . A totalGO functional annotation analysis found the top 9 GO terms with the most obvious enrichment see Fig. .Fig. 3GOKEGG pathway enrichment analysis found the top 20 most significantly enriched related pathways see Fig. . pathwayAfter enrichment results of clearly related diseases are removed, the signal pathways clearly involved are included as shown in Table P = 2.81 × 10–16), and its expression was up-regulated by 3.92 times . Followed by RETN (adjusted P = 1.63 × 10–7), and its expression was up-regulated by 8.306 times . SELE gene was secondly significant (adjusted P = 5.81 × 10–7), and its expression was down-regulated by 22.05 times .In this study, genes associated with CKD-PEW for which expression profile data were obtained included: CRP, IL6, TNF, IL1B, CXCL8, IL12B, IL12A, IL18, IL1A, IL4, IL10, TGFB2, TGFB1, TGFB3, ADIPOQ, NAMPT, RETN, RETNLB, LEP, CD163, ICAM1, VCAM1, SELE, NF-κB1, NF-κB2, a total of 25 genes (Table Chronic Kidney Disease (CKD) is a worldwide epidemic disease. In children with CKD, Protein Energy Wasting (PEW) is common, which affects the outcome of children and is an important cause of poor prognosis , 13. ResInflammation is an important component of chronic kidney disease (CKD), which has been recognized since the late 1990s and is associated with PEW and mortality . In the The current mainstream view is that CKD-induced muscle atrophy is closely related to inflammation. In chronic diseases, inflammatory factors can activate different cellular signals upon binding to their receptors, including camp-dependent protein kinases (PKA), protein kinase C (PKC), phosphatidylinositol 3-kinases (PI3-K), and mitogen-activated protein kinases (MAPKs).The stimulation of these proteins kinases leads to the activation of different transcription factors, of which NF-κB is one of the important signaling pathways , 21. NF-The transcription levels of RETN and SELE genes in patients with CKD were also significantly higher than those in the control group. Because the proportion of up-regulation was the same, it is speculated that RETN and SELE genes may play a synergistic role. KEGG only found that SELE was involved in the "Fluid shear stress and atherosclerosis" signaling pathway, but RETN was not found to be involved in the signaling pathway. According to the literature search, only one article showed that RETN and SELE polymorphisms were significantly associated with coronary artery disease, but the relationship between RETN and SELE polymorphisms and CKD was not reported .Further studies are needed to explore the association between Retn and SELE polymorphisms and CKD-PEW.Inflammation is one of the important factors leading to protein-energy wasting in chronic renal failure, especially muscle wasting . NF-κB iThe sample size of this study is relatively small, and more samples need to be included for further study. The result of transcriptome sequencing in this study needs further biological validation which is the reseach we are currently conducting now.


No significant difference was observed for any parameter once the keratinocytes were cultured from P1 to P4. The CFE and K19 expression decreased from P0 to P1 in both protocols, probably due to the culture process. This study shows that both protocols enable the efficient isolation of skin dermal and epithelial cells and subsequent culture to produce grafts destined for the treatment of patients.For the development of advanced therapies, the use of primary cells instead of cell lines is preferred. The manufacture of human tissue-engineered skin substitutes requires efficient isolation and culture protocols allowing a massive expansion of the cells in culture from an initial specimen of a minimal size. This study compared two skin cell isolation protocols, routinely applied in two clinical laboratories. Epithelial (keratinocytes) and dermal (fibroblasts) cells were isolated and cultured from three human skin biopsies (N = 3). The two-step digestion protocol (LOEX-Protocol) firstly used thermolysin to enzymatically disrupt the dermal–epidermal junction while, for the one-step digestion protocol (UPCIT-Protocol), mechanical detachment with scissors was applied. Then, the epidermal and dermal layers were digested, respectively, to achieve cell isolation. The cell size, viability, yield and growth were analyzed over five passages (P). The colony-forming efficiency (CFE) and Keratin 19 (K19) expression of epithelial cells were also assessed after P0 and P1. Regarding the dermal cells, no significant differences were observed in the tested parameters of isolation and culture. However, for the epithelial cells, viability was higher (93% vs. 85%) and the number of cells extracted per cm Immortalized or commercial cell lines are widely used in research due to their cell growth potential and well-known characteristics . HoweverTo establish a primary cell culture, different aspects such as the cell isolation protocol, cell attachment to plastic surfaces and culture media must be considered . To initTherefore, it is important to optimize and use a successful isolation protocol that preserves the properties and characteristics of the different native human cell types as much as possible. Moreover, these methods should be robust, standardized and report a good cell yield to be able to reduce the manufacturing time in the case where the cell/tissue grafts are required to treat patients ,6. Manufacturing tissue-engineered skin substitutes (TESSs) for clinical purposes, such as burn treatment, requires the use of autologous skin cells, commonly keratinocytes and fibroblasts, the main cell types of the epidermis/appendages and the dermis, respectively . For thiDespite the similarities between most skin cell isolation procedures, different protocols exist depending on the number and type of enzymatic digestions or the addition of mechanical disaggregation or not . Among tThe aim of this study was to compare these two isolation protocols in terms of the cell yield and size, cell proliferation and population doubling time of keratinocytes and fibroblasts over five passages (P0 to P4). The isolation protocols compared were as follows: (i) the one applied at the LOEX Tissue Engineering Laboratory (LOEX-Protocol) and (ii) the one used at the Unidad de Producción Celular e Ingeniería Tisular (UPCIT-Protocol). Moreover, the presence of proliferative and stem cells in the cultures of keratinocytes was evaluated by colony-forming efficiency (CFE) assays and flow cytometry analysis during the primary culture and first passage .p-value < 0.001) smaller than the cultured keratinocytes (P0 to P4)  of the epithelial cells isolated with both the LOEX- and UPCIT-Protocols was similar at each time point (freshly isolated and at each passage). For both methods, the freshly isolated cells were significantly (0 to P4) A.p-value < 0.01). However, after cell culture over several passages, no significant differences were observed. The cell viability varied between 89% and 95% for the cultured keratinocytes (P0 to P4) for both protocols  with the LOEX-Protocol (3.28 × 106 ± 0.92 × 106 cells/cm2 biopsy) than with the UPCIT-Protocol (9.52 × 105 ± 4.81 × 105 cells/cm2 biopsy) (2 for a given passage between P0 and P4 (The cell yield after isolation from the skin biopsy was significantly higher ( biopsy) D. No sig0 and P4 E. p-value < 0.001 and p-value < 0.0001). No significant difference was observed between the protocols at each time point   for the LOEX- and UPCIT-Protocols (0 to P4) A.p-value < 0.05; The cell viability of the freshly isolated cells B and culp-value < 0.05; Regarding cell yield, although a higher number of freshly isolated dermal cells was obtained using the LOEX-Protocol compared to the UPCIT-Protocol, no significant difference was reported D. Althoup-value < 0.01)   B. The ne < 0.01) B. For ce and P4) B. Represp-value < 0.01 for the LOEX-Protocol and p-value < 0.05 for the UPCIT-Protocol)  B. The phrotocol) C.Epithelial stem cells are known to be clonogenic. To evaluate the clonogenicity of the epithelial cells isolated from both protocols, the size and number of colonies formed by the keratinocytes were monitored. Since the size of a colony reveals the proliferation potential of the epithelial cell that generated it, the diameter (Ø) of the colonies was measured to determine the percentage of holoclones (Ø ≥ 4.5 mm), meroclones (4.5 mm > Ø > 1.5 mm) and paraclones (Ø ≤ 1.5 mm). The proportion of holoclones, meroclones and paraclones was similar regardless of the cell isolation protocol applied A,B. Betwp-value < 0.05 for the LOEX-Protocol and p-value < 0.01 for the UPCIT-Protocol), although no significant difference was observed regarding the isolation protocol used (A similar colony-forming efficiency (CFE)—defined as the percentage of holoclones from the initial number of epithelial cells seeded—was observed for both the LOEX- and UPCIT-Protocols but decreased from P0 to P1 C. Howevecol used D. RepresK19 is a marker associated with skin epithelial stem cells . The proIn this study, we compared two human skin cell isolation protocols that are used to manufacture human bilayered tissue-engineered skin substitutes for the treatment of severely burned patients. Each of these protocols was developed independently at the LOEX (Canada) ,10 and tThe main difference between both protocols is the first step, which is a mechanical preparation of the skin or an added digestion step. The protocols involving only one digestion step have been described, in which skin biopsies (epidermis–dermis) are incubated with a trypsin enzyme ,14,15,16Compared to one-step digestion protocols, the two-step digestion protocols are preferred mainly for epithelial cell isolation , and disFor dermal cell isolation, the protocols are more similar and based on collagen digestion. Once the epidermis is digested or detached, the dermal tissue is incubated with a single ,13,20,222 of biopsy (1 × 106–2.6 × 106 cells/cm2) [5–6.6 × 105 cells/cm2) [6–5 × 106 cells/cm2) when skin biopsies are directly digested without trimming the dermis [The higher epithelial cell yield after isolation with the LOEX-Protocol than the UPCIT-Protocol is consistent with previous studies where the application of the two-step digestion protocol allowed for isolating a higher number of epithelial cells/cmlls/cm2) ,31 than lls/cm2) , which ie dermis ,14,15,31e dermis  and, thee dermis ,31, at tThe colony-forming efficiency and total number of colonies were similar in both protocols, although slightly higher using the two-step digestion protocol (LOEX-Protocol) compared to the one-step protocol (UPCIT-Protocol) at P0. Similar results have been reported previously ,32,33. TIn addition, the total number of colonies was increased from P0 to P1 in both protocols, probably due to the keratinocytes’ culture conditions (specific medium and feeder layer), which allow a good proliferation of keratinocytes, through maintaining Sp1 expression, and reduce the growth potential of the residual fibroblasts ,34. A loFinally, culturing keratinocytes through passages can cause stress and reduce the number of epithelial stem cells . TherefoThe clinical implications of these results are that, regardless of the skin cell isolation methodology applied, only a minor significant difference was observed in terms of epithelial cell yield just after isolation. However, during the cell culture of keratinocytes and fibroblasts, similar results demonstrate that both protocols are robust and comparable and allow for obtaining a sufficient number of cells, in an appropriate period of time, to be able to manufacture human bilayered tissue-engineered skin substitutes for clinical applications as fast as possible. Therefore, this information could be useful for clinical laboratories with the same purposes since they could adapt their own protocols or use these as a reference to develop and optimize their preclinical and clinical research. Human keratinocytes and fibroblasts were extracted from three human adult skin biopsies (abdomen (n = 2) and face (n = 1)) from healthy women . Before starting LOEX and UPCIT isolation protocols, adipose tissue and/or blood traces were removed.®, Waltham, MA, USA) with the epidermis facing up and cut into several pieces 1–2 mm wide. Tissues were digested for 16 h at 4 °C in 10 mL of a solution composed of 0.5 mg/mL thermolysin (Sigma-Aldrich) dissolved in HEPES 1X  and 1 mM CaCl2 (Sigma-Aldrich) (pH 7.45). Next day, the epidermis and dermis were separated using tweezers, kept in washing solution and prepared for the second enzymatic digestion [Human skin samples were washed in 30 mL of a solution constituted of PBS 1X, 100 U/mL penicillin , 25 μg/mL gentamicin  and 0.5 µg/mL fungizone  by shaking for 2 min at room temperature (10 times). Then, human skin biopsies were transferred to a 60 × 15 mm Petri dish /0.01% EDTA stirring solution . Then, cell suspension was neutralized, filtered using a Cell Strainer of 100 µm (Fisherbrand®) and centrifuged (10 min/24 °C/300× g). Finally, epithelial cells were counted, seeded at 40,000 cells/cm2 on a feeder layer of irradiated human fibroblasts (8000 cells/cm2) and cultured in keratinocyte medium (Dulbecco–Vogt modified Eagle medium (GibcoTM): Ham’s F12 (GibcoTM), ratio 3:1, supplemented with 24.25 μg/mL adenine (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 0.4 μg/mL hydrocortisone , 0.212 μg/mL isoproterenol hydrochloride (Sigma-Aldrich), 5% bovine HyClone FetalClone II serum , 10 ng/mL human epidermal growth factor , 100 U/mL penicillin (Sigma-Aldrich) and 25 μg/mL gentamicin (Gemini Bio)) [Fragments of the epithelium were incubated for 25 min at 37 °C in 20 mL of a 0.05% Trypsin (Gibconi Bio)) ,10. ThisTM) supplemented with 10% Avantor Seradigm FB Essence , 100 U/mL penicillin (Sigma-Aldrich) and 25 μg/mL gentamicin (Gemini Bio)). After incubation, cell suspension was filtered using a Cell Strainer of 100 µm (Fisherbrand®), separated into four 50 mL tubes (5 mL/tube) and digestion was neutralized with 45 mL/tube of fibroblast medium. After centrifugation of the cell suspensions (10 min/24 °C/450× g), the supernatant was discarded until 10 mL were left in each tube; pellets were resuspended and combined to have two tubes with 20 mL in each. These were centrifuged (10 min/24 °C/450× g) and previous steps were repeated once to obtain one tube with 20 mL of cell suspension. Finally, this tube was centrifuged (10 min/24 °C/450× g). Dermal cells were counted and seeded at 40,000 cells/cm2 in fibroblast medium [Dermal tissue was digested for 3 h at 37 °C by stirring in 20 mL of a solution constituted of 0.125 U/mL Type H collagenase  diluted in fibroblast medium  with the epidermis facing down. Most of the dermis was mechanically detached using scissors and a scalpel and kept for fibroblast isolation [Firstly, human skin biopsies were washed for 30 min, submerged in a solution constituted of PBS 1X, 100 U/mL penicillin (Sigma-Aldrich), 25 μg/mL gentamicin (Gemini Bio) and 0.5 µg/mL fungizone (Bristol-Myers Squibb) at room temperature. Then, the samples were transferred to a 60 solation . TM) at 37 °C (8 cycles). After each cycle, digested solution containing the cells and pieces of tissue was filtered using a Cell Strainer of 100 µm (Fisherbrand®). The filtered cell suspension was neutralized using 10 mL of keratinocyte medium and kept while remaining tissue was digested again. After the 8th cycle, the cell suspension was centrifuged (10 min/24 °C/300× g) [2 on a feeder layer of irradiated human fibroblasts (8000 cells/cm2) and cultured in keratinocyte medium. This primary culture was named passage 0 (P0). The estimated time and cost (considering only the price of the enzymes) for the epithelial cell isolation were 5 h and USD 81.00, respectively.Epidermis was mechanically fragmented into small pieces using scissors and incubated for 15 min in a stirring 5 mL of TrypLE Select Enzyme 10X (Gibco/300× g) . Cells wTM) in Dulbecco–Vogt modified Eagle medium (GibcoTM) for 20 h at 37 °C. The digested tissue was filtered using a Cell Strainer of 100 µm (Fisherbrand®), neutralized using fibroblast medium and centrifuged (20 min/24 °C/450× g) [2 in fibroblast medium. This primary culture was named passage 0 (P0). The estimated time and cost (considering only the price of the enzymes) for the dermal cell isolation were 22 h and USD 11.00, respectively.Dermis was fragmented using scissors and digested using a stirring solution of 2 mg/mL of Type I collagenase (Gibco/450× g) . Finally2) and fibroblasts (8000 cells/cm2).The culture medium of keratinocytes and fibroblasts was changed every two days until cells reached 90–95% confluence; then, cells were trypsinized and cultured again until they reached P4. From P1 to P4, cell seeding density in both protocols was the same for keratinocytes . Cell size was evaluated just after isolation (Fresh Cells) and at the end of each passage from P0 to P4. Setting parameters were established between 7 µm and 21 µm for fresh cells and between 8 µm and 24 µm for cells cultured from P0 to P4. The mean size value was used for statistical analysis.The number of human epithelial and dermal cells and their diameter (Ø) were measured using a Z2 CoulterTM). The viable cell yield per cm2 was calculated using the total number of cells and the area of each human skin sample for fresh cells, and the surface area of the culture flasks for cells recovered from P0 to P4. Cell viability was determined by trypan blue staining  were seeded with 1000 epithelial cells (keratinocytes)/flask and cultured for the same number of days for both protocols. Keratinocyte medium was not changed during the first 5 days of culture, and then medium was changed every two days until colonies started to join with each other.The size  and the number of colonies were evaluated for epithelial cells seeded at P0 and P1. For a given biopsy, passage and isolation protocol, 5 culture flasks   for 30 min at room temperature. Then, colonies were stained with 1.5 mL of Rhodamine 1%  for 15–20 min at room temperature, rinsed with distilled water and dried.Colonies were manually counted and classified by their diameter size, measured using ImageJ software , into i) holoclones: Ø ≥ 4.5 mm, formed by stem cells that rapidly grow; ii) paraclones: Ø ≤ 1.5 mm, constituted by cells programmed for limited growth, which consequently form uniformly small, terminal colonies; and iii) meroclones: 4.5 mm > Ø > 1.5 mm, containing cells with different proliferative capacities .Using this data, CFE  and the 6 cells). Then, cells were washed again with cold PBS 1X (3 times) and kept at 4 °C in 1 mL of PBS 1X/106 cells until flow cytometry analysis.The number of K19-positive keratinocytes after P0 and P1 was evaluated by flow cytometry. Cells were recovered after trypsinization, washed in PBS 1X (3 times) and fixed in filtered formaldehyde 3.7% (pH = 7) (Chaptec) for 30 min at 4 °C  diluted in MilliQ water. Then, cells were immunolabeled for 45 min at room temperature in darkness with mouse IgG2a antihuman K19 primary antibody . For negative controls, keratinocytes were incubated with mouse IgG2a antibody . After 3 washes, samples were immunolabeled with PE-conjugated goat antimouse IgG2a secondary antibody  and incubated for 45–60 min at room temperature in darkness. After 3 washes, each sample was resuspended in 200 μL of PBS FACS solution  and 2% Serum) and analyzed using BD FACSMelody™ Cell Sorter (BD Biosciences).For each protocol and passage, two samples of at least 400,000 cells were immunolabeled. Cells were centrifuged at 450× t-tests and Holm–Sidak’s method, were used to analyze the results. All values were presented as mean value ± standard deviation (SD). The significance threshold was set at 0.05.Microsoft Excel software (v2308) was used for primary analysis of raw data, and GraphPad Prism 8  and FlowJoTM v10 (BD Biosciences) were used for statistical analysis and graphs. Normal distribution of the data was corroborated by Shapiro–Wilk test. Then, depending on the comparison, one-way ANOVA and Tukey’s multiple comparisons tests, or multiple This study was conducted according to the Declaration of Helsinki and was approved by the research ethics committee for human subjects of the CHU de Quebec-Université Laval. All tissue donors provided informed written consent. This comparative in vitro study demonstrated that the application of the two-step digestion protocol (LOEX) or one-step digestion protocol (UPCIT) does not differentially affect the isolation and culture of human dermal fibroblasts. However, the use of the LOEX-Protocol increased the viability and cell yield of the skin epithelial cells after the isolation process, but no differences were observed once the keratinocytes were cultured through different passages. Therefore, both isolation protocols are useful for human skin cell isolation and their subsequent use for the clinical fabrication of human bilayered tissue-engineered skin substitutes.


The solute carrier family 25 member 1 (SLC25A1) is currently the only known human transporter for citrate in the mitochondrial membrane. However, its role in cancer development remains to be elucidated. We aim to analyze the expression profile, prognostic value, potential immunological significance, and effect on tumor growth of SLC25A1 at a pan-cancer level.Herein, the role of SLC25A1 in tumorigenesis and progression was investigated based on the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Genotype-Tissue Expression (GTEx), Clinical Proteomic Tumor Analysis Consortium (CPTAC), GeneMANIA, STRING and Cancer Dependency Map Project (DepMap) database via online websites or the R software. The protein expression levels were validated in tissue microarrays, and the effects on tumor cell lines were accessed through MTS and colony formation assays.SLC25A1 was linked to clinical outcomes across multiple tumor types, particularly in lung adenocarcinoma, where its high expression predicted poor prognosis. Moreover, SLC25A1 was positively associated with MSI, TMB, and CD276 and tightly correlated with tumor-infiltrating immune cells. Furthermore, the knockout of SLC25A1 demonstrated inhibitory effects in most cancer cell lines in the DepMap project. Cellular experiments showed that SLC25A1 knockdown significantly reduced the proliferation of lung adenocarcinoma cells.The expression of SLC25A1 increased in most cancers, and the upregulation of SLC25A1 in colon adenocarcinoma and lung adenocarcinoma was further confirmed by immunohistochemistry. Meanwhile, Our findings suggest the potential of SLC25A1 as a prognostic biomarker for cancers and a therapeutic target for precise antitumor strategy and cancer immunotherapy.The online version contains supplementary material available at 10.1007/s12672-023-00830-z. Cancer ranks as a leading cause of death worldwide, with almost 10.0 million cancer-related deaths estimated to have occurred in 2020 , 4. In aSLC25A1 gene located on chromosome 22q11.21 [SLC25A1 gene expression and its biological function in different types of human cancers remains elusive.The solute carrier family 25 member 1 (SLC25A1), originally known as citrate transporter protein (CTP) or mitochondrial citrate/isocitrate carrier (CIC), is encoded by the 22q11.21 . It is t22q11.21 . Citrate22q11.21 . As such22q11.21 . Of note22q11.21 , 9. SLC222q11.21 –12. More22q11.21 , 14. Fur22q11.21 , 10, 15.In this study, we systematically analyzed the SLC25A1 expression across various human tumors using public databases and immunohistochemistry. Meanwhile, the associations of SLC25A1 with prognostic value, molecular pathways, immune infiltration, and other immune-related biomarkers in pan-cancer were also explored based on online bioinformatics tools and R program. In addition, in vitro functional validation showed that SLC25A1 knockdown attenuated the growth rate of LUAD cells. The findings of this study offer a basis for understanding the roles and potential mechanisms of SLC25A1 in human tumors, and provide a novel perspective for the application of SLC25A1 in cancer therapy.https://www.proteinatlas.org/humanproteome/pathology) [SLC25A1 status in all TCGA tumors was performed by the cBioPortal web tool  [SLC25A1 gene in normal and tumor tissues, gene expression profiles and clinical information of 33 tumors and normal tissue samples were obtained from the TCGA database [https://portalgdc.cancer.gov/) and the GTEx database [https://gtexportal.org/) combined. The abbreviations and full names of 33 tumors are shown in Table http://ualcan.path.uab.edu/index.html) was used to analyze the SLC25A1 protein expression between different cancer tissues and normal tissues [P < 0.05 was considered to be significant.The SLC25A1 expression data across different tissues and cells under physiological conditions were obtained from the Human Protein Atlas (HPA) dataset (thology) . The genal.org/) . To compdatabase   were purchased from Shanghai Outdo Biotech Co., Ltd. HOrgC120PG05 contains a core/case of normal human organs from thyroid gland, esophagus, stomach, colon, liver, pancreas and lung; as well as 3–5 cores/cases of carcinoma and paired adjacent nontumor tissues from papillary thyroid carcinoma, esophageal squamous cell carcinoma, colon adenocarcinoma, rectal adenocarcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma and clear cell renal cell carcinoma. HLugA150CS03 contains 75 cores/cases of LUAD with matched adjacent normal lung tissue, and HColA150CS02 includes 73 cores/cases of COAD with paired normal colon mucosa. Studies using human tissues were approved by the human ethics Review Committee of Shanghai Outdo Biotech Co., Ltd.  in agreement with the guidelines set out by the Helsinki Declaration. The SLC25A1 antibody for IHC was obtained from Proteintech (15235-1-AP) used at 1:1000 dilution. The sample staining score was determined by multiplying the score of staining intensity and the percentage score of positive cells. The score of staining intensity was graded as follows: negative (score 0), weak (score 1), medium (score 2), or strong (score 3). The percentage of positive cells was categorized into four grades: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). Tissue scan and image analysis were proceeded using Aperio ImageScope software. GraphPad Prism 8 was used for statistical analysis. Student’s t-test identified the differences between the two groups. Error bars indicated means ± S.D. SLC25A1 expression with the prognosis of various cancers. Forest Plot was used to represent the hazard ratio, 95% confidence interval, and associated P-values between SLC25A1 and survival. The cutoff for SLC25A1 levels was determined using P-value minimization, and the corresponding Kaplan–Meier curves were then plotted within the R environment. Next, the Kaplan–Meier plotter  was used to perform survival analyses, including OS and first progression (FP) in lung, gastric, breast, and ovarian cancers [P were generated by selecting the “auto select best cutoff” function. Additionally, the association between SLC25A1 expression and survival in pan-cancer was analyzed in PrognoScan (http://dna00bio.kyutech.ac.jp/PrognoScan/index.html) microarray datasets [P-value < 0.05.Survival information of each TCGA sample, including overall survival (OS) and progression-free interval (PFI), was extracted from the TCGA database . Cox reg cancers –25. Kapldatasets . The thrhttp://sangerbox.com/), a cloud-based platform for TCGA data analysis, was used to explore the relationship between SLC25A1 expression and MSI, TMB, and ICP genes. The correlation between SLC25A1 and either MSI or TMB was calculated based on the Spearman method, and the results were visualized using radar plots. Pearson’s rank correlation coefficient was used to describe the association between the SLC25A1 expression and the expression of 60 common immune checkpoint genes [The SangerBox online tool (nt genes .SLC25A1 and immune infiltration was determined and investigated by the TIMER (http://cistrome.org/TIMER/) [SLC25A1 expression and the abundance of six categories of immune invading cells, including CD4 + T cells, CD8 + T cells, neutrophils, myeloid dendritic cells, macrophage, and B cells. In addition, the relationship between SLC25A1 expression and the immune score or matrix score was analyzed using the Stromalscore, Immunescore, and ESTIMATE score using SangerBox online tool.The association between /TIMER/) . The TIMhttp://www.genemania.org), which is a helpful web tool for providing PPI networks, generating hypotheses about gene function, and detecting genes with similar functions [https://string-db.org/) was also used to investigate SLC25A1-binding proteins [SLC25A1-related genes of all TCGA tumors were obtained through the “Similar Gene Detection” module in GEPIA2 [P-value were indicated in the plot. Moreover, the heatmap of the selected genes was gained via the “Gene_Corr” module of TIMER2, and the P-value and partial correlation (cor) were calculated by Pearson's correlation test. The Venn diagram was used to display intersections of SLC25A1-binding proteins and related genes based on the above data from GeneMANIA, STRING, and GEPIA2 databases. Finally, the selected genes were combined to perform Gene Ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis using R package  under R software v4.0.3 [P < 0.05 was considered statistically significant.Protein–protein interaction (PPI) network of SLC25A1 was first constructed using GeneMANIA  portal  [The dependency of a panel of tumor cell lines on the portal/) . The genThe human lung adenocarcinoma cell lines, A549 and H1299, were obtained from the American Type Culture Collection . Cells were cultured in standard conditions according to the Repository’s instructions.SLC25A1 with targeting sequence: 5′- GGAGATTGTGCGGGAACAA-3′ was obtained from Ruibo Company. Lipofectamine 2000  was used to transfect siRNA or plasmids into cells at 70% confluence, according to the manufacturer’s instructions.Small interfering RNA (siRNA) targeting human SLC25A1, forward: 5ʹ-CCCCATGGAGACCATCAAG-3ʹ, reverse: 5ʹ-CCTGGTACGTCCCCTTCAG-3ʹ; ACTB, forward: 5ʹ-GGCTGTATTCCCCTCCATCG-3ʹ, reverse: 5ʹ-CCAGTTGGTAACAATGCCATGT-3ʹ. The relative gene expression for each sample was normalized to that of ACTB. An independent Student’s t test identified the differences between the groups. P < 0.05 was considered statistically significant.Total RNAs were extracted using TRIzol reagent (Thermo Fisher) and reversely transcribed into cDNA using a cDNA synthesis kit (Marligen Biosciences) according to the manufacturer’s instructions. The Power SYBR Green PCR Master Mix (Takara) was used for quantitative real-time PCR (qRT-PCR). The sequences of the primers were: Collected cells were homogenized in lysis buffer . Protein concentrations were measured using pierce BCA protein assay . Proteins were run on a standard SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk, incubated with primary antibody at a concentration of 1: 1000, then incubated with secondary antibody at a concentration of 1: 10 000 and read using ECL detection system . The anti-SLC25A1 (15235-1-AP) and anti- alpha-tubulin (11224-1-AP) were purchased from Proteintech.P-value < 0.05 was regarded as statistically significant.For the MTS assay, cells were seeded in a 96-well plate at a density of 2000 cells per well. Cell viability was determined using a MTS assay system  every day for 4 days, following the manufacturer’s instructions.For the colony formation assay, 1000 cells were seeded and incubated in 6-well plates. After 2 weeks of culture, the cells were fixed with icecold methanol, stained with crystal violet, imaged and quantified using AlphaImager HP system (Alpha Innotech). Data were presented as mean ± SD. The statistical significance of differences was determined using the Student's t-test. SLC25A1 expression in normal tissues using the HPA database. As shown in Fig. SLC25A1 mRNA expression was prominent in the liver, breast, small intestine, duodenum, and adipose tissue, all of which have a high content of lipids. We next used cBioPortal to explore genetic aberrations of SLC25A1 based on mutation, fusion, amplification, deep deletion, and multiple alternations across 32 types of cancer. The results revealed that gene amplification accounted for the most common alteration, while gene mutation rates were generally low  was significantly higher. In comparison, its expression in 3 types  was lower. Meanwhile, the area under the curve (AUC) values for ROC analysis of SLC25A1 mRNA expression was performed in each cancer . The results indicated that SLC25A1 had certain accuracy (AUC > 0.7) in predicting 16 cancer types, including BLCA, COAD, DLBC, GBM, HNSC, LAML, LGG, LIHC, LUSC, PAAD, PRAD, READ, STAD, THYM, UCEC, and UCS. Among them, SLC25A1 had high accuracy (AUC > 0.9) in predicting GBM, LAML, LGG, and PAAD. We also explored the correlation between SLC25A1 expression and the pathological stages of cancers using TCGA data  in tissue microarrays containing several kinds of human normal tissues and cancers. High SLC25A1 protein expression was observed in normal liver tissue Fig. A. This rSLC25A1 across 33 cancer types in the TCGA database , ACC , LUAD , HNSC , LIHC , MESO  and SKCM   and PCPG  .We examined the prognostic values of ase Fig. A. Our st95% CI 1.4–3.40, PSLC25A1 expression and tumor prognosis was checked based on GEO datasets. Using the gene chip data in Kaplan–Meier Plotter, we found that SLC25A1 was a high-risk gene in lung cancer , gastric cancer  and breast cancer   . The impact of SLC25A1 on survival was also evaluated through PrognoScan. The results are summarized in Supplementary Table 1. Similar to our findings in Kaplan–Meier Plotter and TCGA database, SLC25A1 played a detrimental role in breast cancer, lung cancer (adenocarcinoma), and skin cancer (melanoma). Meanwhile, SLC25A1 had a protective role in brain cancer. Notably, patients with ovarian cancer displayed the opposite trend in DUKE-OC and GSE8841. These findings suggested that SLC25A1 expression is differentially related to the prognosis in pan-cancer and may be a potential prognostic marker in certain types of tumors.Then, the relationship between SLC25A1 expression and MSI/TMB in diverse cancer types of TCGA. The results revealed that SLC25A1 was positively associated with the MSI in DLBC (P = 1e-06), LUAD (P = 0.0053), PRAD (P = 0.00032), UCEC (P = 6.3e-11), TGCT (P = 2e-06), ESCA (P = 0.0079), STAD (P = 4.2e-05), KIRC (P = 0.0036) and HNSC (P = 2.8e-05) , LUAD (P = 6.4e-08), PRAD (P = 0.013), UCEC (P = 0.0044), BLCA (P = 0.0045) and STAD (P = 2.9e-06)  and Tumor mutation burden (TMB) were considered to be important biomarkers in predicting the response to ICIs, which are represented by anti–programmed death-1/ligand-1 (PD-1/PD-L1) and anti–cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitors. Hence, we analyzed the correlation between  P = 1e-0, LUAD  in pan-cancer from the TIMER database. The top three tumors where SLC25A1 expression showed the most relevance to immune infiltration levels were PRAD, PAAD, and LUSC , macrophage and B cells  but negative correlation with CD4 + T cells , neutrophils  and myeloid dendritic cells  in PRAD. In PAAD, SLC25A1 expression has a significant positive correlation with the immune infiltrating levels of CD4 + T cells  but negative correlation with the infiltrating levels of CD8 + T cells, neutrophils  and myeloid dendritic cells . In addition, SLC25A1 expression was significantly and negatively correlated with infiltrating levels of CD8 + T cells , neutrophils , myeloid dendritic cells  and macrophage  in LUSC.Tumor-infiltrating immune cells are an essential part of the tumor microenvironment (TME) and have been identified as potential biomarkers for predicting prognosis and response to treatment in cancer patients. Following that, we searched the relationship between USC Fig. A. Among SLC25A1 gene expression was negatively correlated with Stromalscore, Immunescore, and ESTIMATE score , STAD (r = − 0.33), and BLCA (r = − 0.30) and significantly negatively correlated with ImmuneScore in LUSC (r = − 0.47), TGCT (r = − 0.43), and CESC (r = − 0.43). Similarly, There were negative correlations between SLC25A1 expression and ESTIMATE score in LUSC (r = − 0.44), CESC (r = − 0.39) and STAD (r = − 0.38). These results indicated that SLC25A1 expression was closely related to the tumor microenvironment in certain tumor types.The Estimation of Stromal and Immune cells in Malignant Tumor tissues using the Expression data (ESTIMATE) algorithm could help to determine the fraction of stromal and immune cells in the tumor microenvironment. The present study found that ore Fig. B. The exSLC25A1 expression was statistically related to that of the top 5 genes, including DGCR6L (R = 0.45), DDT (R = 0.44), PCYT2 (R = 0.42), TXNRD2 (R = 0.4) and COMT (R = 0.4)  Fig. C. The heCGA Fig. D.Fig. 8SAccording to the Venn diagram, three common genes of the STRING group and GeneMANIA group were screened out as ACLY, ACO2, and G6PD; while six common members were located in both STRING and GEPIA2 list, including DGCR2, DGCR14, MRPL24, MRPL40, UFD1L and TXNRD2 Fig. A. CombinSLC25A1 in large panels of human cancer cell lines using the DepMap project data . Negative scores of Gene Effect (dependency scores) reflect reduction in cell growth and survival after depletion of a particular gene and the score of -1 corresponds to the median of all common essential genes.In order to comprehensively study SLC25A1 expression on cancer cell growth, we analyzed the vulnerability to CRISPR/Cas9-mediated perturbation of SLC25A1 knockout showed an inhibitory effect in most cancer cell lines of all cancer types with a median dependency score < 0 , endometrial/uterine cancer cell line (JHUEM1), sarcoma cell lines (RH30 and RHJT), leukemia cell lines (ROS50 and HB1119) and breast cancer cell line (KPL1), all of which exhibited a dependency score less than − 0.85  to knock down the expression levels of SLC25A1 in A549 and H1299 cell lines . Olga Catalina-Rodriguez et al. showed that expression levels of SLC25A1 increased in human breast cancer [SLC25A1 mRNA in BRCA, which extracted and assessed by the GTEx and TCGA database combined. The heterogeneity between studies may be due to differences in analytic methodology, and study sample sizes. It is worth noting that the expression of SLC25A1 was significantly reduced in LAML compared to normal controls. As an hematologic malignancy, LAML have an atypical metabolic profile characterized by a greater reliance on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) [SLC25A1 mRNA high expression may be an unfavorable prognosis indicator for LAML, ACC, LUAD, HNSC, LIHC, MESO, SKCM, BRCA, and STAD. Among them,, the prognostic implication of SLC25A1 in LUAD was highly consistent in different databases. Further in vitro verification experiments demonstrated that inhibition of SLC25A1 could suppress cell proliferation in LUAD cells. Notably, our data also implicates that SLC25A1 might be a favorable prognosis indicator for LGG, PCPG, and OV. This difference may be due to the complexity of the tumor metabolic microenvironment, genetic context, and the intrinsic properties between types of tumors. Future functional studies and independent validation will help to determine whether SLC25A1 can be used as an independent predictive factor in cancers.The expression of SLC25A1 has been found to be regulated by many factors. The transcriptional activation of SLC25A1 was promoted by p53 mutants and Myc, while it was repressed by the tumor suppressor gene PTEN . Previou studies , 12. Amot cancer . Our daton (FAO) . Intriguon (FAO) . We statSLC25A1 expression was positively related to MSI in 9 cancers and TMB in 6 cancers. Among them, LUAD, PRAD, UCEC, and STAD were significantly correlated with MSI and TMB with overlapping relationships.Mounting evidence demonstrates that cancer metabolism not only participates in bioenergy, biosynthesis and signaling transduction for tumorigenesis and tumor maintenance but also regulates antitumor immunity and response to immunotherapy through the release of metabolites and modulating the expression of immune-related molecules . RecentlSLC25A1 expression and common immune checkpoint genes. Interestingly, the correlation of SLC25A1 and CD276 expression was highly consistent in a variety of tumors. CD276, also known as B7-H3, belongs to the B7 ligand family and is a newly identified immune checkpoint molecule [Furthermore, the study presented a complexity of the associations of molecule . It has molecule . Numeroumolecule , 47. TheSLC25A1 expression and CD4 + T cells, CD8 + T cells, neutrophils, myeloid dendritic cells, macrophage, and B cells was systematically evaluated at the pan-cancer level in this study. SLC25A1 expression was found to be associated with levels of the six immune cell types mentioned above in multiple cancer types. Especially, SLC25A1 was consistently negatively associated with levels of neutrophils and myeloid dendritic cells across different cancers including PRAD, PAAD, and LUSC. Neutrophils are a double-edged sword in cancer immunology. Although it facilitates cancer progression by remodeling the extracellular matrix and stimulating angiogenesis, neutrophil-mediated cytotoxicity also leads to tumor cell-killing [SLC25A1 and the immune score. These findings suggest that SLC25A1 expression is closely related to the tumor microenvironment, which may affect the tumor cell behavior and influence the prognosis and efficacy of immunotherapy for various cancers.As a crucial component of the TME, tumor-infiltrating immune cells (TIICs) can regulate tumor growth, invasion, metastasis, and the host antitumor immune response by altering the immune status . Increas-killing . Dendrit-killing . In addi. To better understand the underlying mechanisms of SLC25A1 in cancers, the information on SLC25A1-related proteins and genes for a series of analyses was integrated. ACLY, ACO2, and G6PD were common components of the String and GeneMANIA SLC25A1-PPI networks. ATP citrate lyase (ACLY) is the key enzyme for the de novo synthesis of fatty acids, which converts the conversion of citrate to acetyl-CoA [Several lines of evidence have demonstrated that SLC25A1 enhances cancer growth by promoting mitochondrial oxidative metabolism and de novo lipid synthesis , 52. Theetyl-CoA . Mitochoetyl-CoA . Glucoseetyl-CoA . Similarin vitro and in vivo. Catalina-Rodriguez et al. showed that inhibition of SLC25A1 in breast cancer by shRNA or specific inhibitor hampers tumor activity of MBA-MD-231 cells [SLC25A1 deletion by CRISPR/Cas9 blunted growth in both monolayer in two-dimensional (2D) culture and spheroids in three-dimensional (3D) culture of lung cancer cells [SLC25A1 knockdown suppressed the growth of colorectal cancer cells and induced cell apoptosis [A growing body of research investigated the effects of the genetic suppression or pharmacologic inhibition of SLC25A1 alone and in combinatorial therapies on cancers 31 cells . Jiang eer cells . Yang etpoptosis . Moreovepoptosis . Additiopoptosis . Hence, poptosis .SLC25A1 genetic inhibition by CRISPR/Cas9 at the pan-cancer level via the Dependency Map (DepMap) project. The cancer dependency screens showed that the majority of cancer cell lines display varying degrees of reliance on SLC25A1. Overall, lymphomas were most vulnerable to SLC25A1 knockout, specifically SUDHL10 and SUDHL5 diffuse large B-cell lymphoma (DLBCL) cell lines. So far, there have been no reports on the role of SLC25A1 in DLBCL. However, recent studies have suggested that certain DLBCL highly depends on OXPHOS and lipid synthesis [SLC25A1 in cancer were also examined using the DepMap data. Four  out of the top five dependencies are genes required for the TCA cycle, which may be attributable to their cooperative function with SLC25A1 in maintaining mitochondrial homeostasis. All of the above suggest that SLC25A1 could be a novel and promising target for cancer therapy.In this report, we first analyzed the impacts of ynthesis , 58. GivHowever, there were potential limitations in our report. Although the SLC25A1 expression across various cancers was investigated using public databases and immunohistochemistry, the sample size of tissue microarray in this study was small, resulting in limited information. Importantly, the prognostic significance, immunological role, and biological effects of SLC25A1 in cancers require follow-up appropriate models in vitro or in vivo and large clinical samples to testify. Deep studies focusing on the functions of SLC25A1 in oncogenesis may facilitate the discovery of new prognostic biomarkers and the development of effective targets for cancer treatment.Our analysis is the first comprehensive study of the expression levels, clinical prognosis, as well as its immunological and biological implications of SLC25A1 on a pan-cancer basis. This study shed new light on the role of SLC25A1 in tumor immunity. These findings may contribute to a better understanding of the functional significance of SLC25A1 in tumorigenesis and progression, and provide a valuable resource for personalized immunotherapy and precision medicine.Supplementary file1 (TIF 1681 KB)Supplementary file2 (DOCX 594 KB)


Semen cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of sperm membranes, resulting in a reduction in sperm motility and decreased fertilizing ability. We investigated the effects of bioactive peptides from palm kernel meal protein hydrolysates (PKMPHs) on sperm viability, motility, acrosome and mitochondrial function in frozen–thawed boar semen. PKMPH improved the total motility, progressive motility, viability, acrosome integrity, and mitochondrial function of sperm and diminished lipid peroxidation. Therefore, we suggest that PKMPH plays a crucial role as an antioxidant during the freezing of boar sperm.n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations  in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality.Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates ( Boar sperm is sensitive to particular conditions during cryopreservation, including oxidative damage, intracellular and extracellular ice formation during the semen cryopreservation and thawing processes, excessive formation of reactive oxygen species (ROS), and osmotic stress . It is wUntil now, palm kernel meal protein hydrolysates (PKMPH) have gained much attention not only because this digested protein has antimicrobial properties but also because of its antioxidant properties ,16. PKMPn = 17) were collected from a commercial pig farm from 10 boars, including the Landrace, Large White, and Duroc breeds (their ages ranged from 1.5 to 3 years old). They were kept in individual pens in an evaporative cooling system. Fresh, clean water was provided ad libitum with automated watering; the feeding level was adjusted to meet the semen production requirements of 3 kg/day. Boars were used for routine semen collection for artificial insemination.Semen ejaculates (v/v) glycerol and 1.5% (v/v) Equex-STM® .To prepare the PKMPH stock solution for the experiment, PKMPH powder  was diluted in distilled water at 5 mg/mL. In this study, there were three extenders used for boar semen cryopreservation, as follows: Extender I was a commercial semen extender for the Beltsville Thawing Solution . Extender II was composed of 20% egg yolk and 11% lactose solution supplemented with different concentrations of PKMPH  in distilled water. Each aliquot was diluted with or without (control) PKMPH substance with an equal extender volume. Extender III was composed of 89.5% extender II, with 9%  with the BTS extender shortly after collection. The diluted semen was transferred to 50 mL centrifuge tubes and equilibrated at 15 °C for 120 min before being centrifuged at 15 °C at 800× g for 10 min . After centrifugation, the supernatant was discarded, and the sperm pellet was re-suspended (approximately 1–2:1) in Extender II at a concentration of 1.5 × 109 spermatozoa/mL [9 sperm/mL. The processed semen was loaded into 0.5 mL straws . The straws were placed in contact with nitrogen vapor (4 cm above the level of the liquid nitrogen) for 20 min (−20 °C/min) in a polystyrene box and plunged into the liquid nitrogen tank (−196 °C) for storage prior to analysis [All samples of sperm were frozen using the traditional nitrogen technique. In brief, the semen was diluted  with a pre-warmed BTS extender at 37 °C for 15 min .®, Minitube, Tiefenbach, Germany). Briefly, 3 µL of semen sample was carefully transferred into a disposable counting chamber  and maintained at 37 °C throughout the analysis. A minimum of 600 sperm cells were counted in each analysis by examining five fields from each sample. The results were represented as the percentage of total sperm motility, progressive motility, and motility patterns, including curvilinear velocity , average pathway velocity , straight-line velocity , amplitude of lateral head displacement , straightness , and linearity . Motile spermatozoa were defined with VCL ≥ 24 µm/s and ALH > 1 µm. Progressive motility (PMOT) was interpreted as the presence of a VCL ≥ 48 µm/s and a VSL < 10 µm/s. The total motility (MOT) is the summation of sperm motility subpopulations that were determined by VCL thresholds, including local motility (VCL ≥ 24 and < 48 µm/s), slow motility (VCL ≥ 48 and < 80 µm/s), and fast motility (VCL ≥ 80 µm/s) [The sperm motility was conducted using computer-assisted sperm motility analysis (CASA) (AndroVision80 µm/s) .Sperm morphology was evaluated using William’s staining method (carbolfuchsin-eosin) under a light microscope at 400× magnification, and the results were expressed as the percentage of normal spermatozoa .The sperm viability was assessed using the staining techniques of SYBR-14  and Ethidiumhomodimer-1 (EthD-1). Briefly, 10 µL of semen was combined with 2.7 µL of SYBR-14 (0.54 µM in DMSO) and 10 µL of 1.17 µM EthD-1. The mixture was incubated at 37 °C for 15 min. Under a 400× magnification fluorescence microscope, 200 sperm were assessed. The SYBR-14/EhtD-1-stained sperm were classified into viable and non-viable sperm. The nuclei of live sperm with an intact plasma membrane fluoresced green, whereas the nuclei of dead sperm or damaged plasma membranes fluoresced red . The perArachis hypogaea) agglutinin (FITC-PNA) staining. A 10 µL specimen of diluted semen was mixed with 10 µL of 1.17 µM EthD-1 and incubated at 37 °C for 15 min. Then, 5 µL of the sample was smeared onto a glass slide and allowed to air dry. The sample was fixed with 95% ethanol for 30 s and air-dried. Then, 40 µL of FITC-PNA (100 µg/mL in PBS) was spread over the slides and incubated in a moist chamber at 4 °C for 30 min. The slides were rinsed with cold PBS and air-dried. Under a 1000× magnification fluorescence microscope, 200 sperm were evaluated, and the sperm with intact acrosomes were shown in green color with a smooth contour in the acrosomal region, while the damaged acrosomes were shown in green with a rough contour , FITC-PNA (1 mg/mL in PBS), and 5,5’,6,6’-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1 (1.53 mM) diluted with DMSO (ratio 1:10)  staining. Then, 150 μL of frozen–thawed semen was mixed with 2.5 μL of PI and incubated at 37 °C for 5 min. Then, the mitochondria were labeled using 2 μL of JC-1 and incubated at 37 °C for 10 min. Finally, the acrosomes were labeled using 2 μL of FITC-PNA and incubated at 37 °C for 15 min in the dark . Under a®, Cambridge, UK) following the manufacturer’s instructions. Briefly, 250 µL of post-thawed sperm in each treatment was diluted with BTS (1:4), centrifuged at 1300× g for 5 min, the supernatant was removed, and the sperm pellet was resuspended with cold PBS (repeated twice). The sperm pellet was lysed by lysis buffer and homogenizer on ice and then centrifuged at 13,000× g for 10 min. The supernatant was collected and analyzed. The MDA in the supernatant sample (200 µL) reacted with 600 µL of thiobarbituric acid (TBA) solution to generate an MDA-TBA adduct that was quantified. The MDA-TBA product was measured immediately in a microplate reader  at 532 nm. For the colorimetric assay, a 2 mM MDA standard was prepared and serially diluted for the standard curve. The levels of MDA were calculated from the MDA standard curve and expressed as µMol/L.Lipid peroxidation was evaluated by measuring the concentration of malondialdehyde (MDA) using a colorimetric lipid peroxidation assay kit . The normal distribution test of the data was examined using the Shapiro–Wilk test and the parameters included total motility, progressive motility, sperm motility patterns, sperm viability, acrosome integrity, mitochondrial membrane potential, and MDA, which were presented as mean ± SEM. The means were analyzed with a one-way ANOVA according to Randomized Complete Block Design (RCBD). Each treatment (different concentrations of PKMPH in a freezing extender) was the fixed factor, and the boars were the random block factor. The comparison of sperm parameters among treatment groups was performed by Duncan’s multiple-range test. A statistically significant difference was defined as The results of sperm viability in the treatment groups were higher than in the control group, as shown in ± 2.2%), which was higher than the control group by 18.2%.A variation in acrosome integrity was observed among the control and treatment groups . However± 1.6%) was found when compared with the other groups.The post-thawed sperm mitochondrial membrane potential is presented in p = 0.2). However, PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared with the other groups.The effect of PKMPH on lipid peroxidation during cryopreservation is shown in During the process of mammalian sperm cryopreservation, cryoinjuries  of sperm cause oxidation stress and the overproduction of ROS, which leads to an imbalance of the antioxidant system , subsequently affecting semen qualities such as sperm motility, mitochondrial activity, membrane permeability, sperm functions, and survival rates ,28. To our knowledge, there is no information on using PKMPH, which contains bioactive peptides, for boar semen cryopreservation. This is the first study to observe the effect of PKMPH and its optimal concentration on frozen boar semen qualities. The present results clearly showed that adding PKMPH to the freezing extender significantly improved the post-thawed boar semen parameters, including total motility (10.7%), progressive motility (7.8%), sperm viability (12.3%), acrosome integrity (18.2%), mitochondrial membrane potential (12.7%), and kinetic motility patterns such as VCL, VSL, VAP, ALH, and LIN. Inferior MDA levels, indicating lower lipid peroxidation, were found in the treatment groups compared to the control, particularly when supplemented with PKMPH at a concentration of 2.5 µg/mL. This might be explained by the fact that spermatozoa absorbed and utilized bioactive peptides from the freezing extender to neutralize ROS, which, in turn, reduced lipid peroxidation in the plasma membrane and inner organelles ,30. A hi50 values of 5.73 ± 0.23 μg/mL (PKMPH) versus 28.25 ± 0.97 μg/mL (L-glutathione), and ABTS radical scavenging activity of 7.84 ± 0.89 μg/mL (PKMPH) versus 14.03 ± 0.58 μg/mL (L-glutathione). In this study, we utilized PKMPH, whose function as an antioxidant was previously elucidated by Surangkulwattana et al. [It has been documented that the antioxidant activity of protein hydrolysate depends on its peptide and amino acid compositions . There aa et al. . Besidesa et al. . Howevera et al. ,36. ThisFrom these results, it can be concluded that PKMPH, with its bioactive peptide, is capable of reducing ROS generation during cryopreservation, inhibiting lipid peroxidation, and improving sperm motility, viability, intact acrosomes, and the mitochondrial membrane potential of frozen–thawed boar sperm. The PKMPH supplementation at an optimal dose of 2.5 µg/mL in a freezing extender improves the post-thawed boar semen quality. However, the supplementation of more than 5 µg/mL could lower the post-thawed semen quality.


O’nyong-nyong virus (ONNV) is a member of the reemerging arthritogenic alphaviruses that cause chronic debilitating polyarthralgia and/or polyarthritis via their tropism for the musculoskeletal system. Thus, the discovery of dual antiviral and anti-inflammatory drugs is a great challenge in this field. We investigated the effects of the common plant-derived alkaloids berberine (isoquinoline), matrine (quinolizidine), and tabersonine (indole) at a non-toxic concentration (10 μM) on a human fibroblast cell line (HS633T) infected by ONNV (MOI 1). Using qRT-PCR analyses, we measured the RNA levels of the gene coding for the viral proteins and for the host cell immune factors. These alkaloids demonstrated multifocal effects by the inhibition of viral replication, as well as the regulation of the type-I interferon antiviral signaling pathway and the inflammatory mediators and pathways. Berberine and tabersonine proved to be the more valuable compounds. The results supported the proposal that these common alkaloids may be useful scaffolds for drug discovery against arthritogenic alphavirus infection. Togaviridae family; they are globally reemerging arthropod-borne viruses (arboviruses) transmitted to mammalians by mosquitoes. They typically cause fever, rash, and/or myalgia [Alphaviruses belong to the enveloped single-stranded positive RNA viruses of the  myalgia ,2,3. Alp myalgia . O’nyong myalgia . ONNV is myalgia ,7. The l myalgia ,9,10,11. myalgia ,13,14. F myalgia .During infection, the host immune system operates via a complex array of mechanisms for viral detection and clearance, as well as for the protective inflammatory response that is normally regulated to prevent the aforementioned disorders . After tThe management of arthritogenic alphavirus infection currently consists of symptom relief by conventional analgesics  and non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, during the acute phase or alleviation of arthritic disorders during the chronic stage using disease-modifying antirheumatic drugs (DMARDs) such as methotrexate ,28. DespAlkaloids are nitrogen-containing secondary metabolites, mainly found in plants. They display a wide range of biological activities, including, notably, those against the most prominent human health disorders  . ThroughIn addition to this common antiviral strategy based on virus-targeting inhibitors  or host-targeting inhibitors to abrogate the essential host cell functions for the virus life cycle , immunomodulatory drugs have gained growing interest as regulators of the immune response ,38. In pBerberis (Berberidaceae family), with a wide range of biological activities [Sophora L. (Fabaceae family) with biological activities, including anti-inflammatory and antiviral effects, but no specific anti-alphaviral activity has yet been described [Catharanthus roseus (L.) G.Don (Apocynaceae family), which is endowed with potent anti-inflammatory activities [Berberine (BER) is an isoquinoline alkaloid derived from various plants, mostly from the genus tivities ,43. The tivities ,45. Matrescribed . Tabersotivities .The viability of HS633T cells was assessed under infection by ONNV (MOI 1) or treatment by each of the three studied alkaloids (10 µM) in comparison with CHL (15 µM). The microscopy images A showed −1 ± 7.69 × 10−2, ****: p < 0.0001; E2: 1.70 × 10−2 ± 9.83 × 10−3, ***: p < 0.001; capsid: 6.13 × 10−5 ± 3.54 × 10−5, ***: p < 0.0001; and nsP2: 2.35 × 10−3 ± 2.39 × 10−4, ****: p < 0.0001) when compared to the control cells (CTRL). When compared to the infected untreated cells , chloroquine (CHL) significantly decreased the expression of all the studied viral genes . Downregulation of the viral E1 and E2 genes occurred with almost all the chemical treatments, but counterintuitively, MAT significantly increased the expression of the capsid and nsP2 genes. The above results were consistent with the significant reduction in the viral progeny production  ; BER ; and TAB  when compared to the non-treated and infected cells (ONNV).We performed qRT-PCR to analyze several RNA coding expressions of the viral proteins during infection alone or together with the different chemical treatments. As shown in s, PFUs) B observeThe host cell intrinsic antiviral response was first studied from its initiation with the viral receptor MxRa8, the PRR RIG-I, the downstream signals of the IFNAR-dependent pathways (IFN-β), including the IRF3-dependent JAK/STAT pathway, and the resulting production of ISG-15.−4 ± 3.34 × 10−5. This expression was significantly upregulated by ONNV infection  by a factor  of 2.3. The RIG-I expression was 1.49 × 10−2 ± 1.29 × 10−3 in the CTRL and was upregulated (FC: 9.5) in response to ONNV .As shown in −4 ± 5.55 × 10−5, ##: p < 0.01), BER , and TAB , but it was drastically increased when using MAT , with an FC that was 3-fold higher when compared to ONNV. The RIG-I gene expression was downregulated by the all the tested compounds .When compared to the infected cells (ONNV), the MXRA8 gene expression was downregulated upon treatment by CHL (2.08−4 ± 3.34 × 10−5), ONNV increased the gene expression of IRF3 ; IFN-β ; and ISG-15 , but it reduced the gene expression of STAT1 .When compared to the control cells  and ISG15 . The gene expression of IFN-β was upregulated when using CHL  or BER , but it was significantly downregulated by MAT  and TAB . CHL and the other alkaloid treatments had no effect on the downregulation of the STAT1 gene expression by ONNV infection.In comparison with the infected cells (ONNV), we observed a distinct effect of MAT that significantly upregulated the gene expression of IRF3  and AMPK  by ONNV. From the chemical treatment, only CHL showed a significant upregulation for PI3K and AMPK .Autophagy is assumed to be a part of the host cell antiviral defense and is mainly mediated by PI3K/AKT . In our −2 ± 1.25 × 10−2; ONNV: 1.10 × 10−1 ± 3.61 × 10−3, ****: p < 0.0001; FC: 2.5); CCL5 ; and TNF-α .The anti-inflammatory potential of the studied alkaloids was first assessed on the key inflammatory mediators. When compared to the CTRL, as shown in −3 ± 1.06 × 10−3, ###: p < 0.001), CCL5 , and TNF-α . The other alkaloids had no significant effect on the regulation of CCL5 expression. Interestingly, BER and TAB exerted substantial downregulation (####: p < 0.0001) on both the CCL2 and the TNF-α gene expression. MAT upregulated the gene expression of CCL2 , while decreasing the TNF-α level . None of these treatments significantly affected the gene expression of CXCL8.When compared to the infected cells, CHL proved to be efficient in decreasing the expression of CCL2 , which was substantially downregulated by CHL . Surprisingly, ONNV downregulated the MAPK gene expression  when compared to the CTRL, and this effect was significantly attenuated only by TAB . ONNV infection did not affect the gene expression of STAT3 . Interestingly, BER (p < 0.05) and TAB (p < 0.01) significantly downregulated the STAT3 gene expression when compared to the infected cells (ONNV). ONNV infection upregulated the gene expression of NRLP3  and caspase-1 , and this upregulation was heightened in the infected cells by BER . MAT only downregulated the NLRP3 gene expression , while TAB downregulated the NLRP3  and caspase-1  gene expression, as already observed with CHL .When compared to the CTRL, the ONNV infection increased the gene expression of NF-κB  by ONNV infection was remarkably suppressed by CHL , BER , and TAB .The robust upregulation of the COX-2 gene expression , as established by the expression of critical viral E1, E2, capsid, and nsP2 genes A. E1 andAs revealed by this study, ONNV infection triggers a robust host immune response from HS633T cells. The upregulation of MXRA8 grants effective viral entry . The eleAcute to chronic inflammation is a result of alphavirus infection with mild to severe disorders, as supported for CHIKV ,58,59,60The analyses of the various inflammatory signaling pathways  revealed2 and were allowed to grow until 80–90% confluence. An isolate of O’nyong-nyong virus (ONNV) was obtained from the National Reference Center  and titrated at 107 PFU.mL−1 on Vero cells.The HS633T cells were obtained from the European Collection of Authenticated Cell Cultures  and were grown in Minimum Essential Medium Eagle , supplemented with 5% decomplemented fetal bovine serum (FBS) , L-glutamine 2 mM , 100 U/mL–0.1 mg/mL penicillin-streptomycin , 1 mM sodium pyruvate , and 0.5 μg/mL fungizone . The cells were maintained in a humid atmosphere at 37 °C with 5% CO2. The cells were allowed to grow until 80–90% confluence. The cells were then infected with ONNV to a multiplicity of infection 1 (MOI 1) and/or co-treated with CHL, BBR , MAT , or TAB  for 24 h.The HS633T cells were cultivated in 6- or 96-well plates  and maintained at 37 °C in a humid atmosphere with 5% CO®Non-Radiaoactive Cytotoxicity Assay, G1782, Promega, Madison, WI, USA, G1782) was added. The reaction was stopped 30 mn later, and the optical density was measured at 490 nm . For the MTT assay, the cells were grown in a 96-well culture plate  and were exposed to CHL (15 μM), BER (10 μM), MAT (10 μM), or TAB (10 μM) for 20 h. Twenty microliters of sterile filtered MTT solution (5 mg/mL) was added to each well. After 4 h, the plate was centrifugated , the medium was removed, and 200 µL of dimethyl sulfoxide was added to resuspend the formazan crystals. The optical density was measured at 560 nm with a reference at 670 nm .Lactate dehydrogenase (LDH) and 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed. For the LDH assay, the cells were grown in a 96-well plate ; then, they were exposed to CHL (15 µM), BER (10 μM), MAT (10 μM), or TAB (10 μM) for 24 h. The plate was centrifugated ; 50 μL of supernatant was put in a new plate, and 50 μL of reagent from the commercial kit  and the QuantStudio 5 Real-Time PCR System  in a final reaction volume of 5 μL . GAPDH was used as a housekeeping gene. Analyses were performed on the results of three independent experiments. The primers used for qRT-PCR are listed in the following table , BER (10 μM), MAT (10 μM), or TAB (10 μM) using the Zymo kit . The cells were stabilized with 200 µL of RNA Shield, then lysed with 800 µL of lysis buffer. qRT-PCR analyses were performed using one-step RT-PCR with the SYBRng table .p-value was calculated from variance analysis followed by a Bonferroni multiple comparison test.The data were expressed as mean ± SEM of the three independent experiments. Statistical analysis was achieved using GraphPad Prism version 6.0 software. The Our overall results support the paradigm that the ONNV infection of fibroblasts drives a robust antiviral and inflammatory response via the essential type-I IFN pathway and canonical inflammatory signaling pathways . Our fin


Purpose: Currently, there is no interventional approach to increase the intensity of Surya Namaskar a popular hatha yoga sequence used worldwide. Therefore, this study investigated how tempo-based high-intensity interval cardio yoga (HIICY) and traditional interval hatha yoga (TIHY) affects cardiometabolic fitness in active adults.Methods: Twenty physically active male and female individuals were randomly separated into HIICY  and TIHY  groups. The intervention included twelve exercise sessions for 4 weeks in both groups. Participants conducted a ramp test to determine their maximal oxygen uptake (2max), maximal velocity at 2max (v2max), and maximal heart rate (HRmax). Afterward, they performed a 10-min high-intensity cardio yoga test (HICYT) to determine heart rate (HRpeak and HRmean), oxygen uptake (2peak and 2mean), respiratory exchange ratio (RER), blood lactate concentrations (La−peak and ∆La−), fat and carbohydrate oxidations , and energetic contributions .Results:2max and v2max showed time and group × time interactions . 2max after HIICY was significantly higher than in pre-testing and following TIHY . 2peak, 2mean, RER, HRpeak, and HRmean during the 10-min HICYT showed significant time effects (p < 0.05). ∆La− indicated a group × time interaction (p < 0.05). Group x time interaction effects for FATox at the fourth and sixth minute were observed . Absolute (kJ) and relative (%) WOxi, WGly, and WTotal showed time and group × time interaction effects . Furthermore, %WGly was reduced following HIICY (p < 0.05). Additionally, 2max and v2max were highly correlated with WOxi in kJ . Moderate to high correlations were observed among CHOox, FATox, and absolute 2max .Conclusion: A 4-week period of HIICY improved cardiometabolic fitness, oxidative capacity, and metabolic flexibility compared with TIHY, in physically active adults. Therefore, HIICY is suitable as HY-specific HIIT and time-efficient approach for relatively healthy individuals. This guideline consists of at least 150 min of moderate-intensity or 75 min of high-intensity aerobic activity per week . It is n females . Further2max)  . V˙O2m science . Moreovee groups . Furthere groups .2max, the most effective intervention is high-intensity interval training (HIIT) (2max or peak oxygen uptake (2peak) and thus provide the most effective stimulation to increase 2max (In terms of improving g (HIIT) . It is wV˙O2max .max), the second ventilatory threshold (>VT2), over the second lactate threshold , and >85% of 2max and 2peak  . Previou˙O2peak .In terms of metabolism, metabolic flexibility is defined as the ability to rapidly convert to generate adenosine triphosphate (ATP) from efficient fat and carbohydrate utilization based on physiological demands . EfficieIn this regard, yoga is a mind-body practice consisting of physical postures (Asana), breathing techniques (Pranayama), and meditation (Dhyana) . Notably−1  values during high-intensity HY (HIHY), that reached 95.6%, 88.7%, 10.54, and 8.67 METs, and 8.31 mmol·L−1 La−, respectively. This HIHY indicated suitable levels for HIIT. However, no interventional approach to HIHY was performed in this study. It was unclear therefore, whether high-intensity interval cardio yoga (HIICY) as HIHY could improve 2max, energetic contributions, and metabolic flexibility in physically active individuals.The average metabolic cost during HY focusing on various postures, alignments, and breathing for different times is less than 3 METs and show results similar to walking on a treadmill at 3.2 km·h−1 . However−1 . A recen2max, energetic contributions, and metabolic flexibility.Therefore, the aim of the study was to investigate how at least 4-week tempo-based HIICY and traditional interval hatha yoga (TIHY) practices affected cardiometabolic fitness parameters, such as −2. The anthropometric data of HIICY and TIHY groups were not significantly different  which considered: effect size = 0.3, alpha error probability = 0.05, and statistical power = 0.8. The effect size was determined based on previous studies . The totifferent . Partici2max on a treadmill . Afterward, a high-intensity cardio yoga test (HICYT) (one set) was performed on the same testing day. 2max and HICYT post-testing were conducted the same as pre-testing . All participants rested for 2 h after lunchtime and conducted a ramp test for -testing .2 and 5% CO2  and the turbine volume transducer was calibrated using a 3-L syringe . An initial warm-up was conducted for 10 min by running at 70% of the estimated HRmax  >1.0 were reached, or until volitional exhaustion by the participant (−1·min−1) that has been explained in previous studies  were determined . The gas analyzer was calibrated before each test with 15% Oed HRmax . The ramus study . The initicipant . The pla studies . Further plateau . The HR V˙O2max . Finallytermined .2max testing, the participants were advised to continue low-intensity jogging until blood lactate levels were below 2.0 mmol·L−1 (2peak), metabolic flexibility  and contributions from the three-energy system. Upon completion of the pre-test, all participants were randomly assigned to either HIICY or TIHY groups. They performed three interventional HIICY and TIHY sessions per week over 4 weeks     . All traessions) . Tempo-bessions) . The entessions)  HIICY orus study . The posus study .2 and carbon dioxide (2) production were used to calculate metabolic flexibility in terms of fat (FATox) and carbohydrate (CHOox) oxidation, using stoichiometric equations according to previous studies  were calculated by the PCr-La−-O2 method  were assessed at 5-min rest, during, and after 10-min HICYT (6 min), by the breath-by-breath method using a mobile gas analyzer. 2mean and 2peak were determined as the average and highest values during the 10-min HICYT, respectively. Capillary blood was collected from the earlobe (20 μL) before, and at 1-min intervals (1st to 10th) after 10-min HICYT to determine resting and maximal blood lactate concentrations  using an enzymatic-amperometric sensor chip system .The contributions of three energy systems  was calculated as the difference between La−peak after 10-min HICYT and La−rest before . The slow component of the bi-exponential model was negligible. Thus, 2 values after 10-min HICYT were fitted using a mono-exponential model and WPCr was estimated by calculating the integral of the exponential domain  vs absolute WOxi, absolute 2max vs CHOox, and absolute 2max vs FATox (g·min−1).All parameters were analyzed using GraphPad Prism 9.4.1  and data are presented as mean ± standard deviation (SD). Normality of the data was analyzed using the Shapiro−Wilk test. All physiological variables, energetic contributions, and metabolic flexibility during HICYT were analyzed using a two-way (group x time) repeated-measures analysis of variance (ANOVA) with the Greenhouse-Geisser correction for violation of the sphericity assumption. If the main effect was significant, a Bonferroni post-hoc test was performed to compare among different conditions. The significance level was set at ectively . Additio2max and v2max indicated significant time and group × time interaction effects    . The absctively) . Further]: 0.33) .2peak, 2mean, HRpeak, HRmean, and RER indicated significant time effects  (Two-way repeated-measures ANOVA showed significant time effects in absolute (kJ) for ctively) . The rel]: 0.68) .p = 0.0312; effect size [p = 0.0412; [p = 0.0386; [Two-way repeated-measures ANOVA showed group × time interaction effects at the fourth and time and group × time interaction effects at sixth minute of FATox (ctively) . OtherwiWOxi in kJ, and absolute 2max, as well as v2max   and moderate positive correlation between FATox and absolute value of 2max  were found (High positive correlations were found between ctively) . Furtherre found .2max, energetic contributions, and metabolic flexibility in physically active adults. To the best of our knowledge, this is the first study to investigate how different intensities during 4-week tempo-based HY practices (HIICY and TIHY) affected 2max, energetic contributions, and metabolic flexibility. Our major findings indicate that time and group × time interaction effects in absolute and relative 2max after 4-week HIICY and TIHY training regimens were observed. 2max was improved between pre- and post-testing in the HIICY but not in TIHY. During the HICYT, time and group × time interaction effects among WOxi in kJ, WTotal, WGly in kJ, WOxi in %, and WGly in % were found. The value of WGly in % was decreased only between pre- and post-tests in the HIICY group. Furthermore, time and group × time interaction effects in FATox showed at the fourth and sixth minute after HIICY and TIHY, and moderate to high correlations among 2max vs WOxi, v2max vs WOxi, 2max vs CHOox, and 2max vs FATox were found.Training effects of HIIT and HY on health, oxidative capacity, and metabolism have been well reported previously. To date, however, there was no interventional approach to HIICY. It is unclear whether HIICY as HIHY can improve cardiometabolic fitness parameters such as 2max and v2max while 10-min TIHY showed no increase in 2max between pre- and post-tests in physically active individuals. In this regard, at least 10-min of Surya Namaskar B (SS) compared with general HY achieved an intensity that could improve cardiometabolic fitness . These outcomes suggest that fast tempo-based HY practice is necessary, if the goal is to increase endurance performance and cardiometabolic fitness. The adjustment of exercise intensity and duration between work and recovery intervals alters the relative demand for specific metabolic pathways within muscle cells, oxygen delivery to the working muscle, and subsequent adaptations. These changes occur at the cellular and systemic levels depending on the specific nature of the training program  mRNA  mRNA . Regular1α) mRNA . Greater1α) mRNA . PGC-1α 1α) mRNA . Downstr1α) mRNA .− at a given intensity  1 and 4 after HIIT . Higher ntensity . Accordiecreased . Becauseecreased . Indeed,ecreased . Also, haptation .2max vs CHOox, and 2max vs FATox were found  for 4 weeks compared with TIHY in physically active adults. Moreover, we investigated whether HIICY for at least 4 weeks significantly affected the cardiometabolic biomarkers described above. Therefore, we suggest that the positively affected outcomes likely depend on exercise intensity influencing the activation of PGC-1α, the master regulator of mitochondrial biogenesis in human skeletal muscle (2max (In sum, this study has demonstrated increased cardiometabolic fitness, including l muscle . Also, tV˙O2max . TherefoThis study does have some limitations. First, it only targeted one cohort of physically active adults and the HIICY approach should be investigated in more diverse populations such as in athletes, sedentary people, and clinical populations in further studies. Second, the small sample size might have influenced our results. Therefore, more participants should be recruited in a future study. Third, experimenting with efficient periodization to progressively increase HY intensity from TIHY to HIICY should be considered. Finally, more direct measurements during training using proteomics and metabolomics, and the measurement of fluorescent protein tools should be investigated to determine molecular responses in the future.2max. While HY is an effective practice in many respects, significant and efficient improvements to cardiometabolic fitness require fast-tempo HIICY. Moreover, a proportionately higher 2max strongly signals greater cardiovascular health and life expectancy at any age. Thus, this substitute system of practice may prevent cardiac/metabolic diseases in the general population. However, HIICY has a very high intensity for exercise beginners. Therefore, it is recommended to start practicing with 3 s tempo-based TIHY during an initial period within the typical linear periodization model for an aerobic base, and then gradually increase the intensity to HIICY.Our findings indicate that 4-week of HIICY compared with TIHY improved cardiometabolic fitness, oxidative capacity, and metabolic flexibility in physically active individuals. Therefore, HIICY is a suitable training regimen for HY-specific HIIT, and is appropriate for relatively healthy individuals who may have HY experience but need time-efficient exercise options. Through our study outcomes, it is once again confirmed that exercise intensity during HY is important to improving


Almost all light treatments significantly decreased TSS/TA  and soluble sugar/TA ratio, which is exacerbated by the addition of sucrose. Blue or red light in combination with sucrose notably increased total phenolic content and decreased malondialdehyde (MDA) accumulation. In addition, blue or red light combined with sucrose increased abscisic acid (ABA) content and promoted ABA signaling by inducing ABA-INSENSITIVE 4 (ABI4) expression and inhibiting SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 2.6 (SnRK2.6) expression. The strawberries exposed to blue and red light significantly improved auxin (IAA) content compared to the control (0 d), whereas the addition of sucrose inhibited IAA accumulation. Moreover, sucrose treatment suppressed the AUXIN/INDOLE-3-ACETIC ACID 11 (AUX/IAA11) and AUXIN RESPONSE FACTOR 6 (ARF6) expression under different light qualities. Overall, these results indicated that RL/BL + 100 mM sucrose might promote the detached ripening of strawberries by regulating abscisic acid and auxin signaling.The regulation of detached ripening is significant for prolonging fruit shelf life. Although light quality and sucrose affecting strawberry fruit ripening have been widely reported, little information is available about how they co-regulate the strawberry detached ripening process. In this study, different light qualities  and 100 mM sucrose were applied to regulate the ripening of initial red fruits detached from the plant. The results showed RL-treated samples (RL + H Fruit ripening is the initiation of fruit senescence, accompanied by genetically programmed and highly coordinated changes in flavor, color, texture, and aroma. Fruit can supply humans with several health-promoting benefits, so the regulatory mechanism underlying fruit ripening has been attracting great scientific interest ,2. FleshStrawberries, a model for non-climacteric fruit, have enormous dietetic and economic value. However, one of the restrictive factors that affect its economic value is the very short senescent period and shelf life . The regManipulation of light quality is capable of improving vegetable and fruit postharvest quality ,7. Blue,Light quality and sucrose are environmentally friendly and safe factors to regulate the postharvest ripening of fruit. However, little information on their application for strawberry postharvest ripening is available. Here, the ripening-related physiological and molecular indicators of strawberry fruits that were subjected to white, red, and blue light and sucrose treatments after harvesting at the initial red stage were quantified, to better understand the mechanism underlying the regulation of detached strawberry fruit ripening and provide a supplementary strategy to minimize strawberry postharvest loss.2O, RL + 100 mM Sucrose) was higher than other treatments; however, their a* values had no significant difference. The b* value showed a fluctuating trend in the treatment process. Eventually, the b* value of RL-treated samples was higher than the WL-treated , and BL-treated  samples  and RL-treated  fruit contained more titratable acid (TA) than WL-treated  fruit (As shown in e) fruit D. Almoste) fruit E,F.2O, RL + 100 mM Sucrose) exhibited a higher content than other samples, while WL + 100 mM Sucrose and BL + 100 mM Sucrose treatments manifested significantly lower content than WL + H2O, BL + H2O, and 0 d samples, indicating that sucrose may negatively regulate ascorbic acid production  was performed to obtain a comprehensive overview of variation in the quality indexes of strawberries subjected to different treatments. The first and second principal components (PC1 and PC2) explained 29.6% and 19.7% of the total variance, respectively. The samples subjected to RL + 100 mM sucrose and BL + 100 mM sucrose can be obviously separated from other samples along PC1 loaded in the negative direction of PC1 A. Additi BL+ H2O B.Fleshy fruits are important in the human diet due to their nutritional value. Fruit ripening is synergistically controlled by intrinsic developmental factors and various environmental cues . Light, Fruit ripening is a complex process that has dramatic changes in color, texture, etc. In this study, different light qualities  promoted fruit skin coloration with an increasing a* value after 5 days of continuous irradiation during strawberry ripening; however, there was no significant difference among them, which also could be reflected from total anthocyanin content. This was consistent with a previous study on another strawberry cultivar ‘Tokun’ . HoweverThe firmness of strawberries, regardless of treatment, declined; however, compared to WL and other treatments, a higher firmness was observed in BL-treated fruit. Also in tomatoes, blue light caused the slowest softening trajectory as reported by Li, et al. ; howeverABI4 expression and decreasing SnRK2.6 expression. In addition, the two treatments inhibited the IAA signaling by decreasing AUX/IAA11 and ARF6 expression.The ripening processes of fruits involve complex cross-talks among multiple plant hormones . A decreFragaria × ananassa Duch, ‘Benihoppe’) at the initial red stage were obtained from a local farm in Wenjiang District, Chengdu, China. The detached fruit with 30 mm of peduncle was transported to the laboratory within two hours. After eliminating damaged and diseased ones, 105 strawberries with uniform size and maturity were selected, 15 of which were immediately stored at 80 °C for analysis of 0 d, and the remaining 90 strawberries were randomly divided into six treatments: WL (white light) +H2O, WL + 100 mM Sucrose, BL (blue light) + H2O, BL + 100 mM Sucrose, RL (red light) +H2O, RL + 100 mM Sucrose. Each treatment with 3 replicates of 5 fruits per replicate was immersed in a respective solution for 10 min and placed in plastic containers after drying for about 30 min at room temperature. After that, each treatment was sent to an incubator installed with white, blue (450 nm), or red (730 nm) light, respectively. All treatments were stored in the surroundings under 16 h of 100 μmol·m−2·s−1 light and 8 h of dark each day at 20 °C with (85 ± 5)% relative humidity for 5 d. The skin color of treated samples was evaluated every day.Strawberries  and expressed in the CIE color space , where value L* indicated the lightness from black (0) to white (100), value a* ranged from green (−60) to red (60), and value b* varied from blue (−60) to yellow (60). Chromaticity (C*) depicted color purity from grey or dull (0) to color vividness (100).The TSS was detected using a pocket refractometer , and the results were presented as °Brix. Firmness was measured using a fruit sclerometer  with a probe 3.5 mm in diameter and a penetration depth of 10 mm and expressed as Newton (N). Measurements were made around the equator zone of each strawberry twice.The determination of soluble sugar was performed using the anthrone colorimetric method . Approxi−1 FW. Total flavonoids and phenolics were articulated by the aluminum chloride colorimetric method and Folin–Ciocalteu method, respectively, which had been described previously by Zhang, et al. . A certaThe endogenous ABA and IAA content were assayed by the ABA and IAA determination kit , respectively. The sample (0.1 g) was extracted with 0.9 mL of PBS and centrifuged at 3000 rpm for 20 min. The supernatant was used for the ABA and IAA determination according to the manufacturer’s protocol. The optical density of the mixture was monitored at 450 nm. The IAA level was displayed as μmol of Auxin per liter of sample solution. The ABA level was expressed as ng of Abscisic acid per milliliter of sample solution.2−), hydrogen peroxide (H2O2), and malondialdehyde (MDA) were measured according to the method described by Yang, et al. [w/v) of polyvinylpolypyrrolidone (PVP). The homogenate was centrifuged at 12,000× g for 10 min at 4 °C. The upper phase (0.5 mL) was mixed with 10 mmol L−1 hydroxylamine hydrochloride (0.5 mL) and incubated at 25 °C for 30 min. After that, the mixture was added to 1 mL of 17 mM p-aminobenzenesulfonic acid and 1 mL of 7 mM α-naphthylamine and kept for a further 30 min at 25 °C. The absorbance was immediately recorded at 530 nm. A standard curve with sodium nitrite was employed to qualify the production rate of O2−, which was expressed as mmol min−1 kg−1.Superoxide anion  TiCl4–HCl and 0.2 mL of concentrated ammonia and reacted for 5 min. The sediment after centrifugation at 12,000× g for 15 min at 4 °C was resolved by 3 mL of 2 M H2SO4. The optical density was read at 412 nm. A standard curve with hydrogen peroxide was used to calculate the H2O2 content, which was expressed as mmol kg−1.Fruit sample (0.5 g) was extracted by 3 mL pre-cooled acetone and subsequently centrifugated at 12,000× −1 pre-cooled trichloroacetic acid. After centrifugation at 12,000× g for 10 min at 4 °C, the supernatant (1 mL) was mixed with 0.67% 2-thiobarbituric acid (3 mL). The mixture was placed in the boiling water for 10 min and then immediately cooled on ice. The absorbance was spectrophotometrically measured at 450 nm, 532 nm, and 600 nm after centrifugation at 12,000× g for 10 min at 4 °C. The MDA content was expressed as μmol kg−1 of fruit fresh weight basis.Fruit sample (0.5 g) was homogenized with 3 mL of 100 g L −1 fresh weight. Alongside, the fruit extract was mixed with 60 μM DPPH and then the mixture was immediately placed in the dark for about 30 min at room temperature. The absorbance of the blend was taken at 517 nm using the spectrophotometer. The scavenging activity was evaluated by the inhibition percentage of DPPH.The total antioxidant activity was presented as ferric reducing antioxidant power (FRAP) and 1,1-Diphenyl-2-picryl-hydroxyl (DPPH). The detailed determination methods of FRAP and DPPH were described by Zhang, et al. . The ass−ΔΔCT method. Primers used for qRT-PCR are listed in The total RNA was extracted using the improved CTAB method . The firp ≤ 0.05. A principal components analysis (PCA) was carried out using Origin 2021 software .All experiments were conducted in a completely randomized design (CRD) with three replicates. The data were exhibited as mean ± standard error (SE), analyzed using IBM SPSS Statistics 23.0 in one-way analysis of (ANOVA). To test the differences between treatments, Duncan’s multiple range test was performed at the significance level of Taken together, both light quality and sucrose can regulate to some extent the physiological characteristics during detached strawberry ripening, but not in a simple synergistic way. Specifically, red light or blue light in combination with 100 mM sucrose may promote detached ripening via the positive regulation of abscisic acid signaling and the negative regulation of auxin signaling; however, a detailed investigation is needed.