This study investigated the effects of school climate and students’ aesthetic experience on their professional identity and innovative behavior. A survey was conducted with 385 students from hospitality-related departments of colleges and universities in Hainan, China, and the data were analyzed using a hierarchical linear model (HLM). Using the criteria constituting the students’ aesthetic experience scale proposed by Chang, it was found that teacher support can improve students’ professional identity; school climate and students’ understanding of beauty and full experience contribute to the development of students’ innovative behavior; students’ understanding of beauty and full experience have mediating effects between teacher support and professional cognition; students’ understanding of beauty and full experience have mediating effects between student support and innovative behavior; student support positively moderates the relationships between full experience with professional cognition and students’ appraisal of the hospitality industry; and teacher support positively moderates the relationship between students’ full experience and professional emotion. Therefore, teacher support under school climate and students’ understanding of beauty and full experience under aesthetic experience were the most important factors in enhancing hospitality department students’ professional identity and innovative behavior. The COVID-19 pandemic severely impacted employment in the tourism and hospitality industries, with an estimated 100–120 million people worldwide becoming unemployed at the start of the pandemic . AccordiA core trend in the hospitality industry is the application and development of innovations that provide a strong impetus for this industry’s development . DiversiThe conceptual development of professional identity is an inherent aspect of professionalism . The devHospitality and tourism department students’ innovative behaviors are nurtured in school , which iHowever, as students are nested within classes or schools, appropriate analytical methods such as multilevel modeling and hierarchical linear modeling (HLM) must be used; otherwise, the data will be confounded by hierarchical relationships and lead to misleading findings . TherefoProfessional identity can be theorized in terms of emotions, attitudes, and values . The devAccording to The school climate includes social processes such as teacher support, student support, and opportunities for autonomy in the classroom . TeacherSchools provide the foundation for stimulating and nurturing students’ aptitude for knowledge and innovation, and help them meet future employment challenges . IndividThis study aimed to investigate the effects of school climate and students’ aesthetic experiences on professional identity and innovative behavior. We used SIT as the theoretical basis and HLM to conduct analysis. After the literature review, we proposed the research framework as follows .The tourism industry in Hainan, China, has been developing rapidly in recent years—particularly the hospitality industry, which requires a large number of human resources, and hospitality department students are the main force of this industry. It is important that students be able to enter the hospitality industry smoothly and sustain their careers in the industry.This study selected students from hospitality-related departments of colleges and universities in Hainan, China, as the sample. The survey was conducted with the consent of teachers, who were asked to explain the purpose and procedure of the study to students willing to take the survey; further, teachers informed the participants that the survey was anonymous and voluntary. The survey was conducted in 25 classes of hospitality-related departments in 15 colleges and universities, with 15–20 students per class. A total of 426 students were surveyed. After removing the invalid questionnaires, there were 25 classes with 385 valid questionnaires.2/df = 5.057, root mean square error of approximation (RMSEA) = 0.103, goodness fit index (GFI) = 0.837, adjusted goodness of fit index (AGFI) = 0.789, parsimony goodness of fit index (PGFI) = 0.646, normed fit index (NFI) = 0.896, incremental fit index (IFI) = 0.915, comparative fit index (CFI) = 0.915, parsimony normed fit index (PNFI) = 0.773, and parsimony comparative fit index (PCFI) = 0.789; thus, most of these values meet the criteria  critical values ranging from 50.603 to 87.22, all reaching the significance level of 0.05. Thus, it was suitable for multilevel analysis. Before conducting HLM analysis, it was necessary to detect the presence of intergroup variation  so as to confirm the need for multilevel analysis . The anaariation  in the dTo understand whether students’ aesthetic experience has a direct effect on professional identity and innovative behavior respectively, we conducted analysis using the following model. To keep the paper reasonably concise, we took only professional cognition as an example to elaborate. The effects of the rest of the dimensions on innovative behavior were analyzed in the same way.ij = βj0 + βj1 × (pleasure of beautyij) + βj2 × (aesthetic attitudeij) + βj3 × (understanding of beautyij) + βj4 × (full experienceij) + rijLevel-1: professional cognitionj0 = γ00 + uj0Level-2: β40 = 0.501, p = 0.000). In the professional appraisal section, understanding of beauty and full experience reached a significant level ; in the professional emotion section, full experience reached a significant level . In the innovative behavior section, understanding of beauty and full experience reached a significant level . As for the explained variation (R2) for aesthetic experience, at the individual level, it was calculated that in level-1, the R2 values of professional cognition, professional appraisal, professional emotion, and innovative behavior were 41.39, 36.54, 38.74, and 52.28%, respectively.According to the analysis results , we conducted analysis using the following model.ij = β0j + β1j × (pleasure of beautyij) + β2j × (aesthetic attitudeij) + βj3 × (understanding of beautyij) + βj4 × (full experienceij) + rijLevel-1: professional cognitionj0 = γ00 + γ01 × (teacher supportj) + γ02 × (opportunities for autonomyj) + γ03 × (student supportj) + uj0Level-2: β01 = 0.758, p = 0.000); and in the professional appraisal section, teacher support reached the significant level ; and in the professional emotion section, teacher support reached the significant level . In the innovative behavior section, teacher support, opportunities for autonomy, and student support all reached the significant level . From the above, it is clear that in all cases, teacher support has a direct effect on professional identity, especially innovative behavior.According to the analysis results ; further, γ01 and γ03 of innovative behavior reached a significant level . As shown in 01 and γ03 reached a significant level ; in the aesthetic attitude section, γ01 and γ03 reached a significant level ; in the understanding of beauty section, γ01 and γ03 reached a significant level ; and finally, in the full experience section, γ03 reached a significant level .According to the analysis results (as shown in c′01 (Z→Y) in Equation 3a. The model was as follows:The second step was to further put both the higher-level explanatory variables and the mediating variables into the equation to test the explanatory power of school climate and aesthetic experience on professional identity and innovative behavior. Our focus was on the significance test of γij = βbj0 + βbj1 × (pleasure of beautyij) + βbj2 × (aesthetic attitudeij) + βbj3 × (understanding of beautyij) + βbj4 × (full experienceij) + rbijProfessional cognitionc′01 of teacher support decreased to 0.758 and also reached a significant level , indicating a partial mediating effect. Among the mediated variables, only understanding of beauty and full experience reached a significant level . Thus, understanding of beauty and full experience had partial mediating effects between teacher support and professional cognition. In the professional appraisal section, γc′01 of teacher support increased to 1.023 and also reached a significant level ; thus, it has no mediating effect. For the professional emotion section, γc′01 of teacher support decreased to 1.024 and reached a significant level ; thus, it has a partially mediating effect. Among the mediating variables, only full experience reached a significant level . Thus, full experience has a partial mediating effect. In the innovative behavior section, γc′01 of teacher support increased to 0.335 and also reached a significant level ; thus, it has no mediating effect. γc′03 of student support decreased to 0.24 and reached a significant level ; thus, it has a partial mediating effect. Among the mediating variables, understanding of beauty and full experience reached a significant level ; thus understanding of beauty and full experience have partial mediating effects.After adding mediating variables, the results were obtained from the intercept prediction model, as shown in in To verify whether the presence of the slope term could be explained by the level-2 variable , we conducted analysis using the following model.ij = β0j + β1j × (pleasure of beautyij) + β2j × (aesthetic attitudeij) + β3j × (understanding of beautyij) + β4j × (full experienceij) + rijLevel-1: professional cognition0j = γ00 + γ01 × (teacher supportj) + γ02 × (opportunities for autonomyj) + γ03 × (student supportj) + u0jLevel-2: β1j = γ10 + γ11 × (teacher supportj) + γ12 × (opportunities for autonomyj) + γ13 × (student supportj) + u1jβ2j = γ20 + γ21 × (teacher supportj) + γ22 × (opportunities for autonomyj) + γ23 × (student supportj) + u2jβ3j = γ30 + γ31 × (teacher supportj) + γ32 × (opportunities for autonomyj) + γ33 × (student supportj) + u3jβ4j = γ40 + γ41 × (teacher supportj) + γ42 × (opportunities for autonomyj) + γ43 × (student supportj) + u4jβ21 = −1.457, SE = 0.55, p = 0.016). This indicates that teacher support in school climate has a negative moderating effect on the relationship between aesthetic attitude and professional cognition at the individual level. The interaction coefficient between full experience and student support was significant . This indicates that student support has a positive moderating effect on the relationship between full experience and professional cognition. In the professional appraisal section, the interaction coefficient between full experience and student support was significant . This indicates that student support has a positive moderating effect on the relationship between full experience and professional appraisal. In the professional emotion section, the interaction between full experience and teacher support is significant . This indicates that teacher support positively moderates the relationship between full experience and professional emotion; that is, teacher support reinforces the relationship between full experience and professional emotion. The interaction coefficient between full experience and opportunities for autonomy is significant . This indicates that the variable opportunities for autonomy has a negative moderating effect between full experience and professional emotion; that is, opportunities for autonomy at the organizational level would weaken the relationship between full experience and professional emotion. In the innovative behavior section, there was no interaction of school climate with aesthetic experience and innovative behavior.As shown in The study’s results show that full experience in students’ aesthetic experience helps improve students’ professional identity, professional cognition, professional appraisal, and professional emotion. In other words, if students remember good things related to the past while engaging in creation and discuss good experiences with others, they would have a higher sense of professional identity, better understand their profession, and be more compatible with and enjoy their profession. Students can gain a stronger sense of professional identity through experience, reflection of experience, and their own disciplines . StudentStudents’ understanding of beauty and full experience as part of aesthetic experience can enhance their innovation behaviors. Students can see the special and subtle aspects of beauty, express concepts and reasons for beauty, recall relevant beauty associated with creation, and share and discuss such experiences with others. Students will absorb, internalize, and transform these related aesthetics into personal thinking and experiences that can enhance intrinsic motivation and self-confidence and boost thinking, exploring the occurrence of contemplative, explorative, creative, and innovative behaviors . TherefoTeacher support as part of school climate can enhance students’ professional identity. Teachers’ concern, trust in students, and help provided to students to solve problems make students feel a sense of belonging, thereby enhancing their professional identity . School Students’ understanding of beauty and full experience have partially mediating effects on teacher support and professional cognition. Teacher support can enhance students’ professional cognition through their understanding of beauty and full experience. This means that teacher support can enable students to have a deeper understanding of beauty, share beautiful experiences with others, discuss with others about careers for gaining a deeper understanding, and evaluate and increase their knowledge of the hospitality profession.Students’ understanding of beauty and full experience partially mediates the relationship between student support and innovative behavior. Student support can lead to innovative behavior through students’ understanding of beauty and full experience. This means that mutual trust, liking, respect, and help among students at school make it easier for them to understand the styles, concepts, and special features of beauty, to discuss beauty with others, and perceive the beauty associated with creation—all of which contribute to their innovative behaviors.Teacher support has a negative moderating effect between students’ aesthetic attitude and professional cognition. However, teacher support can enhance students’ professional cognition through students’ understanding of beauty and full experience, while students’ aesthetic attitude has no effect on professional cognition. This may be because when teacher support is stronger, the relationship between students’ aesthetic attitude and professional cognition is weakened, thus causing a negative moderating situation. This also reveals the importance of teacher support for hospitality department students’ professional cognition.Student support has a positive moderating effect on the relationships between students’ full experience with professional cognition and professional appraisal. In other words, when students can help, trust and respect each other, as well as discuss and share good experiences with others, students’ professional cognition and professional appraisal of the hospitality industry would be higher. Teacher support has a positive moderating effect on students’ full experience and professional emotion. This indicates that teachers can help students solve problems, care about them, and believe in them, and students would recall good things about their teachers and share their good experiences with others. Thus, students evaluate the hospitality profession positively and are more willing to engage in hospitality-related work.The variable opportunities for autonomy in school climate has a negative moderating effect on full experience and professional emotion, while full experience has a positive effect on professional emotion. The interaction between opportunities for autonomy and students’ full experience has a negative effect on students’ professional emotion. In other words, when students can decide rules in the classroom and discuss and share their good experiences with others, it decreases their satisfaction and evaluation of the hospitality profession. This may be because hospitality department students have fewer opportunities for autonomy, and opportunities for autonomy has no direct effect on full experience and professional emotion. Therefore, we should pay attention to the moderation of opportunities for autonomy in school climate.It was found that teacher support in school climate and full experience in students’ aesthetic experience are important for hospitality department students’ professional identity and can enhance students’ professional identity, which includes professional cognition, professional appraisal, and professional emotion. Students’ understanding of beauty can also enhance their professional appraisal. School climate can enhance students’ innovative behavior. Students’ understanding of beauty and full experience in aesthetic experience can also enhance students’ innovative behavior.Furthermore, teacher support can enhance students’ professional cognition and professional emotion through students’ full experience. Teacher support can also enhance students’ professional cognition through students’ own understanding of beauty. Student support can enhance innovative behavior through students’ understanding of beauty and full experience. Therefore, understanding of beauty and full experience in students’ aesthetic experience are important mediating variables for hospitality department students’ professional identity and innovative behavior.With regard to the moderating effect of school climate, teacher support negatively moderates the relationship between students’ aesthetic attitude and professional cognition. Student support positively moderates the relationships between students’ full experience with professional cognition and professional appraisal. Teacher support positively moderates the relationship between students’ full experience and professional emotion. Opportunities for autonomy negatively moderates the relationship between students’ full experience and professional emotion. Therefore, teacher support and student support in school climate have an important moderating effect on hospitality department students’ professional identity.In conclusion, for hospitality students, teacher support and student support in school climate and understanding of beauty and full experience in students’ aesthetic experience are crucial for enhancing students’ professional identities and innovative behaviors. Hospitality-related departments in colleges and universities should prioritize teacher support and student support in addition to students’ understanding of beauty and full experience to enhance students’ professional identity and innovative behavior. This will aid students to enter the hospitality industry swiftly after graduation and sustain their careers in the industry. Therefore, it is recommended that hospitality-related departments in colleges and universities should prioritize teacher support and student support and incorporate aesthetic-related courses to give students an understanding and full experience of beauty. The purpose is to improve students’ identification with the hospitality profession, including professional cognition, professional appraisal, and professional emotion, and enhance their innovative behaviors. Accordingly, they can enter the hospitality industry swiftly after graduation and attain sustainable career development in the industry.With respect to research recommendations, this study investigates the effect of school climate on the relation between hospitality students’ professional identities and innovative behaviors among students at hospitality-related departments in colleges and universities during the COVID-19 pandemic in 2022, without directly examining the effect of COVID-19. Accordingly, follow-up studies can conduct investigations into the effect of COVID-19 on hospitality students to facilitate an understanding of the effects of COVID-19 on them.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.WL was responsible for suggesting revision to the concept and writing style of the manuscript. Y-CC was responsible for the conceptualization, investigation, methodology, and writing analyzing data for this manuscript. Both authors have read and agreed to the published version of the manuscript.


Three novel two-dimensional metalloporphyrin COFs  were fabricated by changing the metal atoms in the center of the porphyrin framework. The physicochemical characteristics of MPor−COF−366  composites were fully analyzed by diverse electron microscopy and spectroscopy. Under optimal conditions, experiments on determining butylated hydroxy anisole (BHA) at FePor−COF−366/GCE were conducted using differential pulse voltammetry (DPV). It is noted that the FePor−COF−366/GCE sensor showed excellent electrocatalytic performance in the electrochemical detection of BHA, compared with MnPor−COF−366/GCE and CuPor−COF−366/GCE. A linear relationship was obtained for 0.04–1000 μM concentration of BHA, with a low detection limit of 0.015 μM. Additionally, the designed sensor was successfully employed to detect BHA in practical samples, expanding the development of COF-based composites in electrochemical applications. As a synthetic phenolic antioxidant, butylated hydroxy anisole (BHA) is frequently employed in food packaging, biofuels, and pharmaceutical formulations . It has Several techniques have been developed for determining BHA, including micellar electrokinetic capillary chromatography, gas chromatography, high-performance liquid chromatography, and spectrophotometry ,7,8,9,10Covalent organic frameworks (COFs) are formed by powerful covalent links between light elements with a reticular network of molecular building blocks, and exhibit an exceptionally high surface area and high stability, displaying a wide range of applications ,16,17,184·6H2O, MnSO4·4H2O and CuCl2·6H2O, respectively, to obtain the final composites MPor−COF−366 . Subsequently, to fabricate an electrochemical sensor, the obtained nanocomposites were fixed on the glass carbon electrode (GCE) surface. The sensor exhibited remarkable catalytic activity and selectivity towards BHA, as the synergistic effect of Por-COFs and metal atoms endowed the sensor with unique properties.In this work, we synthesized a series of two-dimensional nanomaterials based on MPor−COF−366. Firstly, Por−COF−366 was produced by the condensation reaction of 5,10,5,20-tetra(4-aminophenyl)porphyrin (TAPP) and 1,3,5-benztriformaldehyde (BDA) under acidic conditions. Strong covalent bonds were constructed by using porphyrin macrocycle molecules as building blocks. Then, Por−COF−366 was impregnated with FeSO2·6H2O, FeSO4·6H2O, MnSO4·4H2O, CH2Cl2, methanol, acetic acid, and 1,3,5-trimethylbenzene. As a supporting electrolyte, phosphate buffer solution was prepared using 0.1 M NaH2PO4/Na2HPO4. Chemicals were used exactly as bought and all were of analytical grade. The following substances were obtained from Shanghai Tansoole: 5,10,5,20-tetra(4-aminophenyl)porphyrin (TAPP), 1,3,5-benztriformaldehyde (BDA), 1,4-dioxane, ethanol, tetrahydrofuran, and acetone. Other chemicals were bought from Sinopharm Chemical Reagent Co., Ltd. , including CuClPor−COF−366 was synthesized according to a previously reported method with some modifications . TAPP  were dissolved in a mixture of 12 mL dichloromethane and methanol. The mixture was stirred continuously for 24 h. After centrifugation, the composites were washed with CH2Cl2 and were dried under vacuum at 60 °C for 12 h to obtain the product FePor−COF−366. After replacing the metal salts with MnCl2·6H2O and CuCl2·6H2O, MnPor−COF−366 and CuPor−COF−366 were obtained by the same synthesis process.Por−COF−366 (20.0 mg) and FeSO2O3 before being sequentially rinsed in water and ethanol and then dried under N2. The GCE surface was coated with 5.0 μL of FePor−COF−366 (1 mg/mL) suspension, and dried using infrared light. After that, 2.0 μL 5 % chitosan solution was selected as binder. MnPor−COF−366/GCE, CuPor−COF−366/GCE, and Por−COF−366/GCE were all modified using the same method.GCE was meticulously polished with α-AlRapeseed, corn, and peanut oils were bought at a nearby grocery. Then, 1.0 g of the material was dissolved in 10 mL of ethanol. The product was centrifuged after being oscillated for 10 min. The supernatant was collected, and filtered through a Millipore nylon filter with a 0.22 μm pore size. A 1.0 mL aliquot of the final product was put into 9.0 mL of pH 4.0 PBS for further analysis.The morphology of the composites was examined using transmission electron microscopy  and scanning electron microscopy . A Cary 610/670 infrared microspectrometer  was employed to obtain the FTIR spectra. Utilizing a D8 Advance X-ray diffractometer , the X-ray diffraction (XRD) patterns were gathered from 1.5° to 80° at ambient temperature. XPS testing was carried out using an ESCALAB 250Xi XPS spectrometer from Thermo Scientific, Massachusetts, USA with an aluminum Kα X-ray source.A CHI852C electrochemical analyzer  was applied for all electrochemical tests. A standard three-electrode system was employed to perform cyclic voltammetry (CV) and differential pulse voltammetry (DPV).The morphologies of Por−COF−366 and MPor−COF−366 composites were characterized using SEM and TEM measurements. In −1, and the characteristic stretching vibrations of C=N at 1623 cm−1 were seen in FTIR spectrum  in 5.0 mM [Fe(CN)ctrolyte . The chatert-butylquinone (TBQ). Compared with CuPor−COF−366/GCE and MnPor−COF−366/GCE, a narrowed redox peak potential separation (ΔpE) and enhanced peak current value were observed after modification with FePor−COF−366/GCE. The ΔpE value in a cyclic voltammogram is one of the important factors for estimating the electron charge-transfer rate at the electrode–electrolyte interface. Generally, the ΔpE value is inversely proportional to the charge-transfer rate constant. Here, the ΔpE values decreased in the sequence CuPor−COF−366/GCE (263.5 mV) > MnPor−COF−366/GCE (232.5 mV) > FePor−COF−366/GCE (229.5 mV). The results indicated that the engineered FePor−COF−366 largely minimized the electron-transfer barrier between the redox species and the electrode. The peak currents of the various modified electrodes followed the order FePor−COF−366 > MnPor−COF−366 > CuPor−COF−366 > Por−COF−366 > bare GCE, indicating that the introduction of Por−COF−366 and MPor−COF−366 can effectively improve the electrochemical activity of the electrode. Among all the investigated samples, the FePor−COF−366 exhibited the best electrochemical response. This phenomenon may be due to the fact that atomically dispersed Fe in the porphyrin macrocycle was coordinated as the catalytically active centre, which was fully exposed to the interaction with BHA, providing a greater specific surface area, increasing the quantity of open active sites, and improving the speed of electron transfer in the composite.The electrochemical behaviours of GCE, Por−COF−366/GCE, and MPor−COF−366/GCE were investigated through CV. As exhibited in Epa) of BHA oxidation exhibited markedly negative movement, showing that protons were implicated in the electro-oxidation of BHA. The linear formula for the relationship between pH and the oxidation peak potential of BHA is: Epa(V) = −0.0572 pH + 0.6832 (R2 = 0.9949). The peak potential and pH are related by:Ep versus pH plot resulted in an m/n ratio of 1, showing that the process involved an equal number of protons and electrons [Substituting the equivalency of the slope values of the lectrons .−1. The anodic and cathodic peak currents and the square root of the scan rates were linearly related, and followed the equations: Ipa (μA) = −0.3314 + 0.3415 1/2, (R2 = 0.9986); Ipc (μA) = −0.1525 − 0.0737 1/2, (R2 = 0.9834) (Inset a in Epc) shifted in a more negative direction as the scan rate increased, while the oxidation peak potential (Epa) steadily moved in a more positive direction. The peak potentials and lnEpa (V) = 0.4346 + 0.0163 ln2 = 0.9967); Epc (V) = 0.0812 − 0.0216 ln2 = 0.9833) nF), respectively. n can be calculated as 2.1. Because the oxidation of BHA is a two-electron transfer process, two electrons and two protons are included in the redox reaction [The linear slopes of reaction . The reaFurthermore, the accumulation step can be considered an effective method for improving the assay sensitivity. As shown in Ipa (µA) = 0.3415 C − 0.3314  and Ipa (µA) = 0.1525 C − 0.0737 . The two linear regions in DPV was applied under optimal conditions to study the relationship between peak current and BHA concentration A. AccordAccording to the data in 2+, Mn2+, Mg2+, Cl−, and SO42−, did not obstruct the measurement of BHA. Furthermore, the BHA measurement was unaffected by ascorbic acid, glucose, and L-cysteine concentrations that were 20-fold greater. Butylated hydroxytoluene (BHT) is another phenolic antioxidant which shares the same hydroxyl group as BHA and can be oxidized close to the potential of BHA. It was found that BHT had no impact on the detection of BHA, even at a 20-fold concentration. Together, these findings demonstrate the great selectivity of the electrochemical sensor for BHA detection. Storage stability was assessed by storing a batch of freshly manufactured electrodes in a refrigerator at 4 °C and taking measurements every three days. After a lengthy storage period of 21 days, it was discovered that approximately 91.2% of the initial value was maintained. The outcomes amply demonstrated the sensor’s ideal stability. Moreover, there was no evident decrease of current response observed after 35 cycles, and 95% of its original value was maintained. Three parallel measurements were taken for each sample, using the standard addition method, to assess the usefulness of the constructed electrochemical sensor. A number of potential coexisting substances were evaluated by DPV in 50 μM BHA, to examine the selectivity of the sensor. These experiments showed that many inorganic ions, including ZnIn conclusion, the pre-designed Por-COFs were successfully used as metallization modification matrixes, and the metal atoms were regulated in the form of coordinated metal-N4 as special functional modulators. The resulting FePor-COFs demonstrated distinctly higher electrocatalytic response for BHA in terms of wide linear range, low detection limit, high sensitivity, superior selectivity, and good reproducibility. This study not only describes an attractive and efficient catalyst electrode for BHA determination, but also opens a new avenue for the design and development of two-dimensional porphyrin COFs as sensors for electrochemical detection of small molecules.


Abstract. This sentence previously stated:A correction has been made to blaNDM−5 , mcr-1 , tet(X4) , and cfr  were also found in these isolates.”“The AMR genes The corrected sentence appears below:blaNDM−5 , mcr-1 , tet(X4) , and cfr  were also found in these isolates.”“The AMR genes The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


The timing of bud break is very important for the flowering and fruiting of longan. To obtain new insights into the underlying regulatory mechanism of bud break in longan, a comparative analysis was conducted in three flower induction stages of two longan varieties with opposite flowering phenotypes by using isobaric tags for relative and absolute quantification (iTRAQ).In total, 3180 unique proteins were identified in 18 samples, and 1101 differentially abundant proteins (DAPs) were identified. “SX” (“Shixia”), a common longan cultivated variety that needs an appropriate period of low temperatures to accumulate energy and nutrients for flower induction, had a strong primary inflorescence, had a strong axillary inflorescence, and contained high contents of sugars, and most DAPs during the bud break process were enriched in assimilates and energy metabolism. Combined with our previous transcriptome data, it was observed that sucrose synthase 6 (SS6) and granule-bound starch synthase 1 (GBSSI) might be the key DAPs for “SX” bud break. Compared to those of “SX”, the primary inflorescence, axillary inflorescence, floral primordium, bract, and prophyll of “SJ” (“Sijimi”) were weaker. In addition, light, rather than a high sugar content or chilling duration, might act as the key signal for triggering bud break. In addition, catalase isozyme 1, an important enzyme in the redox cycle, and RuBisCO, a key enzyme in the Calvin cycle of photosynthetic carbon assimilation, might be the key DAPs for SJ bud break.Our results present a dynamic picture of the bud break of longan, not only revealing the temporal specific expression of key candidate genes and proteins but also providing a scientific basis for the genetic improvement of this fruit tree species.The online version contains supplementary material available at 10.1186/s12870-022-03868-3. Dimocarpus longan) is a subtropical perennial crop, and it is best known for its nutritious fruit, which has a relatively high medicinal value [Longan  paradormancy, where growth inhibition arises from another part of the plant; (2) endodormancy (or true dormancy), which is triggered by internal factors; and (3) ecodormancy, which is controlled by environmental factors , 6. In tArabidopsis sucrose phosphate synthase gene, transgenic poplar lines showed earlier bud break than wild-type lines, which raises the possibility that enhanced sugar and/or starch reserves can promote accelerated bud break [In recent decades, many studies have been conducted on this issue, and many factors have been identified in temperate and boreal trees , 10, witud break . In addiud break . For exaud break , 19.In contrast to boreal and temperate trees, subtropical tree species grow in subtropical regions with short winters and relatively warm temperatures that rarely drop below 5 ℃, which is the typical minimum temperature needed for most temperate plants to meet their winter chilling requirement for bud break and flowering . It seemAccording to the requirements of climate conditions and the variety of characteristics shown by their environments, longan varieties can be divided into either a subtropical longan group or a tropical longan group . Most cuArabidopsis, citrus, and Camellia oleifera [Although several reports have studied floral induction in “SJ” using RNA-seq analysis , 25, theoleifera , 29. Our“SX” and “SJ” longan trees were both cultivated in the same orchard located in Mazhang district , Zhanjiang, Guangdong Province, P. R. China (the identification was undertaken by Pro. Wang ). Three − 1 at 35 °C. For the mobile phase, acetonitrile and twice-distilled water (70:30 v/v) were used along with a refractive index detector. The starch contents of the different flower bud samples were determined enzymatically as glucose equivalents following the method proposed by Chow et al. [The contents of soluble sugars in different flower bud samples were determined using high-performance liquid chromatography (HPLC)  following the description by Shi et al. . In shorw et al. .The total proteins of the flower bud samples of “SX” and “SJ” were extracted following the method of phenol extraction described by Chen et al.  with sli2-fold change in DEGs and DAPs during different flower induction stages in both accessions.RNA sequencing data were obtained in our previous study . To inveThe qRT‒PCR materials and methods are shown in our previous study . The qRT“SX” is a typical “seasonal flowering” (SF) longan cultivar, and it needs an appropriate period of low temperatures to accumulate energy and nutrients for flower induction. Because of these traits, its inflorescence and postdormant bud develop better and more robustly Fig.  A. DiffeTo further study the reason for the different flowering traits of “SX” and “SJ”, a comparative proteome survey was performed on “SX” and “SJ” using the iTRAQ technique. Raw data were deposited into the ProteomeXchange Database (accession number: PXD006710). A total of 419,206, 401,957, and 392,040 spectra were generated in the three biological experiments. A total of 6139, 6407, and 5935 proteins were matched to the longan protein database Fig. . After mAccording to Gene Ontology (GO) enrichment analysis, 2731 of 3180 proteins were classified into three groups Fig. . MeanwhiProteins that underwent a 1.2-fold or 0.83-fold change in abundance with P value < 0.05 between two time points in a particular species (T1–T2 and T2–T3) were identified as DAPs. As shown in Fig. Among the 1101 DAPs, 544 were upregulated DAPs , 552 were downregulated DAPs, and 5 DAPs displayed contrasting patterns in the two longan species. The number of DAPs and the overlaps between the two longan species are summarized in Table In the present study, a transcriptomic analysis of the same samples used in iTRAQ was performed using the RNA-seq method , allowinTo better understand the DAPs involved in the bud break of longan, the unique DAPs belonging to the pathways specifically enriched in the different longan species were analyzed based on our iTRAQ data and RNA-seq data. In total, 38 DAPs that were enriched in “SJ” belonged to five pathways, namely, photosynthesis (8), endocytosis (11), glyoxylate and dicarboxylate metabolism (9), carbon fixation in photosynthetic organisms (9), and oxidative phosphorylation (10) Fig. . Among tDuring the bud dormancy release of “SX”, we found 58 DAPs enriched in seven pathways, namely, amino sugar and nucleotide sugar metabolism (14), porphyrin and chlorophyll metabolism (7), starch and sucrose metabolism (10), ribosome (21), linoleic acid metabolism (3), biosynthesis of unsaturated fatty acids (4), and other glycan degradation (3) Fig. . Among tTo date, the genetic control of bud break has been well studied in model plants . HoweverDifferent from “SJ”, “SX” belongs to typical SF longan cultivars, and it needs winter chilling (an appropriate period of low temperatures) to meet its requirements to induce flowering . This flConsistent with our anatomical analysis and physiological analysis results, we found that most DAPs in “SX” during the bud break process were enriched in assimilates and energy metabolism-related proteins. Numerous experiments have indicated that energy metabolism-related proteins play important roles in flower bud development , 13, 14.Compared to “SX”, the bud break or floral induction of “SJ”, a typical SF longan cultivar, does not need an appropriate environmental factor. Therefore, “SJ” can flower and fruit throughout the year. Due to this trait, “SJ” usually has smaller fruit and lower yields . AlthougTwo proteins were identified as specifically being upregulated  or downregulated  at the transcript and protein levels in “SJ” during the bud dormancy release phases. The first protein is catalase isozyme 1. Catalase (CAT) is an important enzyme in the redox cycle and is a heme-containing compound . The secOur results showed that the key DAPs and enriched pathways in these two longan varieties during the bud break process were quite different based on physiological, anatomical, transcriptome, and proteome analyses. For “SX”, assimilates and energy-metabolism-related pathwayswere important during the bud break process, and the key proteins are starch- and sucrose-metabolism-related proteins (SUS6 and GBSS-I). Different from “SX”, light, rather than a high sugar content, dormancy, or chilling duration, might act as the key signal for triggering the bud break of “SJ”. Most DAPs were enriched in photosynthesis-related pathways, and the key proteins were catalase isozyme 1 and RuBisCO. Taken together, our findings provide a better understanding of the complex regulatory mechanism underlying bud break in longan.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8Supplementary Material 9Supplementary Material 10Supplementary Material 11Supplementary Material 12


This synergistic effect is surprising because TEMPO• alone reacts with dpph• relatively slowly (kS = 31 M–1 s–1 in methanol and 0.03 M–1 s–1 in nonpolar ethyl acetate). We propose a putative mechanism in whicha mutual activation occurs within the acid–base pair TEMPO•/RSV to the resveratrol (RSV) anion and TEMPOH•+ radical cation, both being extremely fast scavengersof the dpph• radical. The fast initial reactionis followed by a much slower but continuous decay of dpph• because a nitroxyl radical is recovered from the TEMPOnium cation,which is reduced directly by RSV/RSV– to TEMPO• or recovered indirectly via a reaction with methanol,producing TEMPOH subsequently oxidized by dpph• toTEMPO•.The reaction of the 2,2′-diphenyl-1-picrylhydrazylradical(dpph Cyclic nitroxides were found to have protective effectsagainst neurodegenerative diseases ,multiple sclerosis, diabetes, ischemia–reperfusion injury,age-related diseases, and many types of cancer .8 The outstanding ability of TEMPO• to remove superoxide doesnot react directly with alkylperoxyl radicals9 (the main chain carrying species during autoxidation). The antioxidanteffect of this cyclic nitroxyl during autoxidation of hydrocarbonsat high temperatures is due to a cycle involving the addition of analkyl radical to TEMPO•, subsequent H atom abstractionfrom alkoxyamine by a peroxyl radical, and β-fragmentation toan aminyl radical, which reacts with another peroxyl radical to regenerateTEMPO•.10 This mechanismdoes not work at lower temperatures, thus, nitroxides must be convertedinto good radical trapping antioxidants (RTA). For example, protonationof the nitroxyl moiety facilitates a fast H-atom transfer from TEMPOH•+ to an alkylperoxyl radical, producing a TEMPOniumcation, which is reduced back to nitroxide through electron transferfrom an alkyl radical R• (• with O2).6 Another example of activation was documented in nonpolar systemswhere peroxidation is mediated by mixed alkyl peroxyl/hydroperoxylradicals or in the presence of molecules capable of transferring thechain carrying radicals from ROO• to HOO•. The inhibition is realized within a cycle of TEMPO• reacting with HOO• .11 In this way, reduction of alkylperoxyl radicals is a side effectof the reaction of TEMPO with hydroperoxyl radicals.Since the discovery of SOD-like activity of nitroxides,12 reported thatnitroxides react easily with alkylperoxyl radicals in water , and they considered two mechanisms, electrontransfer from nitroxide to ROO• or formation ofunstable trioxide: >NOOOR, decomposing to >N=O+ andROO– in the presence of acids. Pratt and coworkers7 proposed another catalytic cycle  measuredunder the same conditions. Moreover, the redox cycles, in which nitroxylsare recovered tens or hundreds of times, make them extremely efficientantioxidants. Nitroxyls are cell-permeable (including Brain–BloodBarrier), nontoxic, nonimmunogenic,8i and thesepersistent radicals are becoming front-line antioxidants.8h The catalytic cycles described above indicate the high redox activityof TEMPO•, TEMPOH, and TEMPOnium cation, which shouldinteract with other antioxidant molecules, and here, we present theradical trapping activity of a model system based on TEMPO• and resveratrol . In contrast to RSV, the nitroxyl moietyin TEMPO• and 4-OH-TEMPO• arenot HB donors; thus, the very slow reaction with dpph• in ethyl acetate (see • (or 4-OH-TEMPO•) are used together (as equimolarmixture), the rate of the reaction with dpph• isnearly the same  ionizationof a phenol and (ii) electron transfer from phenoxide anion to thedpph• radical to form dpph–, whichundergoes protonation (iii) to give dpph-H. The addition of aceticacid to methanol reduces the reaction rate exactly to the level forthe pure HAT process, and kMeOH ≈2kEtOAc, as predicted for Δβ2H = 0.04, see When passing from ethyl acetate to methanol, the reactivityofeach compound greatly increases;• and 4-OH-TEMPO• can be attributed to protonationof the minute fraction of nitroxyls. Such traces of TEMPOH•+ are sufficient to effectively accelerate the process. The experimentwith 10 mM acetic acid added to methanol confirms this hypothesisbecause an additional 4-fold increase in kS is observed . Protonation/deprotonation of the X functional groupin 4-X-TEMPO• is also important for redox properties,for example, the BDE (O–H) in 4-X-TEMPO-H is 5.7 kcal/mol higherfor X = NH3+ than for X = NH2.5 In our system, the expected gain in reactivityafter addition of acetic acid is lost because 4-OH2+-TEMPO• is less active than 4-OH-TEMPO•.Obviously, the above explanation is valid for RSVbut fails fornitroxyl radicals because they are not HB donors, and the SPLET mechanismdoes not exist for the nitroxyl moiety. However, autoprotolysis ofmethanol could be a gentle source of protons interacting with nitroxyls,and the acceleration of reaction observed by TEMPOobserved 1, in ful2.2• reacting with equimolar mixture of RSV and nitroxide. In contrastto kS = 0.97 M–1 s–1 for the reaction in ethyl acetate, which does notvary from kS obtained for the RSV andTEMPO• used separately, the mixture of RSV/TEMPO• in methanol reacts at least 6 times faster than thecompounds used independently. When kMeOH is compared to kEtOAc, a big 1400-foldacceleration is observed for RSV/TEMPO•, see •. This surprising acceleration was also observed when RSV was replacedwith 3,5-DHA, i.e., for a phenol being 50-fold less reactive (towarddpph•) than TEMPO•, the mixtureof 3,5-DHA with TEMPO• reacted much faster thanthese two compounds used separately, see The most surprising effect was observed for dpph23 . The reactionof nitroxyls with some of the most reactive phenolic RTAs in methanolis very slow.25 To be sure that no reaction occurbetween RSV and TEMPO•, we monitored the absorbanceof TEMPO• (at 440 nm) mixed with RSV in methanol,and no change was recorded within 2 h.The accessible literature survey indicatesthat the interactions of the nitroxyl group with phenols in aproticsolvents are limited to the formation of the H bond, quantitativelydetermined by NMR, FTIR, and EPR measurementsKa for RSV and TEMPOH•+ .6a Thus, apartfrom ••, with TEMPOH•+ reactingvia HAT or PCET.The most intriguingis that kcal/mol 2 being a•  reacting with dpph• in methanol, the presenceof 10 mM acetic acid causes 4-fold acceleration, but more reliablecontribution of protonated nitroxyl can be estimated when a weakeracid was used, like hexafluoroisopropanol (HFIP) with pKa = 9.3 comparable to the acidity of RSV. The kS = 61.3 ± 2.1 M–1 s–1 obtained in the presence of 10 mM HFIP is biggerthan kS for TEMPO• inneat methanol. Assuming that •+ and RSV–, and neglecting HAT from nonionized RSVto dpph• , we estimated the contributionof HAT from TEMPOH•+ to dpph• as5% (61 M–1 s–1/1320 M–1 s–1), and the remaining 95% is due to ET fromRSV– to dpph•.3424b estimated the rateof +, e–) from RSV to TEMPO•; that is not the case because,as described above, TEMPO• does not react with RSV.Moreover, if – wouldbe the main scavenger of dpph• and the observedoverall kS determined for the equimolarmixture would be compared to kS obtainedfor the reaction of dpph• with TEMPOH used alone:Aliaga et al.k7 = 35 M–1 s–1 inmethanol  reacting with RSV, TEMPO•, or a mixture of RSV/TEMPO• in methanol during10 h . For 79 μM dpph• reactingwith 15.8 μM RSV, about 30% of the radical was consumed (• during the reactionwith RSV (dpph• is in 5-fold excess), and it canbe concluded that one molecule of RSV does not trap more than tworadicals. This observation (for dpph•/RSV) is inagreement with the rather low stoichiometric factor (n = 1.9) determined by Amorati et al.35 for RSV trapping peroxyl radicals in chlorobenzene, and is muchlower than n ≈ 5 recently observed by us18 for RSV during peroxidation carried out in heterogeneoussystems and explained to be due to dimerization of RSV• in micelles or liposomes, where the local concentration of RSV• is much higher than in a continuous phase. For thesame reasons we exclude dimerization or any other activation/regenerationof RSV in methanol. Formal H atom transfer from RSV to dpph• . In contrast to the fast reaction at the beginning , the long lastingmonitoring of the dpph• decay suggests that afterthe initial fast reaction, TEMPO• alone or mixedwith RSV follows a different kinetics, with drastically slower butcontinuous consumption of dpph•.The results for dpphconsumed 2, line C•. TEMPOH•+ with its high reduction potential (E0red = 955 mV for TEMPOH•+/TEMPOH)36 is an efficient oxidizing agent for TEMPO• (E0red = 750mV for the TEMPOnium+/TEMPO• pair),37 and the process can be presented as a sequence of reversible reactions:30ak10 = (1.4 ± 0.8) ×105 M–1 s–1 (in dilutedsolution).27 It is very likely that • by RSV becauseit consumes TEMPOH•+; thus, it is an additionaldriving force for Such bimodalkinetics can be reasoned as a result of slow evolutionof intermediates, which immediately react with dpphk–10 , rangingfrom 0.23 M–1 s–1 at pH 4.6 to51 ± 1 M–1 s–1 at pH 10 (datafor TEMPO•).31 The resulting hydroxylamineis known as an efficient inhibitor of peroxidation.39 TEMPOH reacts rapidly with peroxyl radicals,with k from 5 × 105 to 3 ×106 M–1 s–1,6 and also should effectively react with dpph• via HAT  is similar to the reactivity of TEMPOH (Sk = 35 M–1 s–1). The main benefit is an increase in overall stoichiometry, regardless k ≈ 1–3 × 1010 M–1 s–1, as presentedin 40 and hydride from THF . 2 and the mechanism is pH dependent43 with bimolecular hydride transfer in acidified and in neutral methanol.44The reverse process,known as comproportionation, is much slower,with pH-dependent •.Thus, • toTEMPOH and TEMPOnium cation , based on the reports by Kerziget al.45 The same researchers determinedthe half-life for  as 50 μs; thus, the reaction of radical anionswith TEMPOnium cations cannot be excluded, and the Coulombic attractionsof the reacting species will further accelerate this process. Theincreased acidity of RSV• compared to RSV also hasan additional consequence for the initial kinetics of dpph• decay because a new acid/base equilibria is established, with anadditional amount of TEMPOH•+ generated in the system.Please note that both m cation 10. On thm cation 2, which m cation 2, line BTable S22, a reaction of TEMPOnium+ with dpph– could be also considered; however, the kinetic significanceof such a reversible process is rather low because [dpph–] ≪ [RSV] ≪ [methanol]; thus, the reaction of TEMPOnium+ with RSV and methanol will be preferred as possible waysof recovery of TEMPO•. Moreover, the reaction ofdpph– with TEMPOnium+ would regeneratedpph•, but inspection of • usedalone or mixed with RSV is a very efficient radical trapping agent.An anonymous reviewer suggested the additional (and not excluding)explanation for the increased number of dpph• radicalstrapped by RSV mixed with TEMPO•  during extended reaction time  and fast HAT from the hydroxylamine radical cationto dpph• (marked in blue). The much slower decayof dpph• during the next several hours was causedby HAT from hydroxylamine formed in at least two reactions  mechanism, which is not relevantto the nonpolar environment in hydrocarbons/polymers/biomembranes.However, recently observed reactivity of nitroxides as inhibitorsof peroxidation in solutions (THF in water) and in dispersed systems(lipid bilayers and ferroptosis)7 revealedparticipation of HAT, PT, ET, and even hydride transfer  mightplay a significant role in the recovery of TEMPO•.Despite many limitations of dpphtransfer 1 as key 3• reacts six times faster withRSV than with TEMPO•; however, when both compoundsare used together as an equimolar mixture, the rate constant for thereaction with dpph• is one order of magnitude biggerthan for any of the components used alone. Acceleration is causedby RSV anions and TEMPOH•+, and both species aremuch more reactive than their neutral parent compounds. Although acidpromoted activation of TEMPO• was previously reported,6 our observation is particularly interestingas an example of a dramatic kinetic consequence of very subtle acidbase equilibria because TEMPO• is an extremely weakbase and the difference in pKa betweenTEMPOH•+ and RSV is about 15 units. Moreover, theobserved acceleration is an effect of a mutual activation of RSV andTEMPO•, with 95% of the dpph• reducedby RSV anions via ET and ca. 5% of dpph• reactswith TEMPOH•+ via the HAT process.In methanol, dpph• (usedin 5-fold excess over TEMPO• and RSV), resultingin the stoichiometry exceeding the number of radicals trapped by typicalphenolic antioxidants. Based on the result for TEMPO• reacting with peroxyl radicals published previously by Pratt andcoworkers,7 we propose a putative cyclic mechanism ofoxidation and reduction of TEMPO• reveals a slow, continuous decay of dpphf TEMPO• 1 includig agents 1. We exp4trans-RSV, TEMPO•, dpph•, 4-OH-TEMPO•, TEMPOH,and 3,5-DHA were used as received .Commercially available 22 and our recent work with catecholamines15 indicates straightforward variation of kS on pH.47 Sinceeven traces of base present in a solvent distilled over CaH2 are kinetically significant,22 methanoland ethyl acetate were distilled over a few crystals of dpph• and a few beads of amberlite resin in order to remove traces ofstabilizers and alkaline impurities. Before each series of experiments,the kinetic purity of methanol was tested by comparison of the rateconstant obtained for 2,4,6-trimethylphenol (reference phenol) witha reference value,22 i.e., kS = (43 ± 15) M–1 s–1.The results of kinetic studies on solventeffects, acid–basecatalysis, and reaction mechanism are extremely sensitive on any impurities,15 The decay of dpph• (concentration between 6 and 9 × 10–5 M)in the stoichiometric excess of the tested compounds (concentrationbetween 1 and 2 × 10–2 M) was monitored at517 nm on an Applied Photophysics SX 20 stopped-flow apparatus, equippedwith a xenon arc lamp source and photodiode array detector. The mixingcell  andthe tubes with the reactants were thermostated at 25 °C. Measurementswere performed in neat methanol, ethyl acetate, and acidified solvents(10 mM acetic acid or 10 mM hexafluoroisopropanol). The initial ratesof reaction were monitored, giving the pseudo-first-order rate constant(kexp), calculated as the average fromat least two sets of measurements. The plots of the kexp vs concentration the tested compounds were linear,and their slopes gave the second-order rate constants, ks.Stopped-flow measurements were made following the proceduredescribedelsewhere.• were monitored with a Varian Cary 50 spectrometerand QS quartzcuvettes with a 10 mm optical path length. A total of 100 μLof dpph•  in methanoland 30 μL of TEMPO•, RSV or TEMPO•, and RSV  were injected to 3mL of methanol in a quartz cuvette. Absorbance data was collectedevery 10 min (517 nm).Extended (ten hours) experiments of the decayof dpph


Nature Communications 10.1038/s41467-019-12081-0, published online 13 September 2019.Correction to: The original version of this article included errors in the Results and These two “merge” commands combined the expression values of all samples of three microarrays. The mistake was that the gene-annotation information-columns were not considered. The “merge” command has the argument “sort”, which by default is TRUE, leading to an alphabetical sorting of the “TargetName” column. This led to a wrong combining of the gene-annotation information-columns and the “TargetName” in a later step.The data has now been reanalyzed, only columns with the expression data were used, and the command “inner_join” was applied, because this command always keeps the row-order. The correct code is: “dataset1_2 <- inner_join dataset<-inner_join and CON and detected 1037 differentially expressed genes . Gene ontology (GO) analysis indicated that several genes are implicated in the biosynthesis of long-chain fatty acyl-CoA and fatty acids as well as reactive oxygen species (ROS) metabolism . GO analysis showed significant enrichments in genes controlling catabolic processes, lipid metabolism, and phosphorylation , lipid metabolism , calcium signaling (PCDH15), protein folding (DNAJC28), and inflammatory processes (CISH) , fatty acid metabolism (SLC27A4), calcium signaling (ATP2C2), transcriptional regulation , protein transport (RAB3D), and inflammatory processes (IL18RAP) ”. now reads “This revealed 70 mitochondrial, 23 lipid metabolism and 60 calcium-related genes with significantly different expression levels ”.MYH3, CCDC87, and ACTC1) , at 24 weeks 709 genes (563 up- and 146 downregulated) in comparison to their expression before surgery . The pathways affected at 12 weeks comprise—among others—glucose homeostasis (GO0042593) and negative regulation of phosphate activity (GO:0006814) . Among differentially expressed genes involved in mitochondrial function, lipid metabolism and calcium signaling, we detected 17, 4, and 15, respectively, which were affected by DNA methylation at 52 weeks ”. now read “This enrichment explains the relatively small overlap between differentially methylated CpGs and differentially expressed genes, as only 2956 CpGs are located in/or close to regions of the 921 differentially expressed genes . Among differentially expressed genes involved in mitochondrial function, lipid metabolism, and calcium signaling, we detected 13, 6 and 30, respectively, which were affected by DNA methylation at 52 weeks ”.The sentence that read “However, at 52 weeks, 1467 CpGs were identified in 430 differentially expressed genes . GO analysis indicated enrichment in genes linked to 12 biological pathways, including cAMP biosynthesis, lysosomal organization, and muscle cell differentiation  signaling in skeletal muscle; PIK3R1, a regulator of the PI3-kinase; MLXIP, involved in transcriptional activation of glycolytic target genes, and ACACB, a mitochondrial enzyme playing a role in fatty acid metabolism . A hypermethylation in the promoter region of PTPRE gene related to lower expression at 52 weeks . A lower DNA methylation in the promoter and a higher methylation in the gene body of PIK3R1 is linked to its higher expression after the surgery , whereas a hypomethylation in the gene body of MLXIP and ACACB associated with their lower expression . In addition, the time course of the expression of the listed candidates was evaluated. PTPRE expression decreased already at 2 weeks, whereas the changes of the expression of PI3KR1 occurred at 12 weeks and ACACB and MLXIP at 52 weeks , whereas hypomethylation of ASPSCR1 in the gene body related to lower expression at 52 weeks . Of note, both genes are involved in glucose transport via glucose transporter 4 (GLUT4)20,21. In addition, both NR4A1 (nuclear receptor subfamily 4 group A member 1) and ELOVL5 (ELOVL fatty acid elongase 5) exhibited hypermethylated promoters and hypomethylated gene bodies corresponding to lower expression . ELOVL5 is involved in the elongation of long-chain polyunsaturated fatty acids 22 and upregulated upon high-fat feeding 23, NR4A1 encodes a nuclear receptor and transcription factor regulating the expression of genes involved in glucose metabolism 24. In addition, the time course of the expression of the listed candidates was evaluated. TBC1D1 expression increased and ASPSCR1 decreased already at 2 weeks, whereas the changes of the expression of ELOVL5 and NR4A1 occurred only at 52 weeks  and ELOVL5  indicate that their interindividual epigenetic alterations are linked to the improvement in insulin sensitivity  and PIK3R1  indicate that their interindividual epigenetic alterations are linked to the improvement in insulin sensitivity , fasting glucose (231), HMW-adiponectin (61), and mitochondrial content (13). GO analysis of the 231 affected genes, which correlated to glucose concentrations, can be linked to regulation of DNA binding (GO:0043388), macromolecule catabolism (GO:0009057), exocytosis (GO:0006887), secretion (GO:0032940), and others (Supplementary Table The sentence “As 1072 transcripts (encoded by 468 genes) were only transiently differentially expressed at 2 weeks  and returned to baseline expression levels, we tested whether epigenetic alterations were responsible for this effect”. now reads “As 1150 mRNAs (encoded by 1126 genes) were only transiently differentially expressed at 2 weeks  and returned to baseline expression levels, we tested whether epigenetic alterations were responsible for this effect”.p < 10−255), including genes involved in mitochondrial function (n = 22), calcium signaling (n = 20), lipid metabolism (n = 10). Representative examples comprise FTO, an obesity-related gene encoding α-ketoglutarate dependent dioxygenase 25, and TOMM7, a translocase of outer mitochondrial membrane 7 involved in translocation of pre-proteins into mitochondria 26,27 , including genes involved in mitochondrial function (n = 12), calcium signaling (n = 11), lipid metabolism (n = 4). Representative examples comprise HMGCS2  of these genes showed changes in DNA methylation at 52 weeks , exhibited differential expression” now reads “Before surgery, as described previously13,19 particularly genes involved in lipid metabolism, such as FFAR4 (free fatty acid receptor 4), exhibited differential expression”.The sentence “Before surgery, as described previouslyABCD3 (ATP-binding cassette sub-family D member 3) or NAPELD (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D) expression at 2 weeks likely reflects the higher FFA uptake45,46. Also, genes related to mitochondria and calcium handling showed higher expression levels, such as SLC25A25  encoding a calcium-dependent mitochondrial solute carrier47”.The sentence “Elevated SLC27A4 (fatty acid transporter 4) expression at 2 weeks likely reflects the higher FFA uptake 50. Also, genes related to mitochondria and calcium handling showed higher expression levels, such as ATP2C2, encoding a manganese-transporting calcium ATPase 51, and MCUR1, a key regulator of oxidative phosphorylation 52, encoding a mitochondrial calcium uniporter protein required for calcium”. now reads “Elevated 54. Interestingly, carriers of polymorphisms in the IL18RAP gene suffer from greater susceptibility to obesity54”. now read “Another set of transiently upregulated genes relates to small GTPase-mediated signaling, such as ARHGAP24, RACGAP1, ITSN1 and to negative regulation of insulin signaling, such as SOCS1, RPS6KB1. The latest mediates TNF-alpha-induced insulin resistance by phosphorylating IRS1 at multiple serine residues, resulting in accelerated degradation of IRS149−51”.The sentences “Consequently, elevated MCUR1 expression can be linked to the evanescent stimulation of β-oxidation. Another set of transiently upregulated genes relates to inflammatory processes, such as IL18RAP, encoding the receptor accessory protein of the pro-inflammatory interleukin 18 (IL18)The sentence that read “Likewise, MTUS1, possibly contributing to protection against pro-inflammatory response of endothelial cells 55, may be involved in the anti-inflammatory defense at 2 weeks. In addition, MTUS1 has recently been shown to be involved in mitochondrial motility, fusion, and in the maintenance of mitochondrial morphology 56 and its reduction at 2 weeks may serve to support the evidence for alterations of mitochondrial content and function early after surgery.” has now been deleted.TBC1D1, contributing to GLUT4 translocation20, ASPSCR1, a tethering protein sequestering GLUT4-containing vesicles21 and ELOVL5, which is essential for fatty acid synthesis22 and known to be upregulated in human skeletal muscle upon overfeeding23”. now read “Nearly 3000 of the differentially methylated CpGs are associated with altered expression of the corresponding 921 genes, pointing towards the relevance of epigenetic mechanisms in response to weight loss. Metabolic surgery remodels DNA methylation of genes involved in insulin signaling, such as PIK3R1 and PTPRE and ACACB , which is essential for fatty acid metabolism59. Mice lacking Acc2 exhibit higher fatty acid oxidation rates in the soleus muscle than control mice60”.The sentences “Nearly 1500 of the differentially methylated CpGs are associated with altered expression of the corresponding 430 genes, pointing towards the relevance of epigenetic mechanisms in response to weight loss. Metabolic surgery remodels DNA methylation of glucose and lipid metabolism-related genes, such as 64. “now reads “The present study identified close to 300 epigenetic changes associated with altered expression of important metabolic genes, likely contributing to improved insulin sensitivity and lipid metabolism. Pathway enrichment analysis of the 921 differentially expressed and methylated genes indicate that several genes are implicated in the regulation of glycogen metabolism, intracellular signal transduction, and cAMP biosynthesis, reflecting improved skeletal muscle activity and metabolism61”.The sentences “The present study identified close to 100 epigenetic changes associated with altered expression of important metabolic genes, likely contributing to improved insulin sensitivity and lipid metabolism. Pathway enrichment analysis of the 430 differentially expressed and methylated genes indicates that several genes are implicated in the regulation of muscle cell differentiation, intracellular signal transduction, and cAMP biosynthesis, reflecting improved skeletal muscle activity and metabolismThe sentence “Moreover, the present study found that changes in DNA methylation are associated with reprogramming up to 70% of the transiently altered transcripts, which possibly normalize their expression levels at 52 weeks ”. now reads “Moreover, the present study found that changes in DNA methylation are associated with reprogramming up to 75% of the transiently altered transcripts, which possibly normalize their expression levels at 52 weeks ”.The original version of this Article contained an error in Tables 2 and 3. The correct version of the ‘Number of genes’ row in Table 2 states 25 for Body mass index, 1219 for Fasting glucose, 76 for FFA suppression, 73 for HMW-adiponectin, 29 for Mitochondria content, instead of the original values of 17, 231, 11, 61, and 13, respectively. The correct version of the row ‘Number of genes’ in Table 3 states 177 for Body mass index, 443 for M-value, 163 for Fasting glucose, 112 for FFA suppression, 70 for HMW-adiponectin, and 27 for Mitochondrial content, instead of the original values of 43, 189, 98, 38, 31, and 9, respectively.The original version of the PIK3R1, HMGCS2, MAPK10…) were changed.All gene ontology terms depicted in Supplementary Fig. ASPSCR1, NR4A1, Elovl5, TBC1D1, TOMM7 and FTO” are replaced by the correct candidates: “ACACB, PTPRE, PI3KR1, MLXIP, IMML2P, HMGCS2”. The correlation plots in Supplementary Fig. ASPSCR1, ELOVL5” to “PTPRE, PIK3R1”).Genes shown in Supplementary Fig. All genes shown in Supplementary Tables In the updated Supplementary Tables In addition, as part of the re-review to resolve this correction, it was also noticed that multiple correction was not applied to the data. This has now been addressed, and in the Statistical Analysis section of the “Methods”, the following text was added “Correction for multiple testing was performed for the methylome data and not for the transcriptome data. The reason for this is to avoid the number of false negatives and oversee relevant effects according to suggestions of John H. McDonald . Sparky House Publishing, Baltimore, Maryland; p. 254–260). However, we provided the results of the multiple correction in the Supplementary Tables from S11 to 15” in the Statistical Analysis section of the “Methods”.p-value and DNA methylation data are adjusted for multiple testing with Benjamini Hochberg correction”. In the legend for Fig. 4 the sentence that read “Up- and downregulated genes are indicated by red and blue signals, respectively. *p < 0.05 ” now reads “Up- and downregulated genes are indicated by red and blue signals, respectively. *p < 0.05 ”. The legend of Fig. 5, where it read “Only significantly differentially methylated CpGs are represented; *p < 0.05 ” now reads “Only significantly differentially methylated CpGs are represented; *p < 0.05 ”. The legend of Fig. 6, where it read “*p < 0.05 ” now reads “*p < 0.05  (b)”.The current version of the manuscript also adds in the Legend of Fig. 3, the text “For gene expression unadjusted The results of the multiple correction testing, requested by the reviewers, are now included in Supplementary Data 1.The original version of Table 2 and 3, and those of Figures 3, 4, 5, and 6 have now been replaced with corrected versions.The previous version of Table 2 was:The correct version appears as:The previous version of Table 3 was:The correct version appears as:The previous version of Figure 3 was:The correct version appears as:The previous version of Figure 4 was:The correct version appears as:The previous version of Figure 5 was:The correct version appears as:The previous version of Figure 6 was:The correct version appears as:These errors have been corrected in the HTML and PDF versions of the article. The HTML has been updated to include a corrected version of the Supplementary information.Supplementary information


C-cap mimics are underdevelopedand suboptimal. Bioinformatic studies described here were undertakento better understand Schellman loops, the most common C-caps in proteins, to design superior synthetic mimics. An algorithm(Schellman Loop Finder) was developed, and data mining with this ledto the discovery that these secondary structures are often stabilizedby combinations of three hydrophobic side chains, most frequentlyfrom Leu, to form hydrophobic triangles. That insightfacilitated design of synthetic mimics, bicyclic Schellman loop mimics(BSMs), where the hydrophobic triumvirate was replaced by 1,3,5-trimethylbenzene.We demonstrate that BSMs can be made quickly and efficiently, andare more rigid and helix-inducing than the best current C-cap mimics, which are rare and all monocycles.Macrocyclic peptidesare the prevalent way to mimic interfacehelicesfor disruption of protein interactions, but current strategies todo this via synthetic  C-caps mimics to inducepeptide helicityare rare and based on monocyclic strategies. We describe more rigid,helix-enforcing, and easily accessible bicyclic designsinspired by a bioinformatic study. The first incorporated a 1,3-xylene linkerbetween residue C3 and C″ at the helix C-terminus with moderate helix-inducingeffect, and the other is a dual-capped peptide  withoutdetailed analyses of how they might pack,24 and as far as we are aware, no studies have followed up on thispoint. As a result, a deeper study using our complete set of uniqueSchellman loops was facilitated to a better understanding of the packingof hydrophobic side chains at C3, C″, and Ccap in common Schellman loops.C3, Ccap, or C″) be less than 4.5 Å to ensure hydrophobicinteractions. “Patches” were previously defined by oneclose interaction: C3–C″. However, our analyses have used a broader definition to include C3–Ccap and C″–Ccap because these are also interactionsbetween two side chains. Data interpretation was facilitated by denotingthe three possible interactions, C3–C″, C3–Ccap, and Ccap–C″, inbinary nomenclature {X,X,X}. Thus, there are eight possibilities from{0,0,0} to {1,1,1} where 1 refers to potential interactions and 0means none. A score of 1 in any position defines a patch. Hydrophobic triangles, a term proposed by us, are for thoseSchellman loops with at least two patches among C3, Ccap, and C″, becausein such cases all three residues participate in the triangulated hydrophobicclusters. Figure S7a shows all eight possible combinations, and Figure S7b gives a detailed breakdown of their relative proportions.A filteringscript to identify potential hydrophobic contacts wasmade in-house. It requires that the nearest carbon atoms between twoside chains (C3 and C″ (38935), hydrophobic triangles comprise the majority of the data set. extended where the three residues are inclined to be stacked,and within this group there are three possibilities: {1,0,1} , {1,1,0} or, conceivably, {0,1,1} . Hydrophobic trianglescan also be compact {1,1,1} . Leucine prevails overall other hydrophobic residues in these packing arrangements vaguelyreminiscent of leucine zippers.25 Hydrophobictriangles stabilize Schellman loops by insulating the inner backboneconformations and intramolecular H-bonds importantfor helix-induction.Of all Schellman loops with hydrophobic Conformations of peptide fragments isolatedfrom their native proteinenvironments can be different to those in the parent protein, especiallyif the fragment contains a hydrophobic motif which could pack againstsimilar regions when buried inside the protein. To be sure that isapplicable in this particular case, we examined hydrophobic trianglesfrom our data set above, and found that 87% were fully buried andonly 2% were completely at the solvent accessible surface of the parentproteins.compact triangles : Ac-WAAAKAAAAKAXAAXGX-NH2 (X’s denote these key hydrophobic residues). Peptides usingLeu,Leu,Leu or Leu,Leu,Ile as triangles presented enhanced helicitiesversus a control peptide with no Schellman loops or triangles, butthese effects were small as anticipated . Thus, hydrophobic triangles could show a small but measurableimpact on linear peptide fragments. This observation motivated usto consider more closely how hydrophobic triangles might be mimickedin constrained helical mimics, where the conformationalrestrictions might overcome the unfavorable hydrophilic–hydrophobicsolvent interactions. Pursuit of that strategy led to the design ofthe mimics we call “BSMs” as described below.It follows from the analysis above that trianglesin isolated modelpeptides would be exposed to solvent, and may not adopt the desiredconformation. Nevertheless, we made short peptides with hydrophobictriangles to test if these motifs could measurably enhance helicitiesfor isolated helical fragments in solution. Specifically, we preparedones having the most abundant residue combinations in C3, Ccap, and C″ replaced by three Cys and then capped with TBMB to enforce the structural integrityof the C-cap conformations. Thus, we aspired to makebicyclic Schellman loop mimics . TMB is an ideal framework for compact mimics because its three methylenegroups are spatially equidistant like the corners of the near-equilateralhydrophobic triangle in these {1,1,1} situations.BSMs manifest the key innovation of this work: unification of theassembled hydrophobic triangles into a single trimethyl-benzenoidring 3a. ThatC-cap to form the 12-mer peptidomimetic in Cs denote Cys capped by a TMB fragment). MD simulations wereexecuted over 1 μs (explicit water box at 300 K); this relativelylong simulation allows the mimic to reach stable conformations. Rootmean square deviations (RMSD) of non-H backbone atomsrelative to the “perfect” starting conformation werequantitated as a function of time to track unwinding of this idealstructure. In the experiment, the unconstrained N-terminus (blue in C-cap integrity dominated throughoutafter fluctuation in the first 150 ns (red line). These observationson a simulated system suggested that the BSM framework is rigid andstable.A starting point wasrequired to test the conformationalrigidifyingeffect of BSMs via molecular dynamics (MD). Consequently, we virtuallyamalgamated coordinates of an ideal α-helix and a BSM  blue in 3b contin27 and it proved effective here. Crude tri-Cys peptide precursors couldreact with TBMB at 25 °C to give mainly  desiredproducts within 15 min. Typical LCMS traces of crude products aftercyclization are in Figure S13.CLIPS is emergingas the dominant click reaction in peptide chemistry,C3–C″ and C3–Ccap hydrophobicpatches, respectively, and can be used to examine if the bicyclictriangle mimic is truly superior to monocyclic patch mimics. The fourthconceivable control  was not considered becausethose three residues in the parent Schellman loop are outside thehelical region and would not be expected to exhibit helicity.Threecontrol compounds were made to compare with the featuredBSM-capped peptide, i.e., “bicyclo 12-mer”  spectra of the BSM-containing bicyclo 12-merand the three controls were recorded to evaluate their helicities.At 25 °C, the bicyclo 12-mer (red line) was significantly morehelical than the others 3d. This 30 1. Both m30 3e; the f30 3e, and c30 3f. HowevC-capped helical conformation of bicyclo12-mer. Four characteristiccontacts anticipated for a C-carboxyamidated Schellmanloop were all observed in the ROESY spectrum of the bicyclo 12-mer exceptfor the acetyl groups at the N-terminus . H-bonds characteristic of common Schellman loops. Five hydrogens (2 CαH and 3 NH) with smallerchemical shifts than their colleagues imply that they are probablyshielded by TMB, which is consistent in the simulated conformers:these five hydrogens are behind TMB and therefore affected by itsshielding effect .NMR studies gave further evidencesupporting the proposeddominant o 12-mer4a,b. Relterminus S27. Figuterminus S27. H-boC2 in the BSM corresponds to Ccap. That residueand others downstream of it toward the C-terminusare expected to break from helicity, and the result matches well: C2, G, and C3 indeed do not have helical φ,ψdihedral angles, whereas all the ones anticipated to be helical do.Even AcA1 at the N-terminus, where mostdeviation would be expected, is mostly helical. Finally, dihedralsof G at C′ accurately fall into the regionof left-handed α-helices, as often observed in Schellman loops.A Ramachandran plot 4f featurH/D exchange experiments wereconducted to measure protection of each carbonyl-amide H-bond. Predicted rates of exchange for the amide H’s in random coils were divided by the measured values togive protection factors. A convex curve is usually anticipated inpeptides since H-bonds at the termini are destabilized.However, in the bicyclo 12-mer, there is no obvious fading at the C-terminus. This again marks the solid waterproof wall builtby the TMB fragment. Incidentally, the NH of C2 is particularly protected . Consequently, substitutions ofthree Cys residues to form BSMs would not deter from their use ininterface mimicry, because residues corresponding to hot spots areusually incorporated outside the C-cap. Besides,BSM can also be applied on helical fragments without Schellman loops(C-capping method in peptides to give mimicswith different physiochemical properties.In conclusion, an loops 2d. This eratures 3. Our bi


APOE e4) carrier increases the risk of atherosclerosis, and others suggest that cardiorespiratory fitness (CRF) could play a key role in atherosclerotic prevention. Our aim was to analyze the association of APOE e4 with carotid atherosclerosis and the association of CRF with atherosclerosis in APOE e4 carriers. A cross-sectional analysis based on a subsample of 90 participants in the Aragon Workers’ Health Study was carried out. Ultrasonography was used to assess the presence of plaques in carotid territory; the submaximal Chester Step Test was used to assess CRF; and behavioral, demographic, anthropometric, and clinical data were obtained by trained personnel during annual medical examinations. APOE e4e4 participants were categorized into Low-CRF (VO2max < 35 mL/kg/min) and High-CRF (VO2max ≥ 35 mL/kg/min) groups. After adjusting for several confounders, compared with APOE e3e3, those participants genotyped as APOE e3e4 and APOE e4e4 showed an OR = 1.60  and OR = 4.29 , respectively, for carotid atherosclerosis. Compared to Low-CRF APOE e4e4 carriers, the odds of carotid plaque detection were 0.09  times lower among High-CRF APOE e4e4 carriers. The APOE e4e4 genotype was associated with increased carotid atherosclerosis. However, CRF is a modifiable factor that may be targeted by APOE e4e4 to decrease the elevation of atherosclerotic risk due to this genetic condition.Some studies suggest that being an apolipoprotein e4 ( However, other potential nonmodifiable risk factors could play an important role in this progression.Atherosclerosis is a chronic arterial disease and a major cause of death. The first functional and pathological changes appear in early to mid-life and slowly progress over timeAPOE e4) is one of the best-investigated genetic factors related to cognitive function and cardiovascular disease (CVD)6, probably due to its association with elevated levels of low-density lipoprotein cholesterol (LDL-c) and plasma triglycerides. Although the presence of APOE e4 has been related to increased carotid intima media thickness (cIMT)8, its association with the presence of peripheral atherosclerosis continues to be the subject of debate. In this context, while some studies have reported no associations between APOE e4 and the presence of atheroma plaques12, others have established that APOE e4 could be a genetic risk factor, especially in men16.The apolipoprotein E epsilon 4 allele  reflects the integrated ability to transport oxygen from the atmosphere to mitochondria to perform physical work and is a strong and independent marker of risk for adverse health outcomes, including all-cause mortalityAPOE e4 carrier status with carotid atherosclerosis, and second, to analyze the association of CRF with carotid atherosclerosis in APOE e4 carriers.Therefore, the aims of this study were, first, to analyze the association of 23. These workers were mainly males (93.5%). Among the total genotyped AWHS sample (n = 5322), those individuals genotyped as APOE e4e4 (n = 46) were invited to undergo CRF, physical activity (PA), and subclinical atherosclerosis measurements and to complete diet and behavior questionnaires. Some of the potential participants refused to participate (n = 16), so a total of 30 participants genotyped as APOE e4e4 were assessed and randomly matched by age, sex, educational level and smoking habits with 30 AWHS participants genotyped as APOE e3e4 and 30 AWHS participants genotyped as APOE e3e3 who were invited to undergo the measurements and complete the questionnaires. Therefore, the final sample subjected to this analysis was composed of 90 participants.This cross-sectional analysis was carried out in a subsample of participants belonging to the Aragon Workers’ Health Study (AWHS), a longitudinal cohort study based on data from the annual health exams of 5678 workers of a car assembly plant24. A plaque was defined as a focal structure that protruded into the lumen of the carotid artery at least 0.5 mm or was ≥ 50% thicker than the surrounding intima media thickness. All measurements were analyzed using electrocardiogram gated frames corresponding to end-diastole (R-wave)25. The presence of subclinical atherosclerosis was defined as the presence of at least 1 plaque in the carotid territory. This methodology has been used and described previously26.A Philips IU22 ultrasound system  was used to assess the presence of plaques in the carotid territory on both sides (right and left), which were considered together as a single site, as the two sides share similar hemodynamics. Ultrasound images were acquired with linear high-frequency 2-dimensional probes , using the Bioimage Study protocol27 for the nondominant wrist: (a) SED: ≤ 2859 counts/min, (b) light PA (LPA): 2860–3940 counts/min, (c) moderate PA (MPA): 3941–5612 counts/min, (d) vigorous PA (VPA): ≥ 5613 counts/min, or (e) moderate to vigorous PA (MVPA): ≥ 3941 counts/min. Participant records were considered valid when they covered at least 10 h/day during ≥ 4 days, requiring at least 3 weekdays and 1 weekend day.PA assessment was performed using an ActiGraph GT3X+ accelerometer . Participants were required to wear the device on their nondominant wrist for 7 consecutive days. Accelerometers were initialized to record accelerations at 30 Hz with a dynamic range of ± 6 G. The acceleration records were downloaded and processed with ActiLife v.6.13.4 software . Time in sedentarism (SED) and PA intensities were classified using thresholds previously proposed by Montoye28. The CST required participants to step up and down on a single step to a metronome beat (a prerecorded audio beat). The stepping rate started at 15 steps/min for 2 min (first level) and increased 5 steps/min every 2 min until the fifth level (end of the test) or until participants reached a heart rate of 80% of the predicted maximum (220—age), whichever came first28. At the end of each level, the heart rate and rating of perceived exertion were recorded. Maximum oxygen uptake (VO2max) was predicted by plotting the recorded heart rates on a graphical datasheet, where a visual best-fit line was drawn between data points, projecting the line up to the predicted maximum heart rate and then estimating the matching oxygen uptake value. Among the 30 e4e4 carriers, two CRF groups were created using the median as a cutoff, referred to as the Low-CRF group (VO2max ≤ 35 mL/kg/min) and the High-CRF group (VO2max > 35 mL/kg/min).CRF assessment was performed through a submaximal multistep test, the Chester Step Test (CST)29.DNA isolation from whole blood was performed by using the FlexiGene DNA AGF3000 kit  on an AutoGenFlex 3000 workstation , and genotyping was carried out in the Genetics Unit-Parque Científico de Madrid . DNA samples were spotted onto 384 plates using a Beckman BioMek 2000 automated liquid handler  and were diluted in a mixture consisting of TaqMan Genotyping MasterMix  and a mixture of premade TaqMan SNP genotyping assays: C_3084793_20 (rs429358) and C_904973_10 (rs7412) (Applied Biosystems). qPCR assays were performed in an HT7900 Fast Real-Time PCR System (Applied Biosystems), and SDS 2.4 software (Applied Biosystems) was used for genotype calling. Samples with known genotypes as well as negative amplification blanks were included within each run to serve as positive/negative controls and assist in genotyping. This methodology has been used and described previously30. The Alternate Mediterranean Dietary Index (aMED) score was calculated based on a scale including nine components: whole grains, vegetables (excluding potatoes), fruits (juices included), legumes, nuts, fish, the ratio of monounsaturated fats to saturated fats, red and processed meats, and alcohol. The total aMED score ranges from 0 to 9, with higher scores reflecting greater Mediterranean diet adherence.Diet was assessed based on a 136-item semiquantitative food frequency questionnaire (FFQ) previously validated in Spain31 when triglyceride levels were < 400 mg/dL. We defined arterial hypertension as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or self-reported use of antihypertensive medication32. Dyslipidemia was defined as total cholesterol ≥ 240 mg/dL, LDL-c ≥ 160 mg/dL, HDL-c < 40 mg/dL, or self-reported use of lipid-lowering drugs33. Diabetes was defined as fasting serum glucose ≥ 126 mg/dL or self-reported treatment with hypoglycemic medication32. Smoking habits were categorized as follows: current smoker if the participant reported having smoked in the last year, former smoker if the participant had smoked at least 50 cigarettes in his lifetime but not in the last year, and never smoker. This methodology has been used and described previously26.Study participants reported their age, sex, and educational level. The clinical data included weight, height, body mass index (BMI), blood pressure, medical history, and the current use of medication. Laboratory measurements were performed on blood samples collected under fasting conditions (> 8 h). Total cholesterol, high-density lipoprotein cholesterol (HDL-c), triglycerides, and fasting serum glucose concentrations were determined via enzyme analysis using the ILAB 650 analyzer of Instrumentation Laboratory . Non-HDL-c was calculated by subtracting the HDL-c value from the total cholesterol value. LDL-c was calculated using the Friedewald formula2max, and aMED score. Odds ratios (ORs) for the presence of carotid plaques for each genotype group were calculated using the APOE e3e3 genotype as a reference. In secondary analysis for linear trends for the presence of e4 alleles, APOE genotypes were analyzed as a continuous variable . Additionally, ORs for the presence of carotid plaques in each CRF group were calculated in APOE e4e4 carriers using the Low-CRF group as a reference. A two-sided p value lower than 0.05 was considered statistically significant. Before conducting the analyses and after considering the missing at random or missing completely at random nature, participants with missing data in VO2max (n = 13) or MVPA (n = 8) .Continuous variables are presented as the mean and standard deviation (SD), and categorical variables are presented as percentages and numerical values. The presence of atherosclerotic plaque in carotid arteries was fitted with logistic regression models depending on genotype and adjusted for BMI, hypertension, dyslipidemia, diabetes, MVPA, VOAll methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Clinical Research Ethics Committee of Aragon (CEICA) (PI07/09), and all participants provided written informed consent.APOE e4e4 participants showed higher systolic blood pressure than the APOE e3e3 participants as well as greater adherence to the Mediterranean diet than APOE e3e4 participants. Baseline characteristics of study participants according to APOE genotype are shown in Table APOE e4e4 participants according to the presence of carotid plaques are shown in Supplementary Table The sample included 90 men . The 30 APOE e3e3 group, 60.0% of the APOE e3e4 group, and 63.3% of the APOE e4e4 group , including 36.7% of the up Table . The oddAPOE e3e4 participants than in the APOE e3e3 participants , the difference was not statistically significant  after adjusting the results for BMI, hypertension, dyslipidemia, diabetes, MVPA, VO2max, and aMED score.Although the odds of having a carotid plaque also tended to be higher in the nt Table . HoweverAPOE e4e4 participants who were in the Low-CRF group, 12 presented at least one carotid plaque, and among the 15 APOE e4e4 participants who were in the High-CRF group, at least one carotid plaque was present in 7 participants (Table APOE e4e4 participants who presented High-CRF (VO2max > 35 mL/kg/min) than those APOE e4e4 participants allocated to the Low-CRF group (VO2max ≤ 35 mL/kg/min) after adjusting for age, BMI, hypertension, dyslipidemia, diabetes, smoking status, MVPA, and aMED score  (Table 2max of these 26 participants as a cutoff (median VO2max = 34.3 mL/kg/min), the ORs for carotid plaques were studied. The odds of having a carotid plaque were 0.003 times lower  among those APOE e4e4 participants who presented High-CRF (VO2max > 34.3 mL/kg/min) than those APOE e4e4 participants who presented Low-CRF (VO2max ≤ 34.3 mL/kg/min) after adjusting for age, BMI, hypertension, dyslipidemia, diabetes, smoking status, MVPA, and aMED score 16  and that although the odds of carotid plaque formation tend to be higher in APOE e3e4 carriers than in APOE e3 homozygotes, the association is not statistically significant 13. Also, the results of our study are similar to those observed by Beilby et al., which reported a linear trend with an increasing risk of carotid plaque per unit change in genotype among men with APOE e3e4 and APOE e4e4 in relation to those with APOE e3e3 men 16.Our results regarding the association between the APOE e4 and atherosclerotic carotid plaques after adjusting for major confounders12. As supported by previous studies, adjusting the results based on important confounders such as smoking or hypertension seems to be crucial35. However, most of these studies have not adjusted their results based on relevant and usually overlooked factors, such as PA, CRF, or adherence to specific dietary patterns. This omission could be relevant to the reproducibility of the results36. Additionally, although the total samples included in these studies seem to be reasonable, the number of participants genotyped as APOE e4e4 is very low. For example, only 10 participants among the total sample (n = 544) of the Djoussé et al. study were APOE e4e4 carriers12. Likewise, most of these studies are mainly limited to presenting data separately for APOE e3e4 and APOE e4e4 carriers12, and as supported by this study and the study conducted by Debette et al. only the APOE e4e4 polymorphism is associated with higher odds of carotid plaque formation13.On the other hand, as mentioned above, other studies have reported no associations between APOE e4e4 carriers to contemporary environmental conditions, such as a Western diet, sedentarism, and longer lifespans, could have led this allele to become a susceptibility allele for peripheral atherosclerosis, coronary artery disease, and several neurodegenerative diseases37. However, some lifestyle factors, such as PA and diet, could modulate genetic expression and seem to be key factors modulating susceptibility and reducing the elevated risks of disease and mortality associated with the APOE e4e4 genotype41.The exposure of 42. However, the role of CRF in atherosclerosis development is underexamined. Only two studies have focused on the association of CRF with carotid plaques, revealing an inverse association20. Although higher CRF seems to be a protective factor against atherosclerosis in the general population20, no studies have investigated this potential effect in subjects with increased genetic risk of atherosclerosis, such as APOE e4e4 carriers. To the best of our knowledge, this is the first study that has investigated the association of CRF and carotid plaques specifically in APOE e4e4 carriers, showing that higher CRF is associated with decreased odds of atherosclerosis development. Bearing in mind that atherosclerosis represents an intermediate step toward CVD and death, this finding has high clinical and public health significance.The health benefits of high CRF in the general population are well described in the literatureAPOE e4e4 carriers and other matched participants by randomization. In addition, the study presents the strength of high-quality data collection methods for obtaining information on subclinical atherosclerosis and other variables. However, several limitations of our study should be acknowledged. First, the cross-sectional design does not allow us to establish causal temporal links. Second, the sample includes only Southern European men, and therefore, the results may not be generalizable to women and individuals of other races or ethnic groups in the population. Third, CRF assessment was performed using a previously validated but indirect technique; therefore, VO2max was estimated and was not directly measured. Fourth, cognitive status, which could modify the associations because APOE e4e4 carriers are more vulnerable to cognitive decline, was not evaluated.This study involved a modest sample size but benefited from the inclusion of a significant number of APOE e4e4 genotype but not the APOE e3e4 genotype was associated with higher carotid atherosclerosis prevalence in men independent of traditional and other atherosclerotic risk factors, supporting the previous observations revealing this association. On the other hand, CRF is a modifiable physiological attribute that may be targeted in APOE e4e4 carriers to decrease their increased atherosclerosis risk due to their genetic condition.In conclusion, the Supplementary Tables.