There was a reduced workload in the second half during the hot condition (p = .016), which coincided with a heightened threat state (p = .007) and more unpleasant feelings (p = .008) experienced in the hot, compared to temperate, condition. Furthermore, plasma normetanephrine (NMET) was higher at half-time (p = .012) and post-CISP (p ≤ .001). Also, plasma metanephrine (MET) was higher post-CISP (p = .009) in the hot compared to temperate condition, reflecting a heightened stress response. Our findings highlight the need for practitioners to consider the detrimental effects heat exposure can have on both physical and decision-making performance when looking to facilitate performance in hot conditions.Global warming and the globalisation of sport has increased the prevalence of sports competitions being held in hot environments. However, there is currently limited research investigating the impact of the heat on soccer-specific decision-making skills during exercise reflective of the physical demands of match-play. Therefore, the effects of heat exposure on physical and soccer-specific decision-making performance, biological markers , appraisal  and affective states, during prolonged high-intensity intermittent exercise were investigated. Nine well-trained male soccer players completed a 92-min cycling intermittent sprint protocol (CISP), whilst simultaneously responding to a series of soccer-specific decision-making trials at various time points, in two temperature conditions: hot  and temperate . Results showed that decision-making score ( Successful performance in soccer is dependent upon the simultaneous execution of a range of physical and cognitive skills. Sport-specific perceptual abilities can distinguish between elite and novice players e.g., ,2, with During the physically demanding nature of competitive soccer, physical fatigue occurs due to a depletion in energy stores , increased thermal strain, dehydration, and the alteration of central mechanisms  . Soccer Research in team sport has shown that high-intensity or prolonged exercise can improve decision-making speed ,13, but C), cardiovascular strain , dehydration, and neurochemical alterations , which could also be linked with physical and cognitive performance. The Theory of Challenge and Threat States in Athletes TCTSA; 28] offers insights into how athletic performance may be impacted by competitive stress and the appraisal of an activity. It is suggested that a challenge state is usually  associated with positively valenced emotions as well as linked with effective decision-making, higher task engagement, and positive performance. In contrast, a threat state is suggested to be associated with  negatively valenced emotions as well as emotions that are perceived as unhelpful for performance, impaired cognitive functioning, reduced task engagement and performance decrements , it is known that fatigue can impair higher level thought processing in soccer players. However, to date, no study has yet to explore how the additional stress imposed by heat exposure may impact such processes. Additionally, research has yet to investigate how heat exposure during prolonged intermittent exercise reflective of physical demands of match-play can affect a combination of biological  and psychological  responses that could have a role to play on performance  in soccer players.1). Second, it was anticipated that during exercise in 32˚C, players’ ability to engage in higher level thought processes would be reduced when retrospectively evaluating their decision, compared to during exercise at 18˚C (H2). Third, it was expected that threat appraisal about the activity demands, and unpleasant affect, would be higher in 32˚C compared to 18˚C (H3). Finally, it was believed that metanephrine and normetanephrine levels would increase in response to exercise, but more significantly in the hot, compared to the temperate conditions, reflecting a heightened physiological stress response (H4).Therefore, the aim of this research was to examine how heat exposure (32˚C) during prolonged intermittent sprint exercise affected physical work output, soccer-specific decision-making, demand and resource appraisals , affective valence , and plasma metanephrines , in comparison to a temperate condition (18˚C). As it was expected that soccer players would be under greater physiological and psychological strain in the heat, the following hypotheses were tested. First, it was hypothesised that power output  and the quality of decision-making would deteriorate during the later stages of the protocol as a result of exercise-induced fatigue, and that this deterioration would be more pronounced in 32˚C compared to 18˚C (Hn = 2), regional (n = 3) and club (n = 4) levels. An estimated sample size of nine participants was determined by a power calculation using G*Power 3.1.9.4 . All decision-making clips were projected onto a television screen positioned outside the viewing window of the environmental chamber, directly in front of the participant. An omnidirectional boundary microphone  was attached to the cycle ergometer to collect verbal responses and was connected via a small port to a laptop located outside of the chamber.Panopto software  was used to sync the video footage which was played from the laptop outside of the chamber, to the verbalised reports. Each recording was then downloaded as an MP4 file and transcribed ready for analysis in NVivo . Inductive content analysis was used to analyse the recordings obtained from the participants ,43, allord, 9th, 15th and 21st sprint in each half. At rest, participants were asked “How demanding do you expect the protocol to be?” and “How able are you to cope with the demands of the protocol?” to measure demand evaluations and resource evaluations, respectively. The tense of these questions was then adapted for during the CISP . These two items were ranked using a 6-point Likert scale anchored from 1  to 6 (extremely). A ratio was calculated by dividing demands by resources, where a value greater than 1 indicated a threat state, and less than 1 indicated a challenge state [To assess challenge and threat states, demand and resource evaluations were measured to determine a cognitive appraisal ratio  pre-CISPge state .rd, 9th, 15th and 21st sprint of each half of the CISP using the Feeling Scale [very bad) through 0  to 5 (very good). Unick et al. [Affective valence and activation were measured pre-CISP and every 3ng Scale  anchoredk et al.  also fouethylenediamine tetraacetic acid (EDTA) tubes from an indwelling venous catheter located in the arm at pre-CISP, half-time, and immediately post-CISP. These were centrifuged immediately  and plasma separated for analysis of plasma metadrenaline (MET) and normetanephrine (NMET), measured as the more stable metabolites of plasma epinephrine and norepinephrine, respectively [For measurement of plasma metanephrines, 3ml blood samples were collected in ectively . These sC was measured using a temperature sensor telemetry system in the form of a small ingestible pill. Participants ingested the thermometer pill ~6 hours prior to exercise [C was first recorded pre-trial following 15-min of seated rest to allow resting values to stabilise. TC was then recorded 1-min into each 2-min block, allowing a period for TC to stabilise following the 5-s sprint [C recordings were averaged across 5-time points for each half of the CISP  for analysis.Texercise . TC was s sprint , and wasAfter sitting for 15-min to allow resting values to stabilise, participants HR (beats per min) was recorded using a polar HR monitor . HR was then recorded immediately after every sprint throughout the CISP, then averaged across 5-time points for each half of the CISP  for analysis.After being towelled off, participants were asked to enter a secure room, undress, and step onto the Tanita scales to measure nude body mass immediately pre- and post-each CISP, to determine fluid loss from the trial . Urine ord, 9th, 15th and 21st sprint in each half of the CISP using Toner et al.’s [unbearably cold) to 8 (unbearably hot) Likert scale. This thermal sensation scale has been shown to be a valid measure of perceived heat stress, where correlations with large effect sizes (r = .72) have been shown between thermal sensation ratings and rectal temperature [Thermal sensation (TSS) was recorded every 3et al.’s  0  were taken every 3rg scale . Previourg scale .p ≤ 0.05. All data were reported as means and standard deviation (SD). For visual clarity, Figure error bars represent standard error mean (SEM).Statistical analysis was completed using IBM SPSS statistics 24.0 (IBM Corporation). After data screening and checking for normality of the data, a series of two-way  repeated measures ANOVAs were performed. Firstly, to analyse differences in indicators of physiological  and perceived psychological  strain. This was to check whether participants were under greater strain in the hot compared to the temperate condition. Then, a series of two-way repeated measures ANOVAs were performed to address our hypotheses. Specifically, to examine differences in physical  and cognitive  performance, perceived challenge and threat appraisals, affective valence, and plasma metanephrines. Homogeneity of variance was assessed using Mauchly’s test of Sphericity. Where homogeneity of variance was unable to be assumed, Greenhouse-Geisser corrections were applied. Where significant results were identified, Bonferroni pairwise comparisons post-hoc tests were performed. Two-tailed statistical significance was accepted at p2) and generalised eta-squared (η2G) are reported as the effect size for ANOVAs. Due to Cohen’s [d was reported as the effect size for pairwise comparisons. In accordance with Cohen [d values of 0.2, 0.5 and 0.8 indicated small, moderate, and large effects for pairwise comparisons, respectively.Partial eta-squared  and an interaction effect , but no effect for condition . Core temperature was higher in hot compared to the temperate condition in the final 6-min of the first half  , but was not different between conditions at any other time-point  (see A condition (2) × time  ANOVA for T.84) see .F1,8 = 13.581, p = .006, ηp2 = .629, η2G = .209), time , and condition (2) × time interaction  effects found for HR . Heart rate was significantly higher in the hot compared to temperate condition at all time-points , apart from in the first 10-min of the first half of the CISP  (See There were condition (.58) See .F1,8 = 47.901, p ≤ .001, ηp2 = .857, η2G ≤ .001) effect, whereby fluid loss was significantly higher in the hot (Mdiff = -1.60 ± 0.30 kg) compared to the temperate (Mdiff = -0.80 ± 0.40 kg) condition. For urine osmolality, no condition , time  nor condition × time interaction  effect was found. Additionally, there was a main condition effect for fluid intake whereby participants consumed more fluid (water) in the hot (1067 ± 440 ml) compared to the temperate (728 ± 168 ml) condition .There was a significant condition  ANOVA identified effects for condition  and time , but no interaction effect . RPE was significantly higher in the hot (14.54 ± 1.65) compared to the temperate  condition. In terms of time, Bonferroni pairwise comparisons identified that RPE was significantly higher toward the end of the first half  , midway through the second half   and at the end of the second half of the CISP  , compared to the start of the first half of the CISP  (10.44 ± 1.55). RPE was also significantly lower at the start of the second half  (11.83 ± 2.45) compared to the end of the second half   (see A 2 (condition) by 8  see .th-min then every 12-min intervals throughout the CISP) ANOVA found significant main effects for condition  and time , but no condition × time interaction effect  for TSS. Specifically, the effect of condition revealed that TSS was significantly higher in the hot (6.10 ± 0.42) compared to temperate  condition. For the time effect, Bonferroni pairwise comparisons identified higher TSS in the 18th  and 90th-min  compared to the 6th-min (4.72 ± 0.47) (see A 2 (condition) by 8  see .Overall, the findings from the preliminary analyses revealed that in the hot condition, participants showed greater cardiovascular strain (increased heart rate), higher fluid loss, felt hotter , perceived the exercise more demanding or exerting (higher RPE). Moreover, although no main condition effect was found for core temperature, core temperature was still higher at the end of each half of the CISP in the hot condition compared to the temperate condition. Therefore, these results are suggestive that participants were under greater physiological and perceived psychological strain during exercise in the hot condition.F1,8 = 4.976, p = .056, ηp2 = .383, η2G = .010) or condition . However, a significant condition × time interaction  was found , but was not significantly different in the second half (739.12 ± 92.73 W) compared to the first half  of the CISP in 18˚C  or second half of the CISP .A 2 (condition) by 2  ANOVA revealed no effect for time  and time , but no condition × time interaction effect    and second  halves of the CISP, compared to pre-CISP (95.17 ± 9.20%).A 2 (condition) by 3  ANOVA revealed significant main effects for condition (001) See . In term.93) see . In termFrom the inductive content analysis, 4 themes and 14 sub-themes were extracted. The themes reflected a) ‘game intelligence’, used for verbalisations which recognised critical information as it occurred such as player movement, goal-scoring opportunities, tactical awareness, and/or showed deeper planning as to what could or should occur following the initial decision; b) ‘risk management’, used for verbalisations which identified potential threat or discussed risk in relation to the available options; c) ‘difficulty’, used to categorise verbalisations which referenced how difficult the decision made would be to execute, or when participants referred to the difficulty, or showed hesitation toward the decision-making process; and finally d) ‘lack of evaluation’, used to reflect responses where the decision made lacked rationale and/or where participants simply re-described the action they would take see . Across F1,7 = 0.476, p = .513, np2 = .064, η2G = .009) or time , nor a condition × time interaction effect  for the number of themes extracted. Additionally, no condition , time  or condition × time interaction  effects were identified for total number of verbalisations , time , nor condition × time interaction  effects were observed. Within the sub-themes underpinning ‘difficulty’, there were no condition , time , nor condition × time interaction  effects for ‘difficult execution’, ‘easy execution’ and ‘uncertainty’.For ‘difficulty’, no main condition , time  or condition × time interaction  effects were noted. For the sub-themes underpinning ‘risk management’, no condition , time  or condition × time interaction  effects were found for ‘perceived threat’, ‘restricted options’, ‘risk assessment’ ‘safe options’ or ‘available time’.For ‘risk management’, no condition , time  or condition × time interaction  effects were observed. For sub-themes underpinning ‘game intelligence’, a main effect for condition  was found for ‘future planning’, but there was no time  nor condition × time interaction  effects. Specifically, a lower number of “future planning” verbalisations were made in the hot (0.99 ± 0.42) compared to the temperate condition . For ‘chance of scoring’ and ‘space and positioning’, no condition , time , or condition × time interaction  effects were found.For ‘game intelligence’, no condition  or condition × time interaction  effect was found. A main effect for time  was identified whereby significantly less ‘tactical awareness’ statements were made in the second half (0.63 ± 0.62) compared to the first half . For ‘player movement’, no main condition  or time  effects were identified. However, a condition × time interaction effect  was observed whereby ‘player movement’ verbalisations were lower in the first half in the hot compared to the temperate condition , though they were not different in the second half .For ‘tactical awareness’, no condition , where more responses lacked evaluation in the second half (0.75 ± 0.48) compared to the first half  of the CISP. There was no effect for condition , nor condition × time interaction .In terms of ‘lack of evaluation’, a main effect for time was found  ANOVA revealed significant main effects for condition  and time , but no interaction effect was identified . Post-hoc analysis revealed a higher threat state in the hot condition (1.20 ± 0.50) compared to a higher challenge state in temperate condition . For the effect of time, no significant differences were observed between time points , apart from between the 6th (.55 + .21) and 66th minute  (see A 2 (condition) by 9  see .th-min, and then 12-min intervals throughout the CISP) ANOVA found significant effects for condition  and time , but no condition × time interaction effect . Post-hoc analysis revealed that affective valence was significantly lower in 32˚C (.41 ± 1.59) in comparison to 18˚C . Bonferroni pairwise comparisons also identified lower affective valence at the start of the second half  , and in the 66th min , and 78th min , compared to rest (3.50 ± 1.50). It was also significantly lower toward the end of the second half in the 66th , 78th  and 90th min  compared to early in the first half in the 4th min (2.39 ± 1.35)  by 9  see .F1,7 = 43.637, p ≤ .001, ηp2 = .862, η2G = .191), time , and condition × time interaction  effects were observed for normetanephrine (NMET) concentration. Specifically, NMET concentration was significantly higher in the hot compared to temperate condition at half-time  and post-CISP , but was not different between the hot and temperate condition pre-CISP  (see Significant condition (.06) see .F1,7 = 5.954, p = .045, ηp2 = .460, η2G = .067), time , and condition × time interaction  effects. MET concentration was significantly higher in the hot compared to temperate condition post-CISP , but not pre-CISP  or at half-time  (see In terms of metanephrine (MET) concentration, there were significant condition (.46) see .1), whereby soccer-specific decision-making scores  was impaired in 32˚C compared to 18˚C. However, no condition × time effects were observed for decision-making scores which was not aligned to our hypotheses. As expected, intermittent sprint performance was impaired in the second half compared to the first half of the CISP in 32˚C, but did not deteriorate in 18˚C. In support of the second hypothesis (H2), ‘future planning’ verbalisations were significantly lower in 32˚C compared to 18˚C, and more responses categorised as ‘lack of evaluation’ were observed in the second half compared to the first half across conditions. This partly reflected lower-level thought processing in the hot condition and following a half of intermittent sprint activity, which partially affirmed our second hypothesis (H2). However, we did not find condition × time interactions as originally anticipated. We also found support for our third hypotheses (H3), whereby players had a heightened threat state and felt more unpleasant in 32˚C compared to 18˚C. Moreover, MET and NMET concentration were significantly higher following exercise, and these increases were greater in the hot compared to the temperate condition, which reflected a heightened physiological stress response when exercising in hot condition thereby supporting our fourth hypothesis (H4). It is worth noting that these findings were similar when analysing data with 8 participants when conditions were fully counterbalanced, and a summary of these findings can be found in the supporting information.This study aimed to investigate how soccer-specific decision-making, power output, appraisal, affect, and plasma metanephrines were affected during prolonged intermittent exercise in a hot, compared to a temperate, environment. There was partial support for our first hypothesis  [C threshold of ~38.5˚C is where cognitive function becomes impaired [C did not exceed this threshold in the majority of participants.It was anticipated that a more pronounced decline in decision-making score would be observed during the CISP in the hot condition compared to the temperate condition due to additional physiological stress experienced  ,29,31. Dhe CISP) . From anhe CISP)  found thimpaired . HoweverA ‘think-aloud’ protocol was also implemented, recording participants retrospective evaluation of each situation to understand what factors were being considered when making the decision, and if this was affected by exercise-induced fatigue or heat stress. Interestingly, thematic analysis revealed that participants verbalised significantly less ‘future planning’ evaluations when in the hot compared to the temperate condition. These findings align with previous research, which found that within 15-min of passive heat exposure  complex planning performance is significantly impaired . This deVerbalisations referencing tactical awareness and execution difficulty were also significantly lower in the second half compared to the first half of the CISP. Additionally, statements which lacked evaluation were higher in the second half compared to the first half, and reference to specific player movement were also significantly reduced in the second half in the hot condition. Support can be drawn from Casanova et al. , who fouAnother possible explanation is that the heat stress in the present research could have also potentially induced some anxiety  that may have contributed in reducing soccer players’ focus during the decision-making task. For example, based on the premises of the Integrated Model of Anxiety and Perceptual-Motor Performance  anxiety C >39°C) [C reached values lower than those typically expected when performing team sport in the heat (>~39˚C) [Heat also impaired athletes’ physical performance in this study. Power output was significantly lower during the second half compared to the first half in the hot condition (-~7%) but was not different between halves in the temperate condition. Previous research has shown that increased perceived exertion and more negative affective states induced by heat exposure can impair self-paced work rate . MoreoveC >39°C) . However(>~39˚C) . This wa(>~39˚C) .C reaching average heights of ~38.4˚C. It is suggested that when players feel their TC increasing more rapidly in hot conditions, they may deliberately or reflexively reduce their high-intensity work to reduce the rate of metabolic heat production and ensure thermal strain does not exceed tolerable limits [The present research also observed higher NMET and MET levels (measured as the more stable metabolites of norepinephrine and epinephrine) at half-time and post- exercise compared to pre-exercise, but such increases were to a much greater extent in the hot compared to temperate condition. This aligns with previous research that has observed that the release of epinephrine and norepinephrine is accelerated in response to acute heat stress . The ince limits . In thisC, HR sweat rate and dehydration) [The present study also identified that participants’ demand appraisals were higher and resource appraisals were lower in hot condition compared to the temperate condition, reflective of an overall higher threat state in the hot compared to the temperate condition. Naturally, when performing in hot environments, individuals are likely to perceive that higher effort may be required than in temperate conditions due to the widely understood exacerbated physiological responses  . Furtherdration) . Based odration) , threat Whilst this study built upon previous methods by integrating a sport-specific visual decision-making task, research should incorporate tasks which require simultaneous visual and auditory attention  to better replicate the demands of match-play. Additionally, a cycling intermittent sprint protocol was used to allow players to modulate their own high-intensity physical work output as per match-play, whilst simultaneously investigating cognitive function and central mechanisms. Despite this being widely employed within team sport literature ,40, it iC was ≤38.5˚C, suggesting that complex, situation-specific decision-making as well as physical work output can be impaired below TC thresholds proposed previously when exposed to heat during exercise [In this research, soccer-specific decision-making was impaired in the hot compared to temperate condition. Physical work output was also significantly reduced in the second half in the heat but was not affected in the temperate condition. These decrements were observed when Texercise . AthleteSoccer players and sports practitioners should consider that performing in more strenuous environments may enhance the likelihood of players making infrequent, but potentially costly, decision-making errors. Sports practitioners could look to reduce potential anticipatory effects of performing in more demanding environmental conditions by improving athletes’ perceptions of such environments, reducing their level of uncertainty, and improving their resource appraisal such as through enhancing self-efficacy and developing perceived control . The impS1 Table(DOCX)Click here for additional data file.


Cuproptosis, a new cell death pattern, is promising as an intervention target to treat tumors. Abnormal long non-coding RNA (lncRNA) expression is closely associated with the occurrence and development of papillary renal cell carcinoma (pRCC). However, cuproptosis-related lncRNAs (CRLs) remain largely unknown as prognostic markers for pRCC. We aimed to forecast the prognosis of pRCC patients by constructing models according to CRLs and to examine the correlation between the signatures and the inflammatory microenvironment. From the Cancer Genome Atlas (TCGA), RNA sequencing, genomic mutations and clinical data of TCGA-KIRP  were analyzed. Randomly selected pRCC patients were allotted to the training and testing sets. To determine the independent prognostic impact of the training characteristic, the least absolute shrinkage and selection operator (LASSO) algorithm was utilized, together with univariate and multivariate Cox regression models. Further validation was performed in the testing and whole cohorts. External datasets were utilized to verify the prognostic value of CRLs as well. The CRLs prognostic features in pRCC were established based on the five CRLs . The utility of CRLs was evaluated and validated in training, testing and all sets on the basis of the Kaplan–Meier (KM) survival analysis. The risk score could be a robust prognostic factor to forecast clinical outcomes for pRCC patients by the LASSO algorithm and univariate and multivariate Cox regression. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) data demonstrated that differentially expressed genes (DEGs) are primarily important for immune responses and the PI3K-Akt pathway. Arachidonic acid metabolism was enriched in the high-risk set by Gene Set Enrichment Analysis (GSEA). In addition, Tumor Immune Dysfunction and Exclusion (TIDE) analysis suggested that there was a high risk of immune escape in the high-risk cohort. The immune functions of the low- and high-risk sets differed significantly based on immune microenvironment analysis. Finally, four drugs were screened with a higher sensitivity to the high-risk set. Taken together, a novel model according to five CRLs was set up to forecast the prognosis of pRCC patients, which provides a potential strategy to treat pRCC by a combination of cuproptosis and immunotherapy. Up to 80% of kidney tumors are renal cell carcinomas (RCCs); The most common type of RCC is clear cell renal cell carcinoma (ccRCC), occupying approximately 75% of all cases, followed by papillary renal cell carcinoma (pRCC) ,2. It haCopper ion is an extremely indispensable metal trace element and cofactor for organisms, which is involved in many biological processes. The amount of copper in the body is tightly regulated and toxic when it exceeds a threshold . It has Long non-coding RNAs (LncRNAs) are molecules longer than 200 nucleotides, which play a key role in many diseases ,17,18. TIn this study, we aimed to develop a prognostic model for an assessment and improvement of the prognosis of pRCC using CRLs, as well as to investigate the differences in the tumor microenvironment (TME), immunotherapy and drug response in pRCC. Therefore, a novel prognostic model was constructed according to five prognostic CRLs , which has outstanding diagnostic value for patients with pRCC and is practical to guide clinicians to personalized immunotherapy and medication for pRCC patients according to individual differences.p < 0.001). A total of 289 pRCC patients were randomly allotted to the training set (n = 145) and the testing set (n =144). The clinical features of the two sets of patients are presented in AC244033.2) + (−0.592113007 × ExpressionLINC00886) + (−2.088352244 × ExpressionAP000866.1) + (0.941720478 × ExpressionMRPS9-AS1) + (1.641000898 × ExpressionCKMT2-AS1). The relationships between the five CRLs and cuproptosis genes are demonstrated in A flowchart of the study is shown in p < 0.001; Training patients were assigned into high- and low-risk sets for survival analysis according to their median risk scores. The results demonstrated that low-risk patients had significantly better OS than those in the high-risk set | ≥ 1, First, the differentially expressed genes (DEGs) based on the average expression of samples from high- and low-risk sets were determined  (p = 0.0024) (p = 0.039) ,  × 10−5) A and RFS 0.0024) B. On the= 0.039) D but not= 0.039) C. Our daIn this study, five prognostic CRLs, including AC244033.2, LINC00886, AP000866.1, MRPS9-AS1 and CKMT2-AS1 were identified by bioinformatic analysis, which had high accuracy in forecasting overall survival (OS). It was found in our study that these five prognostic CRLs were closely related to TME, immunotherapy and drug response in pRCC.Although some targeted agents have been utilized clinically in patients with highly differentiated RCC, their efficacy in pRCC has not been proven ,28. BecaRecent studies have shown that mitochondrial lipid acylated protein accumulation and the loss of Fe–S cluster proteins caused by intracellular copper accumulation could eventually result in proteotoxic stress-induced cell death, known as cuproptosis . StudiesIt has been reported that lncRNAs are critical to the pRCC progression in several studies. Chen et al. found that the up-regulation of lncRNA RP11-63A11.1 could attenuate cell survival and induce apoptosis in pRCC . In anotHere, we divided the TCGA-KIRP cohort into training and testing sets randomly. Using the median risk score from the training set, the training and testing sets were classified as high- and low-risk, respectively. The clinical statistical analysis of the high- and low- risk sets suggested that there was no difference between the two sets on clinical features . Then, hThe study we conducted had some limitations. Since these findings were derived from the TCGA database and further experiments are needed to verify their accuracy. In addition, we could not obtain proper lncRNA and clinical information of pRCC from the GEO database. Thus, two of these lncRNAs were validated using the external Kaplan-Meier Plotter database including GEO, EGA and TCGA.https://portal.gdc.cancer.gov/repository (accessed on 15 October 2022)), which included 291 pRCC tumors and 32 normal tissues. Language Perl ) was employed to extract RNA-seq data from the standardized FPKM format [Clinical and RNA sequencing data of pRCC were downloaded from TCGA database (M format . Patholop < 0.001). Then, the sankey relationship between cuproptosis genes and CRLs was plotted with packages “ggplot2” and “ggalluvial” [The cuproptosis-related expression matrix was obtained using R packages “BiocManager” and “limma” . CRLs exlluvial” .p < 0.05). A total of 5 CRLs were obtained by the LASSO algorithm and univariate and multivariate Cox regression analysis. The related maps were drawn with packages “survival”, “glmnet”, “survminer” and “timeROC” [In a 1:1 ratio, pRCC patients were randomly allotted to the training and testing sets with the package “caret”. The train set was utilized to build the CRLs prognostic model. Model validation was based on the testing and entire sets. Prognostic CRLs were acquired via univariate Cox regression  and Expr (i), respectively. High- and low-risk sets were divided in training, testing, and whole sets, respectively, according to the median risk score for the training set. ROC curves together with C-index were utilized to measure the accuracy of the model with packages “survminer”, “timeROC”, “survival”, “rms” and “pec” . To evalGO analysis was performed with packages “colorspace”, “stringi”, “ggplot2”, “circlize”, “RColorBrewer” and “ggpubr” to demonstrate the DEGs of high- and low-risk sets . KEGG anhttp://tide.dfci.harvard.edu, (accessed on 15 October 2022)) [The package “maftools” was utilized to analyze the differences in TMB between the high- and low-risk sets. Scoring file for TIDE was downloaded from the website (r 2022)) .For low- and high-risk sets, violin plots were created with the packages “limma” and “vioplot”, showing the percentage of 22 types of tumor-infiltrating immune cells. We utilized packages “limma”, “reshape2”, “tidyverse”, “ggplot2”, “ggpubr” and “ggExtra” to survey the correlation among risk score, infiltrating immune cells and immune checkpoints. The heat map exhibited the difference of immune-related functions for the low- and high-risk sets, which was plotted with packages “limma”, “GSVA”, “GSEABase”, “pheatmap” and “reshape2”. Furthermore, the IC50 values of drugs available for treating pRCC in high-risk and low-risk sets were predicted by pRRophetic.https://kmplot.com/analysis/ (accessed on 15 October 2022)). Auto selecting best cutoff was utilized to split patients.CRLs were tested for prognostic value using a KM plotter database (Overall, a novel prognostic model based on five CRLs was developed for pRCC patients. The features were correlated with the immune microenvironment, immune checkpoints and immunotherapy. The findings will contribute to a better understanding of the underlying mechanisms of pRCC emergence and progression, which provide a potential strategy to treat pRCC by the combination of cuproptosis and immunotherapy.


Our society seems to generate the general belief that caring for animals has a positive effect on the well-being of humans, which often creates unrealistic expectations in pet owners. Since there are contradictory results that underline the stressful features of the human–animal relationship, it seems to be highly relevant to investigate this phenomenon in more detail. The present study tries to examine how external stressors such as the COVID-19 pandemic as well as Long-Covid influence the biopsychosocial wellbeing of animal caregivers. The results demonstrate a gap between the subjectively experienced meaning of pets in the context of the pandemic, which is positive throughout, and the calculated findings from several psychometric instruments indicating that animal companionship might be seen as an additional burden in times of crisis. These results underline the existence of the so called “pet-effect paradox” which emphasizes the mismatch between pet owners’ individual perception regarding the importance of an animal and the measurable findings in research. Further research is needed to understand the underlying dynamics in more detail and to prevent false expectations in connection with the human–animal relationship.Studies in the field of human–animal interaction tend to highlight the positive results of the influence of animals on humans, which supports the popular belief that the human–animal bond positively affects humans’ well-being (“pet-effect”). Nevertheless, contradictory results exist that seem especially visible since the COVID-19 pandemic, a prominent external stressor. Despite critical findings, individuals seem to want to believe in the beneficial effects of the human–animal relationship (“pet-effect paradox”). Based on this background, the present study aims to investigate this phenomenon using a mixed-method design. Therefore, animal caregivers were surveyed online and compared using psychometric measurements and open-ended questions. In this context, a special focus was placed on the additional stressor of Long-Covid and related concerns. The results demonstrate once more the existence of the “pet-effect paradox” due to a contradiction in the quantitative and qualitative results. At a quantitative level, the findings show additional burdens on animal caregivers who are confronted with multiple loads. However, the qualitative results indicate a belief in the beneficial effects of pets at the biopsychosocial level. Additionally, the data demonstrate a shift in focus away from the environment to oneself when affected by Long-Covid, which might affect the ability to care for an animal. Since the 1980s, studies in the field of human–animal interactions have suggested that relationships with companion animals are beneficial to humans on a bio-psycho-social level ,3,4. ThiInterestingly, in addition to this general conviction, contradictory results exist regarding the animal’s role in the context of stress management, but they receive little attention in public media as well as research ,14. In twant to believe in the beneficial effects of the human–animal relationship. The latest research results indicate divergencies between the opinions of pet owners regarding the pet’s protective role during the COVID-19 pandemic, focusing on their perception, and the results of standardized measurements [Given these results, a question arises regarding how such contradictory findings are possible. The psychologist Hal Herzog emphasizes the so called “pet-effect paradox”, which explains a mismatch between pet owners’ individual perception regarding the importance of an animal—which seems to be predominantly positive—and the actual findings in research ,20,21. Turements . At a quurements . Yet, fiurements .From these results, it appears that the “paradox of the pet-effect” is particularly evident when individuals are confronted with different stressors. Therefore, the COVID-19 pandemic provided an opportunity for researchers in this field to examine this phenomenon with regard to a global stressor, affecting individuals worldwide, in more detail. In this context, most of the studies indicate a significant and complex influence of animal companionship and related responsibilities in the context of stress experience that seems to be strongly determined by external stressors ,17,18. Tn = 242) and therefore included in statistical and qualitative analysis.Using a cross-sectional mixed methods design, an online survey in the German language was conducted. The survey test battery included closed-ended and open-ended questions regarding demographic data and standardized questionnaires concerning the emotional attachment to the companion animal , symptoms of depression (Beck’s Depression Inventory), the quality of life , loneliness , and social support . Pet owners were recruited via snowball sampling and surveyed online. A total of 242 data sets were fully completed  or the duration of animal husbandry. Furthermore, questions regarding the current COVID-19 situation were asked including possible lockdowns, quarantines, and infections plus the long-term effects. With regard to a Long-Covid affliction, additional data were collected in relation to psychological and physical symptoms.Overall, the sample consists of 83.8% women, 15.4% men, and 0.8% others. The average age of the sample was 36 years . Regarding the marital status, the sample mainly consisted of those living in a relationship (33.8%), single (31.3%), or married (27.1%). Several participants stated that they were divorced (6.3%) or widowed (1.7%). About one third of the sample indicated having finished undergraduate or graduate school . A total of 17.6% stated having graduated from high school and 14.6% completed apprenticeship. To evaluate the current situation with regard to COVID-19, additional questions were asked about lockdown, quarantine, infection, and Long-Covid. In this context 32.7% of the sample stated having strict lockdown restrictions and 3.3% claimed to be in quarantine at the time of the survey. Regarding a COVID-19 infection, 56.5% stated that they were infected with COVID-19 at any time up to the survey and 50.3% indicated that they were suffering from Long-Covid at the time of the survey.The Lexington Attachment to Pets Scale  was usedExample statement: “I feel that my dog is part of my family”.The BDI-II  was usedExample item “Sadness”:“I do not feel sad” to “I am so sad and unhappy that I can’t stand it”.The WHOQOL-BREF  is a selExample question.“How satisfied are you with your ability to perform your daily living activities?”The ESSI  was usedExample Statement.“Is there someone available to you who you can count on to listen when you need to talk?”The Loneliness Scale  is a 20-Example Statement.“I find myself waiting for people to call or write”.Specific pet related stressors/concerns were analyzed based on preliminary investigations . In thisExample Statement.“I’m worried that if I get sick, I won’t be able to take care of my pet.”In addition to animal-related stressors, general stressors/concerns regarding the pandemic were surveyed based on previous studies . In thisExample Statement.“I worry about being socially isolated”.In addition to these closed-ended questions, an open-ended question (“What other concerns are on your mind regarding the corona virus (COVID-19)”) asked participants to raise other concerns that were not covered by the predefined items.Animal caregivers were asked about their personal opinion regarding the animals’ role during the pandemic in general and especially in connection with the management of Long-Covid in an open and closed question format. In this context, participants were asked about their agreement with seven pre-formulated statements using a four-point scale answer format from totally agree to totally disagree. Open questions were used to examine their individual opinions on the human–animal relationship during the pandemic and with regard to Long-Covid .Statistical analyses were computed with SPSS 27.0. Regarding pet-related and pandemic-related concerns, factor analyses were calculated based on the listed items. The aim was to examine latent dimensions and investigate underlying structures of individuals’ associations regarding evaluated concerns. Reliability was assessed with coefficient alpha.Moreover, univariate procedures in the form of paired t-tests were chosen to gain insight into possible differences between animal-caregivers suffering from Long-Covid and animal-caregivers who have not been infected with the COVID-19 virus by the time of the survey with regard to biopsychosocial wellbeing , the human–animal bond (LAPS), and calculated factors of pet-related and pandemic-related concerns. Cohens d was calculated as an effect size measurement. Prior examination required conditions for t-tests and factor analyses were conducted.p ≤ 0.05.Furthermore, correlations were calculated to measure mutual relations between pet-related and pandemic-related concerns, the strength of the human–animal bond (LAPS) and symptoms of depression (BDI-II), the subjectively experienced quality of life (WHOQOL-BREF), loneliness , and social support (ESSI). For every analysis, the significance level was set at In addition to statistical analyses, open-ended questions were examined using the qualitative content analysis according to Phillip A. E. Mayring . IndividThe deductive categorization was also chosen for the thematic analysis of personal opinions regarding the animal’s role during the pandemic in general and especially regarding the management of Long-Covid. Thus, deductive categories were developed based on the theoretical basis of the bio-psycho-social model. To assure reliability, the coding process was carried out by two experts independently and analyses were subjected to a reliability test (Cohens Kappa > 0.60).To investigate the underlying structures of the assessed pet-related and pandemic-related concerns, exploratory factor analyses were calculated. The exploratory factor analysis regarding pet-related concerns resulted in a two-factor solution: responsibility-related concerns and animal-related concerns. All of the initial nine items were retained. The first factor “responsibility-related concerns” describes concerns associated with no longer being able to adequately care for the pet for various reasons. This factor consists of five items  and has a very good reliability (α = 0.855). The second factor “pet-related concerns” describes concerns that explicitly refer to the well-being of the animal. It consists of four items  and has a good reliability (α = 0.805).The exploratory factor analysis regarding pandemic-related concerns also resulted in a two-factor solution: general pandemic-related concerns and infection-related concerns. As previously mentioned, only seven of the eight items have been included in the calculations, as one independent item has been added to the existing list to gain more information. Nevertheless, this item was excluded in the present analysis to avoid distorting the scale calculations. The first factor, “general pandemic-related concerns”, describes concerns associated with daily worries related to the overall situation of the pandemic. This factor consists of five Items  and has a moderate reliability (α = 0.680). The second factor, “infection-related concerns”, describes concerns that explicitly refer to one’s own infection with COVID-19 or the infection of a close person. It consists of two items (No. 1 and 2) and has a very good reliability (α = 0.878).For a better understanding, To determine relationships between pet-related and pandemic-related concerns, Pearson correlations were conducted between the pre-calculated factors of pet-related concerns  and pandemic-related concerns . The results show significant positive correlations between animal-related concerns and responsibility-related concerns as well as between infection-related concerns and general pandemic-related concerns. Moreover, a significant negative correlation was found between responsibility-related concerns and infection-related concerns to the effect that the more infection-related concerns are pronounced the less responsibility-related concerns are perceived, or the other way around.In order to test whether the additional stressor of a chronic disease such as a Long-Covid affliction influences the biopsychosocial wellbeing of animal caregivers during the pandemic, participants suffering from Long-Covid and participants who had not been infected with the COVID-19 virus by the time of the survey were compared via t-test analysis. Additionally, the possible differences were analysed regarding the strength of the human–animal bond and regarding pet-related and pandemic-related concerns. The data show that there are significant differences between animal caregivers suffering from Long-Covid and animal caregivers who have not been infected with the COVID-19 virus by the time of the survey regarding the biopsychosocial wellbeing . In this context, the data demonstrate that animal caregivers suffering from Long-Covid report more symptoms of depression or a stronger expression of these symptoms, a significantly lower experienced quality of life in all the evaluated areas, and a lower sense of social support.Moreover, the data show significant differences between the two samples regarding the assessed strength of the human–animal relationship and pet-related as well as pandemic-related concerns. In this connection, animal caregivers suffering from Long-Covid address a significantly stronger experienced relationship to the animal and significant less distinctive concerns regarding the pet. Furthermore, they report significantly stronger experienced concerns concerning COVID-19 infections and the pandemic in general. No significant differences were analyzed with regard to the perceived feeling of loneliness.In order to investigate the previously mentioned differences in more detail and to gain insight into concerns as possible influencing factors, Pearson correlations between the two factors of pet-related concerns  and the variables “strength of the human–animal relationship” (LAPS), “symptoms of depression” (BDI-II), “quality of life” (WHOQOL-BREF), “loneliness” , and “social support” (ESSI) were calculated separately in the groups “Long-Covid” and “No Infection”. The same procedure has been conducted with respect to the two factors of pandemic-related concerns .The results within the group “Long-Covid” show significant negative correlations between the factor “animal-related concerns” and the overall experienced quality of life, as well as the subdomains “environment” and “social relationships”. Moreover, regarding the factor “responsibility-related concerns”, a significant negative correlation was found with the overall experienced quality of life. r(73) = −0.173, p = 0.143), “physical health” (r(61) = −0.157, p = 0.178), “depressive symptoms” (r(55) = 0.252, p = 0.064), “loneliness” (r(79) =0.160, p = 0.160), “social support” (r(81) = −0.108, p = 0.338), and “human–animal relationship” (r(78) = 0.212, p = 0.063). Additionally, no significant results were found between the factor “responsibility-related concerns” and “psychological health” (r(73) = −0.184, p = 0.120), “environment” (r(73) = −0.208, p = 0.077), “social relationships” (r(75) = −0.156, p = 0.182), “physical health” (r(75) = −0.147, p = 0.209), “depressive symptoms” (r(55) = 0.134, p = 0.328), “loneliness” (r(79) = −0.025, p = 0.827), “social support” (r(81) = −0.114, p = 0.311), and “human–animal relationship” (r(77) = 0.207, p = 0.071).No significant results were found between the factor “animal-related concerns” and the variables “psychological health”  = −0.224, p = 0.080), “environment” (r(61) = −0.188, p = 0.146), “social relationships” (r(61) = 0.082, p = 0.530), “physical health” (r(61) = −0.133, p = 0.307), “overall quality of life” (r(60) = −0.115, p = 0.381), “depressive symptoms” (r(53) = 0.153, p = 0.274), “loneliness” (r(52) = 0.206, p = 0.143), and “social support” (r(54) = −0.142, p = 0.306). Moreover, no significant correlations were found between the factor “responsibility-related concerns” and the variables “psychological health” (r(64) = −0.158, p = 0.212), “environment” (r(63) = −0.123, p = 0.337), “social relationships” (r(63) = −0.103, p = 0.423), “physical health” (r(63) = −0.143, p = 0.262), “overall quality of life” (r(62) = -.0165, p = 0.200), “depressive symptoms” (r(55) = 0.040, p = 0.774), “loneliness” (r(54) = 0.131, p = 0.346), and “social support” (r(55) = −0.160, p = 0.243).No significant results were found between the factor “animal-related concerns” and the variables “psychological health”  = −0.187, p = 0.113), “social relationships” (r(75) = −0.058, p = 0.619), “loneliness” (r(79) = 0.066, p = 0.561), “social support” (r(81) = −0.105, p = 0.345), and “human–animal relationship” (r(79) = 0.127, p = 0.263). Additionally, no significant correlations were found between the factor “general pandemic-related concerns” and “physical health” (r(69) = −0.183, p = 0.133), “social support” (r(75) = −0.129, p = 0.271), and “human–animal relationship” (r(73) = −0.019, p = 0.875).No significant correlations were found between the factor “infection-related concerns” and the variables “environment”  = −0.1651, p = 0.196), “environment” (r(65) = −0.170, p = 0.177), “social relationships” (r(65) = −0.020, p = 0.875), “overall quality of life” (r(64) = −0.198, p = 0.117), “depression” (r(57) = −0.159, p = 0.237), “social support” (r(56) = 0.027, p = 0.844), and “human–animal relationship” (r(76) = 0.024, p = 0.836). Moreover, no significant correlations were found between the factor “general pandemic-related concerns” and the variable “human–animal relationship” (r(74) = −0.161, p = 0.169).No significant correlations were found between the factor “infection-related concerns” and the variables “psychological health”  and “symptoms of depression” (BDI-II), “quality of life” (WHOQOL-BREF), “loneliness” , and “social support” (ESSI).The results within the group “Long-Covid” show significant negative correlations between the variables “human–animal relationship”, “psychological health”, and “overall quality of life” as well as a positive correlation with the variable “symptoms of depression”. r(65) = −0.167, p = 0.182), “social relationships” (r(68) = −0.177, p = 0.150), “physical health” (r(67) = −0.187, p = 0.130), “loneliness” (r(70) = −0.054, p = 0.660), and “social support” (r(72) = −0.174, p = 0.144).No significant correlations were found between the variables “human–animal relationship” and “environment” (r(64) = −0.065, p = 0.611), “environment” (r(64) = −0.085, p = 0.505), “social relationships” (r(64) = 0.169, p = 0.181), “physical health” (r(63) = −0.062, p = 0.628), “overall quality of life” (r(63) = 0.010, p = 0.937), “symptoms of depression” (r(55) = 0.078, p = 0.572), “loneliness” (r(55) = 0.023, p = 0.867), and “social support” (r(55) = −0.020, p = 0.887),.The results within the group “No infection” overall show no significant correlations between the variables “human–animal relationship” and the calculated parameters, i.e., “psychological health”  are presented as follows.Regarding the reflexive self -perception of concerns during the COVID-19 pandemic, the open question “What other concerns are on your mind regarding the Corona virus (COVID-19)” was asked. The method of deductive categorization was used to structure the data. In this context, the participants’ answers were assigned to three main categories and twelve subcategories of an already existing concern scheme according to previous research . Since tTo find out if and how the subjective concerns of the participants differed depending on whether or not they were suffering from a Long-Covid affliction, the responses were analyzed separately within the groups “Long-Covid” and “No infection”. The results of the frequency analysis show that animal caregivers who were suffering from a Long-Covid affliction by the time of the survey mostly stated concerns that could be assigned to the category “own health—physical” (44.87%). Regarding animal caregivers without an infection by the time of the survey, the results of the frequency analysis demonstrate that concerns are most frequently assigned to the subcategories “social division” (23.91%), “restrictions imposed by fundamental rights and freedoms” (19.57%), and “health of related persons” (17.93%).To investigate the subjective opinion regarding the animal’s role during the pandemic in general as well as in connection with coping with Long-Covid, the following open questions were asked: “What does/do your pet(s) mean to you during the COVID-19 pandemic” and “How does/do your pet(s) impact your Long-Covid condition?”. The deductive development of the categories resulted in a three-part categorization including the categories “Biological impact”, “Psychological impact”, and “Social impact”, which were proved to allow for the most appropriate categories to encompass the content of open answers. Again, the content of the answers was analyzed separately within the groups “Long-Covid” and “No infection” to investigate the possible differences. The results of the frequency analysis show that the subjective statements of animal caregivers who were suffering from a Long-Covid affliction by the time of the survey, in relation to their pets’ role during the COVID-19 pandemic, are most frequently assigned to the category “Psychological impact” (60.0%). In this context, the data show the highest frequency regarding the category with positive characteristics (42.5%). A total of 10% percent of the analyzed data included neutral characters and 7.5% included negative characters within the statements. These results are followed by the category “Biological impact”, where the 27.5% positively characterized, 17.5% neutrally characterized, and 7.5% negatively characterized statements were analyzed. Moreover, the category “Social impact” shows the lowest least content allocation and demonstrates 20% positively characterized and 7.5% neutrally characterized statements.Within the group “No infection”, the frequency analysis demonstrates that the stated role of pet(s) during the pandemic could mostly be assigned to the category “Psychological impact” (39.6%). The data show the highest frequency concerning the category with positive characteristics (32.1%) and 7.5% of statements were categorized as neutral statements. These results are followed by the category “Social impact” where the 22.6% positively characterized and 9.3% neutrally characterized statements were analyzed. The category “Biological impact” included 9.4% positively characterized and 3.7% neutrally characterized statements.The present study aims to investigate the influence of animal companionship on different biopsychosocial parameters when one is confronted with multiple external loads such as the COVID-19 pandemic and the additional stressor of a Long-Covid affliction. A special focus was placed on concerns, which were evaluated both as being pet-related and pandemic-related. In this context, the data overall show that there is no evidence of the often-reported animals’ protective role regarding the biopsychosocial wellbeing at a quantitative level. On the contrary, the data might indicate an additional burden caused by the human–animal relationship during the pandemic and especially when one is confronted with multiple stressors. Pandemic-related concerns seem to have a particularly important influence on the biopsychosocial wellbeing of animal caregivers suffering from Long-Covid. Nevertheless, at a qualitative level, the data demonstrate that animal caregivers in both groups (“Long-Covid” and “No infection”) mostly estimate their animals as a biopsychosocial source during the pandemic. For a better understanding, the detailed results are discussed after being separated into two groups.Within the group of animal caregivers suffering from a Long-Covid affliction, the data overall indicate a vulnerability regarding biopsychosocial health. The results demonstrate that affected participants reported significantly more or stronger experienced symptoms of depression as well as a significantly lower quality of life than animal caregivers without an infection by the time of the survey. Moreover, animal caregivers suffering from Long-Covid stated a significantly lower experienced feeling of being socially supported. These results are consistent with previous findings indicating the impaired physical and mental health of individuals with Long-Covid ,32. In aWith regard to the animal’s role during the pandemic and especially when one is confronted with an additional stressor such as a Long-Covid, the findings of the present study once again indicate that there is no sufficient evidence for the often-reported supportive influence of animal companionship. The results at a quantitative level show that animal caregivers suffering from Long-Covid experience a significantly stronger relationship with their animals, but this stronger experienced bond does not seem to have a positive influence on the wellbeing of their owners. On the contrary, the data demonstrate that within the sample of animal caregivers suffering from Long-Covid, a stronger evaluated relationship with the animal is associated with significantly lower levels of the quality of life, psychological health and with higher levels of depressive symptoms. Additionally, no indication could be found that a stronger perceived human–animal relationship influences concerns (positively or negatively) regarding the pandemic. These results suggest that the often-reported positive effects of animals on humans’ wellbeing are not visible on a measurable level within the present study. Nevertheless, on a reflexively perceived qualitative level, companion animals seem to be an important source with respect to the biopsychosocial wellbeing, especially when one is confronted with external stressors. Most animal caregivers suffering from Long-Covid stated a positive influence of the animal especially on a psychological level but also on a biological and social level. Even though some negative statements including negative characteristics such as “It is exhausting to go for a walk with the two twice a day even if you have no power”, the overall reflexive perception of the animal’s influence turns out positive.Since the measurable findings suggest that the participants affected by Long-Covid are suffering from a significantly lower biopsychosocial wellbeing, which can even be linked to the relationship with an animal, it can be assumed that this gap between the qualitative and quantitative findings is a result of the previously described “pet-effect paradox” ,20,21. AWithin the group of animal caregivers without a COVID-19 infection, the data overall indicate that individuals seem to experience a better biopsychosocial wellbeing than animal caregivers suffering from Long-Covid. In this context, as previously stated, the results at a quantitative level demonstrate significant differences between the groups “Long-Covid” and “No Infection” regarding the biopsychosocial health. The only exception in this context is the feeling of loneliness, which does not differ between the two groups. This result might underline the possibility that individuals living with a companion animal experience them as a social source that is not influenced by an impairment of the caregiver’s health.Furthermore, the data within this group overall demonstrate that animal caregivers without an infection seem to be more concerned about others  than themselves. In this context, the results show significantly higher experienced worries regarding the pet and significantly lower pronounced concerns regarding the pandemic when compared with animal caregivers suffering from Long-Covid. Moreover, the data at a qualitative level indicate that the most pronounced worries during the pandemic are concerns regarding social division followed by concerns on the health of related persons, restrictions imposed on fundamental rights and freedoms, and other everyday problems in the context of the pandemic. Looking into the comparison with animal caregivers suffering from Long-Covid, these results might indicate that not being confronted with multiple stressors or acute stressors such as the Long-Covid and related health issues leads to an expansion of concerns away from the individual to the social environment.Regarding the human–animal relationship, no evidence was found that the bond with an animal influences animal caregivers’ wellbeing positively. No significant correlation was found between the strength of the human–animal relationship and any of the biopsychosocial parameters. Therefore, it might be assumed that the aspect of animal companionship alone is not linked to wellbeing during the pandemic when not confronted with an additional stressor. These results are consistent with previous findings that suggest there is no significant influence of pet ownership on the mental or physical health during the COVID-19 pandemic . In contOne limiting factor of the present study is that the sample was biased towards highly educated women as a result of survey recruitment strategies. A more diverse sample might generate results that were not apparent with this sample; however, we chose the random sample to prioritize the speed of response and to gather new information as the COVID-19 pandemic emerged and spread across Austria and Germany. Another critical aspect is that psychometric instruments such as the ones used in the present study might reach their limits due to the extraordinary situation, i.e., a global crisis. Therefore, mixed-methods approaches especially facilitate the generation of hypotheses. In connection with the used qualitative methodological approach, it must be stated that open questions could be widened as the question about “other” concerns might have limited individual answers. Additional interviews examining the reflexive self-perception in relation to the influence of animals during crises could provide more practical insights. This aspect has been considered for our future research. Besides these limitations, our present findings underline the need for further studies that examine the aspect of the contradiction between the inducted ideas regarding the benefits of animals and the actual measurable findings.Overall, the results of the present study indicate the relevant influence of stressors and related concerns on the biopsychosocial wellbeing of animal caregivers. The statistical results demonstrate that multiple external loads  lead to significantly lower levels of quality of life and higher levels of depression. Moreover, the findings suggest that animal companionship may cause an additional burden if someone is confronted with these stressors. Significant correlations were found between higher scores of the subjectively experienced strength of the human–animal relationship and lower levels of the biopsychosocial wellbeing as well as higher levels of psychological distress. Despite these data, the qualitative results, which include reflexive perceptions on the role of pets during the pandemic, paint a very different picture, with almost exclusively positive statements about the importance of the pet. Therefore, a discrepancy between the personal subjective view regarding the animal and the measured results has been identified once more, suggesting that the “pet-effect paradox” is particularly visible when animal caregivers are confronted with various external stressors.Additionally, the results of the present study indicate that the confrontation with existential and acute stressors such as Long-Covid leads to an increased focus on one’s own person and a reduction of the focus on the animal, which might affect the ability to care for an animal in a similar fashion to before the infection. This aspect should be considered in future research and especially in public media since, despite such critical results, there are still recommendations for all people to adopt animals because of their health promoting effects . The indAlthough some statistical results of the present study contradict the existing positive image of pet ownership, the subjective importance of animals for their caregivers cannot be ignored. We propose a closer analysis of the “pet-effect paradox” for a better understanding of how this phenomenon occurs.


In addition, the veterinarians were concerned about the potential for clients to misunderstand information found on the internet, or to develop unrealistic expectations of what is possible in small animal practices. As internet use becomes ever more widespread, we suggest that the types of resources that are available and used by animal owners should be further explored.Owners of dogs, cats, and other companion animals increasingly make use of the internet to find out how to best care for their animals. This may affect owners’ relations with veterinarians in both positive and negative ways. A positive consequence could be that owners are better informed when they approach a veterinarian. However, there can also be challenging situations in which the owners may question veterinarians’ professional advice based on online information. Using a questionnaire, we found that a majority of Austrian, Danish, and UK veterinarians (n = 641). The results show that 48.8% of respondents estimated that 40–79% of their clients use internet resources to find medical information. Further, 70–80% of respondents stated that they are occasionally challenged by clients questioning their advice based on online information. Although veterinarians recognized the potential advantages related to clients’ use of internet resources, such as an increased acceptance of advanced diagnostics and treatments, they also highlighted clients’ increased expectations or false impressions of small animal practices as potentially negative aspects in this context. As internet use increases, it seems likely that these issues will become increasingly important in the future.Veterinary medicine is increasingly affected by animal owners having the opportunity to become better informed on pet health issues by using various internet resources. Using an online questionnaire including a section on clients’ use of internet resources to obtain medical information, this study aimed to investigate veterinarians’ estimates of the percentage of clients using internet resources, how often clients question veterinarians’ professional medical advice based on online information, and veterinarians’ attitudes towards clients’ use of internet resources, across Austrian, Danish, and UK veterinarians ( Internet access has become much more widely available in the last decade. In 2007, 55% of European Union households had internet access; by 2020, this had increased to 91% [In relation to this, Kogan and colleagues surveyed UK pet owners and found that there had been an increase in owners’ use of internet resources to obtain medical information . SimilarWhile clients’ interests in seeking medical information can be viewed as a positive development potentially improving their knowledge and understanding of patient care, other studies have shown that veterinarians are also concerned about the potential negative effects ,4,5,6,7.In addition, veterinarians increasingly experience situations where clients challenge their medical advice based on information they have obtained from the internet ,5,8,9. NClients’ use of the internet to obtain medical information as well as information about specific veterinary services will undoubtedly increase ,6. PreviAlthough a general increase in the use of internet resources can be seen in all European Union households, national differences exist that might influence the use of the internet in the veterinary context. For instance, in 2020, in the UK and Denmark, 94% of the population used the internet on a daily basis ,11, in cFurther, it can be assumed that veterinarians’ attitudes will not only be influenced by socio-demographic and practice-specific factors such as age, employment status (self-employed versus employed), or the type of practice (independently owned versus corporate-owned), but potentially also by any previous negative experiences with clients who come to the practice with information they have obtained from the internet.Therefore, the main aim of this study was to answer the following research questions: (i) What percentage of their clients do veterinarians estimate use internet resources to find medical information prior to consultation? (ii) How often do clients question veterinarians’ professional medical advice based on information obtained from the internet? (iii) What are veterinarians’ attitudes towards their clients’ use of internet resources, and what influences these attitudes? Throughout, we highlight the main similarities and differences between veterinarians in the three countries.The present study forms a part of a larger body of work on different aspects in modern small animal practices including investigations of how veterinarians manage conflicting ethical concerns during decision-making processes , the optIn cooperation with small animal associations in Austria (VÖK), Denmark (DVA), and the UK (BSAVA), the link to the online-questionnaire was sent to 1195 Austrian, 1287 Danish, and 5138 UK veterinarians who worked (mainly) with small animals. For Denmark and Austria, data were collected from 2 March until 9 April 2020. Due to the COVID-19 pandemic outbreak and related organizational challenges, the BSAVA sent out the survey to its members at a later date and it was open between 30 March and 7 May 2020. Reminder e-mails were sent two weeks after opening the survey. The study received ethical approval from the Research Ethics Committee of SCIENCE and HEALTH at the University of Copenhagen (ReF: 504-0114/19-5000).n = 495) in Austria and 35.5% (n = 2812) in the UK. No specific data were available on the actual number of small animal veterinarians in Denmark, but as 90% are members of the DVA, the coverage error is likely around 10%.Since not all small animal veterinarians are members of the relevant veterinary associations in each country, this recruitment method introduces coverage error. Based on comparison with statistics on the study population, the assumed coverage error is 30%  continued on to answer at least one question . Of the Since relevant census data were not available for Denmark, non-response analyses were conducted for Austria and UK only to identify whether the samples deviated from the population of small animal veterinarians . For AusItems related to clients’ use of internet resources to obtain medical information were developed based on results of an Austrian focus group study  and a liIn total, the questionnaire consisted of three sections  and was The first section, A, included 14 closed-ended questions on socio-demographic and practice-specific aspects. In the second section, B, there were three questions including one matrix with six statements related to clients’ use of internet resources. The first question asked veterinarians to estimate how many of their clients use internet resources to find medical information prior to a consultation. Answer options ranged from “None”, over “1–19%”, “20–39%”, and so on up to “80–100%”. The answer option “I don’t know” was also available. Further, six statements were presented to explore veterinarians’ attitudes towards their clients’ use of internet resources by focusing on possible positive  and negative effects . Respondents could indicate their level of agreement with each statement through a 7-point Likert scale : 1 “StroIn addition, section B included a question about several factors that could influence clients’ desire to further pursue therapy beyond veterinarians’ professional recommendations, including the factor that “Medical information obtained using internet resources” influences clients’ desire to pursue further therapy beyond veterinarians’ recommendation. Possible answer options were “Not at all”, “Slightly”, “Moderately”, “Relatively Strong”, and “Strong”. The answer option “I don’t know” was also provided.® . IBM® SPSS® Statistics version 27.0  was used for all analyses. Univariate descriptive statistics were presented in tables, figures, or text. For bivariate analysis, Chi-square tests or Kruskal–Wallis H tests were conducted to test whether the frequency distribution differed between the Austrian, Danish, and UK sub-populations. The significance level was 0.05.All three online surveys were set up using the survey software AlchemerOrdinal regression analyses were conducted to identify the extent to which socio-demographic and practice-specific factors, clients’ use of internet resources, percentage of clients who question veterinarians’ professional advice, and the impact of internet resources on clients’ desire to pursue treatment had an effect on veterinarians’ attitudes towards clients’ use of internet resources . For eacp = 0.301).p < 0.001). Significantly more Austrian veterinarians indicated that they were ‘never’ confronted with clients who questioned their advice compared to Danish (χ2 (1) = 12.487; p < 0.001) and UK (χ2 (1) = 23.925; p < 0.001) veterinarians. In comparison, significantly more veterinarians from Denmark and the UK indicated that they were ‘frequently’ confronted with such clients, in contrast to their Austrian colleagues (DK: χ 2 (1) = 28.533, p < 0.001; UK: χ2 (1) = 27.091, p < 0.001).In all three countries, between 70–78% of the respondents indicated that their clients occasionally questioned their professional advice based on information they had obtained from the internet . SignifiSix statements were presented exploring veterinarians’ attitudes towards clients’ use of internet resources to obtain medical information . In genep = 0.007; p = 0.047) and UK  colleagues. The opinion that clients’ use of internet resources “improves discussion about diagnostic and treatment options as clients have a greater knowledge” obtained less agreement from Austrian veterinarians compared to their colleagues from Denmark (p = 0.001) and the UK (p = 0.017). Further, UK veterinarians more frequently agreed with the statement that the use of internet resources “causes clients to form strong opinions so that veterinarians have to justify their diagnostics and therapeutic steps” than Austrian (p = 0.002) and Danish (p < 0.001) veterinarians.However, significant differences were identified between the countries. Danish respondents significantly more frequently agreed that clients’ use of internet resources led to a “greater acceptance of advanced diagnostic and treatments” and less frequently agreed that it “creates the wrong impression of modern small animal practice” compared to their Austrian  = 11.009, p < 0.001) or only ‘occasionally’ (Austria: χ2 (1) = 8.578, p = 0.003; UK: χ2 (1) = 6.940, p = 0.008) questioned by clients.In Austria and the UK, veterinarians who indicated that their professional advice was ‘frequently/always’ questioned by clients were less likely to agree with the statement, compared to veterinarians who indicated that they were ‘never’ (Austria: χ2 (1) = 12.210, p < 0.001). In addition, the veterinarians who estimated that ‘none–19%’ of their clients make use of internet resources were less likely to agree with the statement compared to those veterinarians who estimated that over 60% of their clients use internet resources (UK: χ2 (1) = 4.679, p = 0.031).In the UK, the likelihood of agreeing with this statement increased with age (UK: χ2 (1) = 5.411, p = 0.020).For Denmark, male veterinarians were more likely to agree with the statement than their female colleagues (DK: χ2 (1) = 4.684, p = 0.030; UK: χ2 (1) = 5.870, p = 0.015).For the UK and Denmark, the likelihood of agreeing with this statement increased with age (DK: χ2 (1) = 21.159, p < 0.001). In addition, UK veterinarians who stated that their professional advice is ‘never’ (UK: χ2 (1) = 12.945, p < 0.001) or only ‘occasionally’ (UK: χ2 (1) = 12.525, p < 0.001) questioned by clients were less likely to agree with the statement, compared to their colleagues who are ‘frequently/always’ questioned by clients.Further, UK veterinarians who agreed more strongly that internet resources impact clients’ desire to pursue treatment were more likely to agree that clients have greater expectations of advanced diagnostics and treatments (UK: χ2 (1) = 4.9730, p = 0.026). In addition, the likelihood of agreeing with the statement increased with age (χ2 (1) = 16.835, p < 0.001).For the UK, female professionals were more likely to agree with this statement than their male colleagues (χ2 (1) = 4.003, p = 0.045; DK: χ2 (1) = 5.7111, p = 0.017; UK: χ2 (1) = 7.828, p = 0.005). Further, Austrian, Danish, and UK veterinarians who indicated that their professional advice is ‘frequently/always’ questioned by clients were more likely to agree with the statement compared to colleagues who indicated that clients ‘never’ questioned their advice (Austria: χ2 (1) = 5.251, p = 0.022; Denmark: χ2 (1) = 13.798, p < 0.001; UK: χ2 (1) = 13.886, p < 0.001).In all three countries, the likelihood of agreeing with the statement decreased with an increasing age (Austria: χ2 (1) = 4.996, p = 0.025; UK: χ2 (1) = 8.228, p = 0.004).Danish and UK veterinarians who stated that their professional advice was ‘occasionally’ questioned by clients were less likely to agree with the statement compared to veterinarians who indicated that this happened ‘frequently/always’ (Denmark: χ2 (1) = 4.561, p = 0.033) or ‘20–59%’ (χ2 (1) = 7.376, p = 0.007). Further, in Denmark and the UK, the veterinarians who indicated that the use of internet resources had a strong impact on clients’ desire to pursue treatment were more likely to agree with this statement (Denmark: χ2 (1) = 11.551, p < 0.001; UK: χ2 (1) = 4.216, p = 0.040).In addition, Austrian veterinarians who indicated that over 60% of their clients used internet resources were more likely to agree with this statement compared to those who estimated the level to be ‘none–19%’ (χ2 (1) = 6.068, p = 0.014). Additionally, veterinarians who indicated that over 60% of their clients use internet resources were more likely to agree with this statement than those veterinarians who indicated that ‘none–19%’ of their clients use them (χ2 (1) = 6.803, p = 0.009). In Austria, female veterinarians were more likely to agree with this statement than their male colleagues (χ2 (1) = 17.682, p < 0.001) or only ‘occasionally’ questioned (χ2 (1) = 4.001, p = 0.045) were less likely to agree with this statement compared to their colleagues who were ‘frequently/always’ questioned by clients. Further, UK veterinarians who indicated that internet resources had a strong impact on clients’ desire to pursue treatment were more likely to agree with this statement (χ2 (1) = 6.080, p = 0.014).In the UK, veterinarians who indicated that their professional advice was ‘never’  and self-employed veterinarians and the lower proportion of veterinarians with 0.5–5 years of work experience compared to Denmark and UK could have influenced the differences we identified. Further, of the 21 veterinarians who were categorized as “Other” in relation to business type, only three indicated that they were retired (one from each country). Due to the small number and mostly short duration of retirement , we did not consider this aspect in our analyses in the context of their ability to recall information relating to their clients and clients’ use of internet resources.Second, to generate an overview of the different aspects that are of relevance in relation to clients’ use of internet resources, we formulated six statements to identify veterinarians’ attitudes toward this issue based on the results of an Austrian focus group study  as well Third, the use of answer options such as “occasionally” or “frequently” is more subjective compared to, for example, a specific percentage. Thus, the respondents may have interpreted these terms differently, introducing variability into the reported occurrence rates.Fourth, veterinarians were asked how many of their clients they thought used internet resources prior to a consultation, which may not reflect the actual number who do so, as not all clients will explicitly share that information with their veterinarians. Future work should collect that information directly from clients for a comparison.Fifth, when we asked veterinarians about their perceptions of clients’ acceptance or otherwise of ‘advanced’ diagnostics and treatments, we did not provide a detailed description of the term “advanced”. Although a generally understood and commonly used term within the profession, it may have been interpreted in different ways by the respondents, perhaps depending on their working background and/or level of specialization.Although our results indicate how often veterinarians are challenged by clients questioning their professional advice based on information obtained from the internet, we did not specify the context, e.g., clients questioning veterinarians on diagnostic or treatment recommendations, costs, vaccination policies, behavioral issues, etc. Further, we did not ask how often veterinarians showed interest in what kinds of information clients have found on the internet. Hence, we recommend that these aspects should be explored in future studies.We conclude that veterinarians generally recognize the advantages of clients’ use of internet resources to become better informed, for example, with respect to making them potentially more accepting of advanced diagnostics and treatments in patient care. However, it can also have a negative impact if clients misunderstand the information or develop unrealistic expectations of what is possible in most small animal practices. As internet use increases, and the available information continues to rapidly expand, such concerns will play an increasing role in modern veterinary practices. Thus, further research is recommended to understand both the attitudes of clients towards internet resources and the types of resources most frequently used to search for veterinary medical information.


O-glucoside and cyanidin-3-O-glucoside in the red fruit of ‘Mixue’ were significantly decreased compared with ‘Sachinoka’. In comparison with ‘Sachinoka’, the expression levels of FaMYB10, FaMYB11.2, FaWD40 and FaTT19 in the turning fruit of ‘Mixue’ were significantly down-regulated, while the expression of FaMYB1 was significantly up-regulated in the red fruit. ‘Sachinoka’ and ‘Mixue’ fruits were found to have 110 volatile components. Among them, 15 volatile components in the red fruit of ‘Mixue’ were significantly increased compared with ‘Sachinoka’, such as nerolidol, benzaldehyde, ethyl hexanoate, ethyl isovalerate, which led to an enhanced aroma in ‘Mixue’ and might result from the up-regulated expression of FaNES1, FaCNL and FaAATs in ‘Mixue’. These results provide useful information on the effect of somaclonal variation on metabolic components of strawberry fruit and lay the foundation for the improvement in quality of strawberry.Plant tissue culture produces a wide range of genetic variations which are useful for quality improvement of the plant species. However, the differences in metabolic components and the key genes responsible for the difference in metabolic components between somaclonal variation and the original parent are still largely unknown. In this study, a mutant named ‘Mixue’ was identified with somaclonal variation of the ‘Sachinoka’ strawberry. The contents of pelargonidin-3- Fragaria × ananassa Duch.) belongs to the Fragaria genus of the Rosaceae family, which is an important economic crop and is widely consumed throughout the world. Antioxidants, vitamins, dietary fibers, and pleasant volatile components are abundant in strawberry. Strawberry also has the potential benefits to human health, such as protective effects in inflammation, neurodegenerative diseases and coronary heart diseases, ageing, cancer, and obesity [Strawberry  and cyanidin-3-O-glucoside (Cy3G) were remarkably increased in the red fruit of ‘Sachinoka’ compared to its somaclonal mutant ‘Mixue’ , acids (9), ethyl esters (10), acetate esters (8), other esters (8), benzene and volatile phenols (15), aldehydes and ketones (23), isoprenoids (17) and furans (3) . Some 70In the red fruit, 15 volatile components were significantly increased in somaclonal mutant ‘Mixue’ with at least 1.5-fold change relative to ‘Sachinoka’, including two alcohols, three ethyl esters, one acetate, on other ester, five aldehydes and ketones, and three isoprenoids . InteresWe analyzed the content of nine categories of volatile components in the fruits of ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ at different developmental stages. In the green fruit stage, the contents of aldehydes and ketones in the somaclonal mutant ‘Mixue’ fruits were significantly higher than those in ‘Sachinoka’. The total contents of 2-hexenal and hexanal in ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ at the green fruit stage accounted for 88.66% and 94.59% of the aldehydes and ketones, respectively. The total contents of 2-hexenal and hexanal in the somaclonal mutant ‘Mixue’ were 4.37 times higher than those in ‘Sachinoka’. In the white fruit stage, the content of acetate ester was increased by 84.79% in ‘Sachinoka’ compared with the green fruit stage and was markedly higher than that in somaclonal mutant ‘Mixue’ . In addiNext, we analyzed the trend of the contents of the nine categories of volatile components in the fruit of the ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ at four developmental stages. In the first three stages of fruit development , the trend of the contents of nine categories of volatile components between ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ was similar . The conVolatile components in strawberry fruit are usually distinguished by the odor activity value  OAV: ra. There wThe characteristic volatile components with higher OAVs in ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ during the red fruit stage were ethyl hexanoate, ethyl butyrate and linalool . The OAVTo further compare the differences in volatile components between ‘Sachinoka’ and its somaclonal mutant ‘Mixue’, a comparative analysis of the unique volatile components in the fruits of ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ at four developmental stages was performed. Nine unique volatile components were identified in ‘Sachinoka’. Among them, seven unique volatile components in the green fruit, one unique volatile component in the white fruit and one unique volatile component in the red fruit were identified, respectively . Eleven To identify volatile component differences between ‘Sachinoka’ and its somaclonal mutant ‘Mixue’, principal component analysis (PCA) based on the contents of nine categories of volatile components was conducted. The first principal component (PC1) and the second principal component (PC2) accounted for 80.50% and 11.28% of the total variance, respectively . PC1 andFaPAL1, FaCHS1, FaCHS2, FaCHI, FaDFR, FaF3H and FaANS had similar expression patterns in ‘Sachinoka’ and its somaclonal mutant ‘Mixue’. The expression levels of these seven structural genes were significantly increased in turning and red fruits in ‘Sachinoka’ and its somaclonal mutant ‘Mixue’, whilst the expression levels of these seven structural genes in turning and red fruits of somaclonal mutant ‘Mixue’ were significantly lower than those in ‘Sachinoka’. In addition, the expression levels of FaTT19, FaMYB10, FaMYB11.2 and FaWD40 in ‘Sachinoka’ at the turning stage were 6.73, 3.02, 2.48, and 2.52 times higher than those in somaclonal mutant ‘Mixue’, respectively  pathway or the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MVA pathway operates in the cytoplasm and the MEP pathway functions in the plastid. The volatile monoterpenes (C10) and sesquiterpenes (C15) are identified in most fleshy fruits . TherefoFaNES1 is one of the crucial genes involved in sesquiterpenoids biosynthesis [FaNES1 was found in the turning stage of ‘Sachinoka’ fruit, while the highest expression of FaNES1 was found in the red stage of the somaclonal mutant ‘Mixue’, which was in accordance with the nerolidol having a 1.69 times higher content in the red fruit stage of the somaclonal mutant ‘Mixue’ than that in ‘Sachinoka’ , 1 or 8 nucleotide insertion, long terminal repeats of transposable element (LTR-TE) insertion [MYB10 between ‘Sachinoka’ and its somaclonal mutant ‘Mixue’. We found the 35th nucleotide in the first exon of the somaclonal mutant ‘Mixue’ was identical to ‘Sachinoka’ and the MYB10 amino acids in the somaclonal mutant ‘Mixue’ did not result in a frameshift and a premature stop codon . Sun et al. found that tt19-7 mutants hardly accumulate anthocyanins [RAP gene (Reduced Anthocyanins in Petioles) in octoploid strawberry using CRISPR/Cas9, resulting in a white-fruited phenotype [FaMYB11.2 and FaTT19 during the turning and red stages in the somaclonal mutant ‘Mixue’, may lead to a reduction in anthocyanin or proanthocyanidin accumulation.The Somaclonal mutant ‘Mixue’ with white flesh in the red fruit stage was a somaclonal variation of ‘Sachinoka’ strawberry. The white flesh of the red fruit stage of somaclonal mutant ‘Mixue’ resulted from the significantly reduced accumulation of Cy3G and Pg3G in the somaclonal mutant ‘Mixue’ B. The exchinoka’ B. MYB10  change GC, 1 or 8nsertion . Thus, wop codon . These r change GC, 1 or 8ic genes . In thiss 99.26% . These r change GC, 1 or 8 FaMYB10 ,37. DNA  in pear ,31,38. Frawberry . We foun ‘Mixue’ . Gene exchinoka’ B. The prs 99.55% . FaWD40 chinoka’ B. FaMYB9wn seeds . Arabidoocyanins . Gao et henotype . TherefoFaMYB10, FaWD40, FaMYB11.2, FaTT19 and FaMYB1 in ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ were correlated with the white flesh of the somaclonal mutant ‘Mixue’ fruit. Further study will be needed to determine whether methylation or large transposon, retrotransposon insertion exist in the promoter of these key factors.These results have demonstrated that the differential expression levels of FaAAT in the somaclonal mutant ‘Mixue’ at the red fruit stage compared with ‘Sachinoka’  Nechinoka’ . Althougchinoka’ . FaNES1 rawberry . Increaschinoka’ B. (3) Inchinoka’ B. In addchinoka’ , which mng stage B. (4) Fuynthesis . FaOMT e to DMMF . HDMF ca to DMMF . In thisectively B. Howevechinoka’ B, which FaMYB10, FaMYB11.2, FaWD40, FaMYB1, and FaTT19. There were significant increases in the characteristic strawberry volatile components in the red fruit of ‘Mixue’, such as nerolidol, benzaldehyde, ethyl hexanoate, ethyl isovalerate, which led to enhanced volatiles in the somaclonal mutant ‘Mixue’ and might result from the up-regulated expression of FaNES1, FaCNL and FaAATs in ‘Mixue’ compared with ‘Sachinoka’. In addition, the significant up-regulation expression of FaQR, FaOMT, FaGT2 and FaGT7 in the somaclonal mutant ‘Mixue’ during the turning and red fruit stages may result in accumulating more furanones in the somaclonal mutant ‘Mixue’  cultivars ‘Sachinoka’ and ‘Mixue’. ‘Mixue’ is a somaclonal mutant produced from the tissue culture of apices of runner tips from ‘Sachinoka’. The somaclonal mutant ‘Mixue’ was observed in the field for 3 years and its phenotype was stable. ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ were grown in the greenhouse of Shenyang Agricultural University, China. For different developmental stages of ‘Sachinoka’ and its somaclonal mutant, fruit at 8 days (green stage), 16 days (white stage), 24 days (turning stage) and 32 days (ripening stage) after pollination were collected for HPLC, HS-SPME-GC-MS and RT-qPCR analysis, respectively.The materials used in this study were cultivated strawberry  hydrochloric acid and extracted at 4°C for 48 h under dark condition. Centrifugated the extracts at 13,000 rpm for 20 min and collected the supernatant. The residues were re-soaked with 2 mL methanol containing 1% hydrochloric acid and the supernatant collected. Then, hydrochloric acid was added to 5 mL.O-glucoside (Cy3G) and pelargonidin-3-O-glucoside (Pg3G) were purchased from Yuanye Bio-Technology Co., Ltd. . These standards were dissolved in methanol to 100 μg/mL. The chromatographic column was Intertsil ODS-35 UM  and Cy3G and Pg3G were detected at 520 nm. The column temperature was 30°C. The mobile phase A was formic acid dissolved in ultrapure water  and mobile phase B was 10% formic acid dissolved in 90% acetonitrile (v/v). The injection volume was 10 μL. The flow rate was set at 1 mL/min and the running time was 75 min. Linear gradient elution was used  . Cyanidin-3-was used . Composihttps://webbook.nist.gov/chemistry/ (accessed on 14 December 2022).The extraction of volatile components from fruit samples was performed following methods previously described un . WeighedTake 4-methyl-2-pentanol as the internal standard  to quantitatively analyze the determined compounds. Two synthetic substrates containing 1% and 11% (v/v) ethanol were prepared in distilled water . Volatile standard solution was dissolved in a 1% and 11% synthetic matrix, and the concentration was usually the concentration of strawberry-clarified juice, respectively. Then the volatile standards were extracted and analyzed under the same conditions as strawberry-clarified juice samples. The quantitative data of volatile components were calculated by the following formula:2O. The first step of thermal cycling denaturation was 95 °C for 10 min, followed by 40 cycles for denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. The relative mRNA levels were calculated using the 2−ΔΔCt method. All data were normalized with the expression level of the 26S and then normalized with the expression of ‘Sachinoka’. Each sample was analyzed in triplicate with three biological replicates.The four developmental stage fruits  of ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ fruits were harvested and immediately frozen in liquid nitrogen. At each stage of fruit development, twenty fruits from twenty plants were mixed and ground to fine powder, respectively. Total RNA from four developmental stage fruits of ‘Sachinoka’ and its somaclonal mutant ‘Mixue’ was extracted using the CTAB method. Then, cDNA was synthesized based on PrimeScript TM RT reagent Kit . Quantitative real-time RT-PCR (RT-qPCR) was conducted using UltraSYBR Mixture  with quantitative primers . RT-qPCRt-test was used to evaluate the significant difference between the two treatments in the nine categories of volatile components . A one-way analysis of variance (ANOVA) was used to analyze the content of volatile substances at different developmental stages employing Duncan’s multiple range tests at a level of p < 0.05. The volatile components and OAVs were presented as the mean ± SD of triplicate measurements. Origin 2021  was used to visualize the data.SPSS 18.0 was applied for statistical analysis . An independent FaMYB10, FaMYB11.2, FaWD40 and FaTT19 and higher expression of FaMYB1 genes. Fifteen volatile components in the ripening fruit of ‘Mixue’ were significantly increased compared with ‘Sachinoka’, such as nerolidol, benzaldehyde, ethyl hexanoate, ethyl isovalerate, which led to an enhanced aroma in ‘Mixue’. The enhanced aroma in ‘Mixue’ was related to the up-regulated expression of FaNES1, FaCNL and FaAATs. These results provide useful information on the effect of somaclonal variation on metabolic components of strawberry fruit and lay the foundation for the improvement in quality of strawberry.The white flesh in the ripening fruit of ‘Mixue’ was due to the lower expression of