Our previous study shows that LINC01278 inhibits the malignant proliferation and invasion of papillary thyroid carcinoma (PTC) cells by regulating the miR-376c-3p/DNM3 axis. However, the regulation mechanism of LINC01278 expression in PTC cells is still unclear.LINC01278 gene. The RNA immunoprecipitation and RNA pulldown were used to determine the enrichment of LINC01278 in β-catenin protein. The proteasome inhibitors (MG132) was used for detecting the β-catenin ubiquitination-proteasome degradation. Wnt/β-catenin specific agonists (LiCI), inhibitors (WiKI4) and TOP/FOP-flash reporter assay were used for detecting the activation of Wnt/β-catenin signal. Western blot was used to detected the expression of target proteins.The luciferase reporter and ChIP assays were used to confirm the binding of LEF-1 to the putative promoter site of LINC01278 gene, and β-catenin enhances the binding of LEF-1 to the LINC01278 gene promoter. Furthermore, LINC01278 negatively regulated the protein accumulation of β-catenin in the cytoplasm, into nucleus, and ultimately inhibited the transcription of downstream target genes activated by Wnt/β-catenin signal. The results of RNA immunoprecipitation and RNA pulldown proved the direct binding of LINC01278 to β-catenin protein. In addition, the combination of LINC01278 and β-catenin promotes the β-catenin ubiquitination-proteasome degradation.The online PROMO algorithm determines a putative LEF-1 binding site on LINC01278 promoter, the LEF-1 binds to the putative promoter site of In summary, we found the transcriptional activation of LINC01278 by the β-catenin/LEF-1 transcription factor, and the negative feedback regulation of LINC01278 onβ-catenin signal. Thyroid cancer is the most common malignant tumor of the endocrine system , 2. In tLong-chain no-coding RNAs (lncRNAs) are transcripts that are more than 200 bp in length and lack protein coding ability . NumerouTypical Wnt signal transduction is conservative and plays a key role in normal development and cancer progression. In the absence of Wnt ligands, the central protein β-catenin binds to the GSK3β/Axin/APC complex. The latter phosphorylates β-catenin, and the phosphorylated β-catenin is degraded by the ubiquitination-proteasomal system . In the LINC01278 gene, and the negative feedback mechanism of LINC01278 on the post-transcriptional regulation of β-catenin.In this study, we will explore the transcriptional regulation of the β-catenin/LEF-1 transcription complex on the 2.Human thyroid cancer cell lines (TPC-1 and BCPAP) were obtained from ATCC (American Type Culture Collection) , and cultured in DMEM (Dulbecco’s modified Eagle’s medium)  supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin , and incubated at 37 °C in a humidified atmosphere containing 5% COLEF-1, β-catenin and LINC01278 overexpression vectors were bought from Kang-cheng Biotechnology Co . Small interfering RNA (siRNA) against LEF-1, β-catenin and LINC01278 were designed and purchased from RiboBio Co., Ltd. . All vectors and siRNAs were transfected into TPC-1 and BCPAP cells using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. The Wnt/β-catenin specific agonists (LiCI), inhibitors (WiKI4) and proteasome inhibitor MG132 were purchased from Sigma .http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) was used to predict the binding of transcription factors to the LINC01278 promoter sequence [The online PROMO algorithm analysis  and mutant promoter sequence of LINC01278 (MUT) in which the LEF-1-binding site was mutated were cloned into a pGL3-Basic vector (Promega), respectively. The recombinant pGL3-Basic vector was co-transfected into TPC-1 and BCPAP cells with LEF-1 overexpression (LEF-1) or negative control (NC) plasmid. After 48 h, the cells were lysed, and the luciferase activity was analyzed using Dual-Luciferase Reporter Kit (Promega) and Varioskan Lux detection system (Thermo Scientific), which was standardized to Renilla.LINC01278 gene and visualized by agarose gel electrophoresis.Cells were fixed and crosslinked in 1% formaldehyde for 10 min at 37 °C and incubated with protease inhibitors. Chromatin was isolated and enzymatically fragmented using an EZ-Zyme Chromatin Prep Kit . Rabbit anti-LEF-1 antibody  or nonspecific IgG  was used to precipitate DNA crosslinked with the LEF-1. The immunoprecipitated promoter fragment containing the LEF-1 response element was probed by PCR using primers targeting the regulatory region of Cells were lysed using RIPA buffer. The lysate was separated by 10−15% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane . The membrane was blocked with 5% nonfat milk and incubated with anti-LEF-1 , anti-β-catenin , anti-CCND2, anti-CyclinD1 , anti-MYC , anti-SOX4 , anti-Ubiquitin  and anti-GAPDH  antibodies overnight at 4 °C. Then the membrane was washed with TBST three times and incubated with the secondary antibody  for 1.5 h at room temperature. The ECL chromogenic solution was used to display the chemiluminescence of the bands. The Quantity One 4.4.0 software was used for densitometry determination.CT) method was used to measure the LINC01278 level, and GAPDH was used as an internal control. The sequence of primers was as follows: LINC01278, F: 5′-CTGTTGCCCTCCTTCACCTA-3′, R: 5′-TGGTCTACAGGGAGTGCAAG-3′; GAPDH, F: 5′-TGTTCGTCATGGGTGTGAAC-3′, R: 5′-ATGGCATGGACTGTGGTCAT-3′.Total cellular RNAs were isolated and purified using TRIzol reagents (Invitrogen) and cDNA synthesis was performed using PrimeScript RT reagent Kit  according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq TM (TaKaRa) in the ABI 7900HT Fast Real-time PCR system . The comparative cycle threshold (CT)  and FlowJo V7 software .1 × 10Cells were fixed and crosslinked in 1% formaldehyde, and lysed using RIPA buffer supplemented with protease-inhibitor cocktail and RNase inhibitor. The cell lysates were incubated with magnetic beads conjugated with anti-β-catenin antibody (Millipore). Mouse IgG (Millipore) was used as the negative control. The immunoprecipitated RNA was extracted from the eluate, and the enrichment of LINC01278 or control ACTB was analyzed by quantitative RT-PCR.The NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Kit  was applied to isolate and collect cytosolic and nuclear fractions.Cells were seeded in a 96-well plate, and the TOP/FOP-Flash (Genechem) was co-transfected into cells along with LINC01278 siRNA or overexpression plasmid. Luciferase activity was measured using the Promega Dual-Luciferase Reporter Assay System at 24 h after transfection. The luciferase activity was normalized to the Renilla luciferase activity.The purified biotinylated LINC01278 transcripts were purchased from Sangon Biotech . 1 mg of cell lysates was incubated with 3 mg of purified biotinylated transcripts for 1 h at 25 °C. The biotin-coupled RNA complexes were isolated by streptavidin agarose beads (Invitrogen), and the protein expression in the pull-down material was detected by western blot.P < 0.05 was considered as statistically significant.Statistical analysis was performed using SPSS 22.0 software . The data came from at least three independent experiments, evaluated by Student’s t test, and presented as mean ± SD. LINC01278 and determine a putative LEF-1 binding site  of TPC-1 and BCPAP cells . It is wIn order to study how LINC01278 regulates the protein level of β-catenin, we added a proteasome inhibitor (MG132) to TPC-1 and BCPAP cells overexpressed by LINC01278, and found that MG132 relieved the effect of LINC01278 overexpression on β-catenin protein levels Fig. C. These In recent years, the important role of lncRNAs has continued to appear in a variety of physiological and pathological processes, including tumorigenesis. Our previous research found that LINC01278 was significantly down-regulated in PTC tissues and cell lines, and exerted a tumor suppressor function in tumor cells . HoweverWe cloned the LINC01278 promoter sequence containing the wild-type or mutant-type putative LEF-1 binding site into the firefly luciferase reporter system vector. The combination of transcription factor LEF-1 and promoter site can regulate the expression of downstream firefly luciferase. This experiment confirmed the binding of transcription factor LEF-1 to the promoter site of LINC01278. ChIP experiments also confirmed the endogenous binding of the transcription factor LEF-1 to the LINC01278 promoter, and showed that β-catenin can enhance the binding of LEF-1 to the LINC01278 promoter. The results of western blot showed that knockdown or overexpression of LEF-1 or β-catenin and specific agonists or inhibitors of the Wnt/β-catenin pathway can affect the expression level of LINC01278. Our results discovered for the first time the regulatory mechanism of Wnt/β-catenin/LEF-1 signaling on the transcriptional activation of LINC01278.In addition, we discovered the negative feedback regulation of LINC01278 on β-catenin signal. Western blot and TOP/FOP luciferase experiments suggested that LINC01278 negatively regulated the protein accumulation of β-catenin in the cytoplasm, into nucleus, and ultimately inhibited the transcriptional of downstream target genes activated by Wnt/β-catenin signals. The results of RNA IP and RNA pulldown proved the direct binding of LINC01278 to β-catenin. Subsequently, the application of proteasome inhibitors suggested that the combination of LINC01278 and β-catenin can promote β-catenin ubiquitination-proteasome degradation. It is currently known that β-catenin has a variety of regulatory mechanisms, such as APC degradation complex and CTNNBIP1. Here, we reported the regulatory mechanism of a new lncRNA on β-catenin signal. In addition, the relationship between LINC01278 and other Wnt/β-catenin signal regulators needs to be further study.In conclusion, we found the transcriptional activation of LINC01278 by the β-catenin/LEF-1 transcription factor, and the negative feedback regulation of LINC01278 on β-catenin signal. This is the first report on the regulation mechanism of LINC01278 expression, as well as a new mechanism report on the β-catenin signal activation. Although, the specific interaction between them needs more research.


Banana peel PPO exhibited the highest affinity towards dopamine (KM = 0.94 mM). Thermal inactivation of ‘Prata’ and ‘Cavendish’ PPO was achieved at 90 °C after 5 and 15 min, respectively, whereas cold plasma treatment did not decrease PPO activity below 46% of the initial enzyme activity. The inactivation behavior of PPO could successfully be described by a two-fraction model indicating at least two types of isoenzymes with different thermal stability. The overall high thermal stability was mainly attributed to membrane-bound PPO. The results may help to prevent enzymatic browning of banana peels and thereby facilitate their valorization as food ingredients.The objective of this work was to characterize banana peel polyphenol oxidase (PPO) and to study the inactivation kinetics during thermal and cold atmospheric pressure plasma treatment. Since varietal differences in enzyme characteristics are a well-known phenomenon, ‘Prata’ banana peel PPO was characterized, and PPO activity and thermal stability of the peel PPO of the two dessert banana cultivars ‘Cavendish’ and ‘Prata’ were compared to identify the cultivar better suited for industrial food applications. A crude extract obtained from the peels of the Brazilian banana variety ‘Prata’ revealed highest PPO activities (46.0–55.2 nkat/mL) at 30–40 °C in a range of pH 6.0–6.5 after addition of 0.5 g/g Musa acuminata x M. balbisiana) has widespread availability in the Brazilian market with rising sales [Bananas are amongst the economically most important fruits worldwide. Even though the Cavendish subgroup dominates international markets, the banana cultivar ‘Prata’ . While the first category catalyzes the oxidation of monophenols to o-diphenols, the latter oxidize o-diphenols to o-quinones in the presence of oxygen [Banana peels represent a promising source of dietary fiber  and secof oxygen . The quif oxygen .Besides the application of anti-browning agents, suffering from a declining acceptance of the consumers, thermal treatment is the most dependable approach to inactivate fruit enzymes and, thus, overcome enzymatic browning . HoweverM. acuminata peel PPO [M. sapientum L. peel PPO [To effectively control the enzymatic browning during the post-harvest processing of banana peels, a deeper understanding of the PPO present in banana peels of different varieties and their affinity towards different substrates is essential. Whereas banana pulp PPO of different cultivars have been characterized ,21,22,23peel PPO  have beepeel PPO ,25,26. Speel PPO ,21 and PTwo major aspects have to be considered during the extraction of PPO from phenol-rich sources like banana peels . First, In the present study, the main objective was to evaluate the potential of different processes on the inactivation of PPO in a crude enzyme extract of banana peels obtained from two varieties (‘Cavendish’ and ‘Prata’) to enable subsequent prevention of enzymatic browning. For this purpose, an adapted extraction protocol for banana peel PPO was established along with characterizing substrate specificity and optimum reaction conditions. Furthermore, the stability of PPO towards thermal and PPA treatment was examined, and inactivation kinetic parameters were derived. These parameters are required to predict suitable process conditions and thereby enable the efficient design of PPO inactivation treatment. This work provides (i) information about the extraction and characterization of ‘Prata’ banana peel PPO, (ii) a comparison of thermal stability of different dessert banana PPO, (iii) insights into the inactivation kinetics of thermally treated crude, soluble, and membrane-bound banana peel PPO, and (iv) data about the inactivation kinetics of banana peel PPO treated with CAPP, for the first time.Musa acuminata x M.balbisiana, genomic group AAB, Latitude 9 Exportação, Petrolina PE, Brazil) and ‘Cavendish’ bananas  were used as raw material. At least 1 kg of bananas was washed. The fruit apex and the stem were removed before the fruits were peeled. The peels of each cultivar were pooled and pre-comminuted with a knife. After freezing the peels at −50 °C they were placed in a freeze dryer  for 48 h. The pressure was adjusted to 1.030 mbar, and the condenser temperature was set to −55 °C. The freeze-dried peels were ground in a Grindomix GM 200 knife mill  for 20 s to obtain banana peel flour and stored at −20 °C until PPO extraction.Brazilian ‘Prata’ bananas , except for disodium hydrogen phosphate and citric acid, which were sourced from Merck , bovine serum albumin (BSA) and Bradford dye, which were from Bio-Rad , and polyvinylpolypyrrolidone (PVPP) obtained from Honeywell Fluka .g for 20 min at 4 °C . Supernatants were collected after filtration through Whatman No. 1 filter paper  and stored as aliquots at −20 °C for up to two weeks. Examined extraction parameters were changed according to a one-factor-at-a-time model. All extractions were performed in triplicate.Previously described extraction protocols applied for banana pulp and apple PPO were specifically adapted to meet the requirements for determining PPO activity in banana peels ,25. For v/v) Triton X-114. After sonication in a Sonorex Digitec DT 100 H ultrasonic bath  for 10 min, the sample was centrifuged again as described above. The resulting supernatant was then subjected to a temperature-induced phase partitioning. While increasing the concentration of Triton X-114 to 8% (v/v), the temperature was kept constant at 4 °C for 30 min. After heating the sample to 35 °C for 15 min, the fluid turned turbid due to the formation of mixed micelles containing hydrophobic compounds. Centrifugation at 20,000× g for 20 min at 25 °C resulted in a detergent-rich phase and a clear supernatant, with the latter containing peripheral membrane proteins being used as crude mPPO extract.To extract fractions of soluble (sPPO) and membrane-bound PPO (mPPO), a method described by Zhou et al.  and Zainv/v) dye concentrate diluted in demineralized water and filtered through Whatman No. 1 filter paper was added. After incubating for 10 min at room temperature, the absorbance was measured at 595 nm with a microplate spectrophotometer . All solutions were analyzed in triplicate.The protein content of the crude enzyme extracts was determined using an assay kit (Bio-Rad Protein Assay) adapted from Bradford  with bov−1 cm−1 were used to quantify the PPO activity. All measurements were carried out in duplicate.The PPO activity was determined spectrophotometrically according to Schweiggert et al. . Unless 2HPO4 until the respective pH was reached. The enzyme activity was measured at 25 °C.To further characterize ‘Prata’ banana peel PPO, the pH optimum of the crude PPO was determined in McIlvaine buffers ranging from pH 4 to 8. The pH was adjusted by adding 0.1 M citric acid to 0.2 M NaThe optimal temperature of the crude PPO was determined in McIlvaine buffer at optimal pH (pH 6.5) in the temperature range from 20 to 70 °C. Reaction buffer and L-proline were incubated in an Eppendorf Thermomixer comfort  at 600 rpm at respective temperatures. After adding the enzyme extract, 4-methylcatechol was added, and enzyme activity was measured.The substrate specificity of PPO was tested for selected mono- and diphenols. Dopamine and chlorogenic acid were diluted in McIlvaine buffer (pH 6.5) and added to the reaction mixture to obtain a final concentration in the range of 0.25 to 10 mM. Due to the limited solubility of L-tyrosine, final concentrations in McIlvaine buffer were between 0.25 and 2 mM. PPO activity was determined at the absorption maximum of each reaction product, which was 475, 400, and 400 nm for dopamine, chlorogenic acid, and L-tyrosine, respectively.N-prolyl-o-benzoquinone, was studied. For this purpose, the concentration of 4-methylcatechol in the final reaction mixture was varied in the range of 0.25 to 10 mM, while a constant L-proline concentration was maintained.In addition, the effect of the concentration on the coupling reaction of 4-methylcatechol and L-proline, which leads to the formation of 5-methyl-4-KM) and the maximum velocity of reaction (Vmax) according to the Michaelis–Menten equation by non-linear regression with OriginPro 2018b . The Michaelis–Menten constant KM and the maximum velocity Vmax represent the PPO’s affinity against a substrate and the maximum product formation rate. The substrate conversion efficiency, or catalytic power (Vmax/KM), was calculated from the resulting values.PPO activity and respective substrate concentrations were used to estimate the Michaelis–Menten constant  described by Kramer et al.  with sliAfter specified treatment times, residual activities of the PPO were used for the estimation of parameters via non-linear regression using non-linear least-squares analysis by OriginPro 2018b. In order to describe the inactivation behavior of banana peel PPO, different models were used.tA and A0 are defined as enzyme activity of the treated crude PPO after time t and the native PPO, respectively, t is the treatment time, and k is the inactivation rate constant.First-order kinetics (Equation (1)) is the most common model applied for the description of the influence of thermal treatment on enzyme activity ,15,31 antA and A0 are the enzyme activities of the treated and the native crude PPO, t is the treatment time, Sk and Lk are inactivation rate constants of the stabile and labile fraction, and α represents the active fraction of the labile isoform in relation to the total activity.Considering that several isoenzymes with different stabilities might be present in the crude PPO extract, biphasic models such as the two-fraction model can also be used to describe the inactivation behavior of enzymes. The two-fraction model displayed in Equation (2) was used to model inactivation kinetics of enzymes such as PPO ,32.(2)AtA and A0 are the enzyme activities of the treated and the native crude PPO, t is the treatment time, b is the scale parameter, and n is the shape parameter. In contrast to Equations (1) and (2), the Weibull model is based on an empiric rather than a kinetic approach. Empiric models tend to be more flexible as they do not follow physical laws [Additionally, the Weibull distribution model, according to Equation (3), was evaluated regarding the approximation of the inactivation kinetic.cal laws ,33.2adj) and root-mean-square error (RMSE), respectively [The fit of the models was evaluated in terms of the suitability of the model to describe the variance of the measured values and the difference of measured values from the predicted values using the adjusted coefficient of determination (Rectively . Besidesectively .n = 3) ± standard deviation. Statistical analysis was performed by one-way analysis of variance (one-way ANOVA) if the normality test (Shapiro–Wilk test) was passed. Means were compared using the Tukey test (p < 0.05) to determine significant differences amongst samples.All values are expressed as the mean of at least three experiments  by adding at least 0.5 g PVP to the extraction buffer. The high content of phenolics, which was previously reported for banana peels [Specific PPO activity in crude enzyme extracts could solely be increased significantly  after the addition of Triton X-100, which indicates unspecific overall solubilization of membrane-bound proteins resulting in increased protein contents of the crude PPO extracts. Varying liquid-to-solid ratio (p ≥ 0.237), extraction time (p ≥ 0.37), and homogenization time (p ≥ 0.22) had no significant influence on the specific and volumetric PPO activities. However, prolonged extraction time of 24 h at 4 °C resulted in a significant specific PPO activity loss (p = 0.035) compared to extraction times between 0 and 10 h in the presence of 0.5 g PVP and 0.5% (v/v) Triton X-100. Additionally, homogenization using an UltraTurrax did not significantly increase PPO activity, which is in contrast to previous findings displaying the necessity for homogenization when using fresh banana samples [At the same time, no significant differences of specific PPO activities were observed  might be explained by variations of the raw materials, despite the use of banana peels with the maturity degree 7 (yellow with a few dark spots) determined by the color chart  and a pov/v) Triton X-100 was carried out without any homogenization or extraction step. Thus, specific PPO activities of 143.6 and 269.1 nkat/mgprotein were achieved for crude PPO extracts from ‘Prata’ and ‘Cavendish’ peels at the adapted extraction procedure, equaling to 222.5 and 417.1 Units/mgprotein, respectively, with one unit of enzyme activity being defined as a change in absorbance of 0.001 per min. In comparison to the specific activity of M. sapientum peel PPO of 2250 Units/mgprotein reported by Yang et al. [In conclusion, the extraction of 1 g lyophilized banana peels in 30 mL McIlvain buffer pH 6.5) with the addition of 0.5 g PVP and 0.5% , while a KM of 7.54 mM was shown for chlorogenic acid. Moreover, the maximum product formation rate represented by a Vmax of 104.5 Units was higher for dopamine. Thus, the resulting substrate conversion efficiency of crude banana peel PPO for dopamine was 110.8 Units/mM, which is more than ten times higher than the one observed for chlorogenic acid with 10.4 Units/mM.For the tested diphenolic substrates dopamine and chlorogenic acid the ‘Prata’ banana peel PPO followed Michaelis–Menten kinetics . The lowKM value for dopamine is in a similar range as the values reported for PPO from banana peels [KM value of ‘Prata’ banana peel PPO for chlorogenic acid was twice as high as the KM of a dwarf banana pulp PPO (4.1 mM) [Vmax of 0.085 and 0.062 ∆A per min, corresponding to a Vmax of 85 and 62 Units for banana root PPO [The observed na peels , banana na peels ,17,18, ana peels . The KM 4.1 mM) , reflectroot PPO . The lowroot PPO , might b.1 mM , rroot PPO .KM, Vmax, and substrate conversion efficiency of 4.01 mM, 390 Units, and 97.3 Units/mM were observed, respectively. Comparing the apparent KM value for the oxidation of 4-methylcatechol in the presence of L-proline to previous investigations using solely 4-methylcatechol, our result is half of the KM value obtained for banana root PPO (8.3 mM). In contrast, the obtained apparent Vmax is comparable to the reported Vmax of 0.417 ∆A per min, corresponding to 417 Units, for banana root PPO [N-prolyl-o-benzoquinone [KM value of the adduct formation is comparable to the values obtained without L-proline as a coupling agent. Additionally, the apparent Vmax of the proline-coupled assay is reduced by one-half compared to the direct assay. Thus, our results for the proline-coupled assay indicated a higher affinity and a higher maximum product formation rate of banana peel PPO in comparison to banana root PPO [To increase the sensitivity of the PPO assay, the product of the coupling reaction of the oxidized diphenol 4-methylcatechol and L-proline was also used to quantify the PPO activity . Similarroot PPO . Becauseroot PPO .p = 0.308) for dopamine and 4-methylcatechol. The sensitivity, indicated by the volumetric PPO activity, was increased to 1673 Units/mL by the coupling reaction of 4-methylcatechol and L-proline probably due to the reaction product’s high extinction coefficient of ε = 1550 L mol−1 cm−1 [However, due to the high apparent maximum product formation rate, the catalytic power of the banana peel PPO did not differ significantly p = 0.8 for dopl−1 cm−1 .The temperature stability profiles of the PPO extracted from ‘Prata’ and ‘Cavendish’ banana peels were investigated for temperatures between 60 and 90 °C . CompariIn general, increasing the temperatures and times resulted in a decreased residual PPO activity of crude PPO extracts from both peels, exhibiting similar inactivation curves. At 60 °C, an almost linear decrease in residual PPO activity was observed, whereas at higher temperatures, the inactivation was fast at short treatment times, while the inactivation velocity decreased over time. For instance, at 60 and 70 °C residual PPO activities were above 5% even after treatment for 120 min. Accordingly, the inactivation of crude peel PPO was insufficient for both banana varieties. In contrast, an almost complete PPO inactivation below 0.5% residual activity was achieved for the thermally more stable ‘Cavendish’ cultivar after incubation at 80 °C for 60 min or at 90 °C for 15 min, respectively.A comparison of the thermal stability of the two cultivars revealed that the treatment of the crude PPO extracts above 60 °C resulted in a faster decrease of ‘Prata’ banana peel PPO activity compared to ‘Cavendish’ banana peel PPO. However, ‘Prata’ banana peel PPO showed less susceptibility to thermal treatment at 60 °C. Therefore, it seems that the ‘Prata’ banana peel PPO contains a higher ratio of thermally stable PPO, which, in turn, is more easily inactivated than the thermally stable ‘Cavendish’ peel PPO.Chaisakdanugull et al.  reported−1 at 60 °C to 0.197 min−1 at 90 °C was observed, reflecting an increasingly fast inactivation.Based on the thermal stability data of crude ‘Cavendish’ banana peel PPO, different models were evaluated regarding their potential to describe the inactivation kinetics. Considering the estimated parameters of first-order models obtained by non-linear regression , an incr2adj and a low RMSE indicate that the inactivation of the ‘Cavendish’ banana peel PPO at higher temperatures (80 and 90 °C) followed a first-order inactivation model, which is also reflected by a good fit of the curve to the data  and membrane-bound PPO (mPPO) from ‘Cavendish’ banana peels.Sequential extraction of soluble and membrane-bound PPO was applied to ‘Cavendish’ banana peels to investigate the presence of different isoenzymes with diverse thermal stabilities. A similar approach was previously described for apple , pear 2, and snaFollowing the sequential extraction, the two PPO fractions (sPPO and mPPO) were subjected to thermal treatments at 70 and 80 °C. We observed that the mPPO is thermally more stable than the sPPO at both temperatures c,d. For Lk value, while the relative amount of the labile fraction of the mPPO decreased from 81% to 0%. Therefore, the inactivation of mPPO resembles a pseudo-first-order kinetics, which is in accordance with the results of the inactivation trials with the crude PPO extract. Despite this pseudo-first-order inactivation kinetics at 80 °C for crude PPO, two fractions could be observed for the sPPO at both temperatures with a constant relative amount of 63%. However, high uncertainty of the estimated parameters and the similarity of the curve fitting obtained by first-order and two-fraction model  was assumed with the highest affinity towards dopamine. As 4-methylcatechol exhibited a similar conversion efficiency like dopamine, and it does not naturally occur in banana peels to a great extent, the coupling reaction of 4-methylcatechol was used to increase the sensitivity of the assay when determining PPO activity in this study. By adapting the extraction protocol for crude banana peel PPO, the volumetric and specific PPO activity could be increased 64.2- and 3.9-fold, compared to the use of McIlvaine buffer without additives. By adapting the extraction process and characterizing the PPO, we could provide optimal extraction and assay conditions for banana peel PPO. A comparison of ‘Cavendish’ and ‘Prata’ cultivars revealed a lower PPO activity and stability of ‘Prata’ banana peel PPO. For this reason, a faster thermal stabilization can be assumed for the latter, which might result in higher retention of nutritional and organoleptic quality.This study indicates that the application of PPA in the chosen experimental set-up does not appear to be a promising approach for the inactivation of banana peel PPO due to insufficient inactivation, even after long treatment times. In contrast, thermal treatment at high temperatures (80–90 °C) resulted in complete inactivation within one hour or less for ‘Cavendish’ banana peel PPO. As the mPPO seems to predominate the inactivation behavior during thermal treatment, future studies on banana peel PPO should pay attention to solubilize the mPPO during the extraction by utilizing an appropriate detergent. Therefore, the results of the present investigations are the basis for complete utilization of banana peels as high-quality food ingredients and a good source of dietary fibers by inactivating endogenous PPO. Besides, further research is needed to evaluate a potential pesticide accumulation during processing, as well as strategies for the removal of pesticides to ensure food safety and the production of high-quality banana peel ingredients.


Epoxy polymer composites embedded with thermally conductive nanofillers play an important role in the thermal management of polymer microelectronic packages, since they can provide thermal conduction properties with electrically insulating properties. An epoxy composite system filled with graphitic-based fillers; multi-walled carbon nanotubes (MWCNTs), graphene nanoplatelets (GNPs) and ceramic-based filler; silicon carbide nanoparticles (SiCs) was investigated as a form of thermal-effective reinforcement for epoxy matrices. The epoxy composites were fabricated using a simple fabrication method, which included ultrasonication and planetary centrifugal mixing. The effect of graphite-based and ceramic-based fillers on the thermal conductivity was measured by the transient plane source method, while the glass transition temperature of the fully cured samples was studied by differential scanning calorimetry. Thermal gravimetric analysis was adopted to study the thermal stability of the samples, and the compressive properties of different filler loadings (1–5 vol.%) were also discussed. The glass temperatures and thermal stabilities of the epoxy system were increased when incorporated with the graphite- and ceramic-based fillers. These results can be correlated with the thermal conductivity of the samples, which was found to increase with the increase in the filler loadings, except for the epoxy/SiCs composites. The thermal conductivity of the composites increased to 0.4 W/mK with 5 vol.% of MWCNTs, which is a 100% improvement over pure epoxy. The GNPs, SiCs, and MWCNTs showed uniform dispersion in the epoxy matrix and well-established thermally conductive pathways. Epoxy resin is preferred as the ideal thermal management material (TMM) for packaging in the power and electronic industries because of its superior electrical insulation, corrosion resistance, adhesion, easy processing ability, light weight, and low cost . HoweverHigh-aspect-ratio nanoparticles have received particular attention in the development of an effective filler for this purpose, since they can effortlessly demonstrate thermally conductive pathways in polymer composites . These nOn the other hand, ceramic-based fillers, such as aluminium nitride, aluminium oxide, boron nitride, and silicon carbide (SiC), have long been regarded as highly thermally conductive ceramic particles with good electrical insulating properties ,14. The Many studies have shown that adding graphite-based, thermally conductive fillers to the polymer matrix improves thermal conductivity ,17,18, bThe dispersion of fillers in the matrix material is one of the main difficulties in the manufacturing process of particulate composites. However, well-distributed nanofillers can lead to new and unique composite properties, such as enhanced stiffness and toughness, with minimal filler loading . DissolvThe novel aspect of this work is the utilisation of methyl ethyl ketone (MEK) as the solvent for the solution mixing method. This simple method was implemented as a form of double dispersion; MEK was used to premix the filler in the ultrasonic first, and later mixed again in planetary centrifugal mixing with the presence of epoxy and the absence of air. Because most of the solvent (MEK) could be removed during the mixing process, this approach is different from others, which require an additional step to completely remove the used solvent after the mixing process ,27. AddiIn this study, the effect of three different nanofillers, MWCNTs, GNPs, and SiCs, on the physical, thermal transport, and compressive properties of epoxy nanocomposites was investigated. Therefore, neat and reinforced nanocomposite bulk samples were manufactured using a simple approach of processing and further compressing them during hardening. Next, the neat and epoxy nanocomposites were investigated for their microstructures and physical, mechanical, and thermal conductivity. The compression strengths of the neat and epoxy nanocomposites were experimentally obtained and compared. Moreover, the fractured surfaces of the composites were analysed using scanning electron microscopy (SEM) fractography.This paper is a preliminary part of a project on developing heat-conductive composites with electrical insulators that are reinforced with nanofillers for high-end applications, such as thermal management materials (TMMs). An ideal TMM must have excellent thermal conductivity to address heat dissipation issues. It must be able to withstand harsh environments, such as high temperature and high humidity, over a long period, without the degradation of its thermal conductivity. Generally, these environments require the materials to have very high thermal conductivity, around >2.5 W/mK. High thermal conductivity, high electrical resistivity, low coefficient of thermal expansion, and non-toxicity are the main reasons why these three materials were chosen as the nanofiller embedded into the epoxy matrix. Although the thermal conductivity values were far short of those at which we aimed, the materials are still under development.3, respectively. The weight ratio of epoxy resin to curing agent was 4:1, as suggested by the manufacturer. Methyl ethyl ketone (MEK) was purchased from Merck Sdn. Bhd  and used as a dispersion solvent. The properties of the fillers utilised in this study are listed in GNPs 0540DX and SiCs 6820HK were provided by SkySpring Nanomaterials Inc. . Hydroxyl-functionalised MWCNTs UCNT-COOH were provided by United Nanotech Innovations Pvt. . The GNP and SiC nanoparticles were used as obtained, without any further functionalisation. An epoxy resin called EpoxAmite 100 was used as a matrix in this study, and the selected curing agent was EpoxAmite 103 Slow. Both chemical products were manufactured by the Smooth-On Inc. . and supplied by Castmech Technologies Sdn. Bhd . The densities of the epoxy resin and the curing agent were 1.10 and 0.96 g/cmThe as-received nanoparticles were dispersed in a solution comprising MEK and epoxy resin through ultrasonication at 40% amplitude for 45 min to break any agglomerations between the nanoparticles. The mixture’s temperature was always maintained below 20 °C using an ice bath to avoid an increase in temperature during the sonication process. After the completion of the dispersion stage, the suspension comprising epoxy, MEK, and nanoparticles was mixed via a planetary centrifugal mixer , at a rotation speed of 2000 rpm for 5 min and a revolution speed of 1000 rpm for 1 min to disperse the particles in the resin. Next, the curing agent was added to the obtained dispersion’s mixture, and the mixture was stirred for 2 min at a rotation speed of 2000 rpm and then for the 30s at a rotation speed of 1000 rpm. As most of the solvent (MEK) can be removed during the mixing process, this method differs from others for making filler-based composites because it ensures good dispersion of fillers as the solvent level decreases . After tdryW) and then weighed after submerging in water, (wetW). From these data, the density (ρ) of the sample was calculated using Equation (1). Further, the theoretical density was calculated for each composite formulation using Equation (2). In these equations, iϕ is used for the weight fraction of the constituents; ‘iρ’ is the density of the constituents, and ‘Theoρ’ is the calculated theoretical density.The sample densities were determined using Archimedes’ principle and an electronic scale (Mettler Toledo). The samples were first weighed while dry  were used to hand-polish the samples. Thermal conductivity of both the pure epoxy and epoxy nanocomposites was measured using Hot Disk TPS 2500s , a hot disc thermal analyser, through the transient plane source (TPS) method. In the TPS method, an electrically insulated flat nickel sensor was placed between two substrate pieces. The sensor served as a heater and a thermometer at the same time. During the measurement, a current pulse was passed through the sensor to generate a heatwave. The rise in temperature as a function of time was used to determine the material’s thermal properties. The time and input power were chosen so that the heat flow was contained within the sample boundaries, and the temperature rise of the sensor was unaffected by the sample’s outer boundaries. A series of four measurements was recorded in each sample with a suitable time interval between the measurements. This time interval allowed the sample to revert to its initial state. Typically, the difference between these measurements was 0.05 W/mK.gT) of the pure epoxy and epoxy nanocomposites was measured using the Perkin Elmer DSC 8000  instrument. The measurements were performed in a dry atmosphere, a temperature range of 50 °C to 250 °C, and a heating rate of 10 °C/min under a nitrogen atmosphere. The nitrogen flow rate was kept constant at 50 mL/min.The glass transition temperature , was used to analyse dynamic thermogravimetry from 25 °C to 800 °C. During the analysis, nitrogen flow was kept at a constant rate of 50 mL/min and a heating rate of 10 °C/min. The sample was weighed at 10 mg and placed in a 100 mL crucible according to the ASTM E1131 procedure.The compression performance of the epoxy nanocomposites was determined through a compression test using Instron-3369 , a universal testing machine. The samples used were in the form of a cylinder with a diameter of 12.7 mm and 5 mm thickness as per the ASTM D 695-15 standard. The compression rate was maintained at 1 mm/min throughout the test. The compression strength and modulus of the composites were determined using five replicates each, and the average values were reported.The morphologies of the fractured surfaces of the epoxy nanocomposites were characterised using SEM TESCAN VEGA 4 . The fractured parts of the samples were trimmed before scanning, and gold was properly coated throughout the samples’ surfaces. SEM images were obtained at accelerating voltages ranging from 5 to 15 kV and magnifications ranging from 100× to 1000×.However, the agglomeration occurred at higher SiC loading and created voids and stress concentration sites that resulted in the failure of the nanocomposites. The epoxy/SiCs 5′s density decreases might have been due to the void formation in the samples. If more SiCs were added to the epoxy nanocomposite mixture, the viscosity of the epoxy nanocomposite mixture would increase dramatically, and it would turn into a stiff paste at around 5 vol.%. More than 5 vol.% of SiC content could not be achieved for the SiC particles because, for the same SiC content, the surface-area-to-volume ratio increased as the particle size decreased because of the high aspect ratio, resulting in the epoxy matrix failing to bind the nanofillers. This could have been attributed to the filler sedimentation in the suspension and unfilled spaces in the samples themselves . With suThe thermal conductivity and diffusivity of various epoxy nanocomposite systems containing MWCNT, GNP, and SiC nanofillers were examined, and the results are presented in It was discovered that the epoxy matrix had a thermal bridge created by the MWCNTs and GNPs, which improved heat transfer. As a result, the thermal chains created between the fillers improved the epoxy nanocomposites’ thermal conductivity. Both epoxy/MWCNTs and epoxy/GNPs presented an increase in thermal conductivity because MWCNTs and GNPs have extremely high thermal conductivities , the gT values of the epoxy composites incorporated with GNPs and SiCs were enhanced at 30.58 °C and 33.04 °C, respectively. With the addition of the MWCNTs, GNPs, and SiCs to the epoxy matrix, the gT values increased, indicating that a robust interface was formed by the reaction of nanofillers with the matrix molecules during the curing process. This generated more obstacles to impede the macromolecular chain motion, resulting in greater gT values and enhanced thermal stability. The addition of nanofillers to the epoxy polymer acted as an obstruction and increased the variability of the crosslinked structure [gT values of the epoxy nanocomposites. The obtained results were also validated by other researchers [g.TIn this study, DSC was employed to compare the igations . The Tg tructure . Variabiearchers ,40. IncoThe thermal stabilities of the pure epoxy and epoxy nanocomposites were obtained using TGA analysis, as shown in −1 determined in the thermogravimetric analysis. This observation is consistent with that of Guo et al. [The second stage of decomposition occurred at approximately 320 °C, representing the decomposition of pure epoxy and epoxy in the matrix , as wello et al. . Accordio et al. . In thisThe third stage of decomposition was initiated at temperatures exceeding 400 °C, which involves the elimination of amorphous carbon. Wei et al.  obtainedAt the last stage, at temperatures above 500 °C, the complete decomposition of the epoxy network was observed for the pure epoxy polymer, which tended to have nearly no remaining components. However, in the case of the epoxy nanocomposites reinforced with MWCNT, GNP, and SiC nanofillers, char was produced throughout under a high-temperature range. Due to greater crosslinking in the presence of nanofillers combined with homogeneous dispersion, the thermal degradation profile of the epoxy nanocomposites was improved by adding nanofillers. As a result, the thermal degradation was higher compared to that of the cured pure polymer . The comCompressive stress–strain curves for the pure epoxy and epoxy nanocomposites filled with MWCNTs, GNPs, and SiCs at a filler loading of 4 vol.% are shown in The compressive modulus of the epoxy/MWCNT 4 was 7.01× greater than that of the pure epoxy, which was 0.498 GPa. With 0.484 GPa, the compressive modulus of the epoxy/GNP 4 appeared to be the highest of the epoxy/GNP composites, demonstrating a 6.82× increment over the pure epoxy. On the other hand, the epoxy/SiC 4 demonstrated the greatest compressive modulus of all the epoxy/SiCs at 0.476 GPa, representing a 6.70× increase over the pure epoxy. Similarly, the compressive strength of the wpoxy/MWCNTs filled with 4 vol.% MWCNTs was found to be the highest, increasing by up to 192% (158.64 MPa) when compared to the pure epoxy. In the case of the epoxy/GNP composites, the compressive strength composite filled with 4 vol.% GNPs displayed the highest compressive strength, with an enhancement of up to 148% (134.50 MPa) above the pure epoxy. Finally, for the epoxy/SiCs, the compressive-strength composite filled with 4 vol.% SiCs demonstrated the highest compressive strength, increasing up to 96% (106.26 MPa) above the pure epoxy. As mentioned above, this indicates that the epoxy nanocomposites created using this method showed greater dispersion and distribution. However, further filler loadings greater than 4 vol.% compressive strength and modulus showed a decreasing trend because the nanofillers formed larger agglomerations in the epoxy matrices, making it easier for the overlapped tubes, sheets, and particles to slide past one another. This led to early crack initiation in the composites due to the nanofillers’ agglomeration, which created difficulty in dispersing the filler at higher loadings, particularly with the large surface area of the MWCNTs, GNPs, and SiCs. This is comparable to the report by Sheshkar et al. . On the The compressive strength of the epoxy/SiCs 5 nanocomposites demonstrated a considerable decrease while the compressive modulus slightly reduced compared with the epoxy/MWCNT 5 and epoxy/GNP 5 nanocomposites. This also proved that SiCs, as ceramic-based fillers, can produce stiffer composites than graphite-based fillers at equivalent filler loadings. The surface area of the SiCs used in this study was lower than those of the MWCNTs and GNPs. Thus, the interfacial area of the SiCs with the epoxy was lower than that of the MWCNTs and GNPs with the epoxy matrix. The lower interfacial contact area for the former meant that the SiC nanofillers could develop much more extensive interconnections than those between the graphite-based fillers due to their nanotubes and platelet morphology. This is discussed in more detail in the following section (on the fracture surface morphology). Consequently, there was less interstitial resin to interfere with the SiC particle interactions, thereby allowing more interactions between the SiC particles under compression. When the epoxy/SiCs 5 nanocomposites lost strength, they were less compliant. Meanwhile, the higher interfacial area between the MWCNT and GNP nanofillers and resins and the greater dispersion of MWCNTs and GNPs on a sub-microscopic level allowed fewer interconnections between the fillers. Therefore, less interconnectivity was required between the nanofillers because the interfacial area between the MWCNT and GNP nanofillers was larger, and the MWCNTs and GNPs were dispersed more evenly. The epoxy could be deformed before the stress was transferred to the fillers, resulting in composites with improved strength and compliance. This comparison suggests that the graphite-based fillers (MWCNTs and GNPs) produced composites with a higher thermal conductivity than those prepared using the ceramic-based filler composites at significantly lower filler loadings, producing more compliant composites than in the latter case. Epoxy resin reinforced with graphite-based and ceramic-based fillers has similar compression strength behaviour, as reported by Hemath and Selvan  and RazaThe fracture surfaces of the pure epoxy and epoxy nanocomposites were evaluated using SEM, as shown in Therefore, optical microscopy and Raman imaging analyses can be used to determine the regularity of the agglomeration and distribution of MWCNTs, while SEM images can reveal whether the MWCNTs are disentangled, as suggested by Gardea et al. . The MWCHowever, at the fracture surface of the epoxy/SiC 5, the agglomeration of nanoparticles and the formation of air voids were visible, as can be seen in Among these nanofiller materials, the MWCNTs showed the highest compressive strength and modulus, followed by up to 4 vol.% filler loadings of GNPs and SiCs, with an increase of 266%, 240%, and 224%, respectively, compared to the pure epoxy. This was because the MWCNTs prepared had a greater dispersion and distribution in the epoxy matrix. Since the MWCNTs were hydroxyl-functionalised MWCNTs, the chemical modification improved the interfacial adhesion through amine linkages; therefore, the MWCNTs fillers could create additional interfaces between the filler-to-filler and filler-to-resin, resulting in a better filler–matrix interaction and enhancing the stress transfer from the epoxy matrix to the MWCNT nanofillers. However, all the nanocomposites above 4 vol.% filler loadings showed a decreasing trend in compressive strength and modulus because many agglomerations occurred in the composites, which became the stress-cracking points of the composites.The thermal stability of the MWCNTs was slightly lower than those of the GNPs and SiCs. The low thermal stability of the functionalised MWCNTs was attributed to the oxygen-containing groups. Despite this, the thermal conductivity of the epoxy/MWCNTs was higher than those of the Epoxy/GNPs and Epoxy/SiCs. Furthermore, the SEM analysis revealed that the fillers were close to each other in the epoxy matrix, forming a promising pathway for phonons to travel in the epoxy matrix. Thus, less scattering of phonons occurred, and more phonons could move at a faster transmission rate through the conductive pathways, leading to an increase in thermal conductivity.The morphological analyses indicated that the nanocomposites failed under compression loading in typical modes, such as filler pull-out, debonding, and matrix cracking. For most of the nanocomposites, the stress-cracking points were initiated by the agglomeration of the nanofillers, propagated through the huge agglomerated fillers and, lastly, transferred to the void-filled epoxy matrix.Thermally conductive epoxy composites have been utilised in various electronic applications, such as thermal interface materials, electronic packaging, and energy storage devices. The effects of different nanofillers on epoxy nanocomposites’ mechanical and thermal properties were investigated. The graphite-based fillers, MWCNTs and GNPs, were more effective reinforcements for the epoxies concerning mechanical and thermal conductivity than the ceramic-based fillers, SiCs. However, these inorganic fillers often became sedimented during the curing reaction of the epoxy matrix. The relatively uniform dispersion of the MWCNT and GNP nanofillers was achieved using ultrasonication combined with planetary centrifugal mixing, but this was not the case for the SiC nanofillers, where significant agglomeration was observed in each of the filler loadings. This was possible in the epoxy/SiCs at very low loadings because of the nanoparticles’ highly dispersive surface energy, which produced spontaneous re-agglomeration. The following conclusions can be drawn from the study’s findings:


The self-report questionnaire was prepared to collect demographic and epidemiological variables of interest and the COVID-19-related information. All participants also completed the Profile of Mood State (POMS), General Health Questionnaire-28 (GHQ-28), and Satisfaction with Life Scale (SWLS). The main result is that the training rate, mental health, life satisfaction, and positive mood have decreased during the home confinement period as compared with the reopening and semi-lockdown phases. However, the need for psychosocial services has increased during the pandemic period. The present study provides the first preliminary evidence that home confinement conditions during the COVID-19 pandemic might have negatively influenced elite athlete’s mood state, mental health, and life satisfaction, as well as training rates. Monitoring the psychological parameters of elite athletes and developing strategies to improve their mental health during the COVID-19 pandemic should be on the agenda. Next studies, therefore, seem reasonable to focus on active interventions for athletes during the ongoing COVID-19 pandemic.Scientific reports notified that the pandemic caused by the Coronavirus disease 2019 (COVID-19) has raised an unprecedented mental health emergency worldwide. Abrupt changes in daily routine, environmental constraints, adopted home confinement measures, and uncertainty about a date for returning to usual activities can potentially affect mental health and sports activities in athletes. Hence, we designed a cross-sectional study with a within-subjects design to investigate the impact of the pandemic on mental health, mood states, and life satisfaction of elite athletes. During the three phases of home confinement (April 14–24,  Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by newly discovered Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). It was firstly reported in Wuhan, Hubei province , causingThe positive COVID-19 tests in world-class athletes at premier leagues presented that no one is potentially safe . MoreoveWHO has also expressed its concern over the pandemic’s mental health and psycho-social consequences . It specIt is noteworthy that after the quarantine, the governments decided to reopen the community and, consequently, sports with some recommendations . HoweverElite athletes as a target group might need specific help for their psychological needs, including maintenance of mental health during outbreaks to minimize the psychosocial toll. An accurate understanding of the psychological impact accompanying the COVID-19 lockdown, reopening, and semi-lockdown is necessary to plan for a crisis-oriented base research and intervention to foster a healthy lifestyle and physical and mental well-being in athletes . ConsistThe first phase (home confinement) of the study was conducted through an online survey between April 14 and April 24, 2020. This time frame was chosen to assess participants’ responses during an early phase of the COVID-19 outbreak, following the Iranian Government declaration of lockdown and the WHO announcement of the COVID-19 as a pandemic . Moreover, at this time, all sports facilities and clubs were closed and all sports events were canceled. The second phase (reopening condition) of this study was performed between May 09 and May 19, 2020. In this phase, athletes were able to continue sports training with some restrictions and more sports clubs opened. Finally, the third phase (semi-lockdown) of our study was launched between July 20 and July 31, 2020. During this period, the Iranian Government announced the semi-lockdown condition for the community and more sports clubs were closed, and the daily routine of elite athletes was difficult and limited. It should be noted that the second and third phases of this study were performed similarly to the first phase procedure.TM, InstagramTM, and WhatsAppTM. It is necessary to mention that athletes and some research and sports organizations were also involved in the dissemination plans of our research through the promotion of the survey in their networks.Efforts were made to recruit participants from all regions in the country, affected by the pandemic to different extents, to obtain a representative population. Eligible participants were in the age range of 18–45 years, were competing in the super league, national, and international levels ; had livThe study was approved by the Ethics Committee of the Tehran University of Medical Sciences (number: 1399-335) and was conducted according to the Declaration of Helsinki. The participant could withdraw from the survey at any moment without providing any justification. We also provided scores and status reports for anyone who wished. The survey was anonymous, and data confidentiality was assured according to the privacy policy. This web-based questionnaire was completely voluntary and non-commercial.h/w, exercise during COVID-19h/w, and coaches follow-up for plans and fitness and COVID-19-related information . All participants completed five standardized Persian versions of these questionnaires including General Health Questionnaire-28 (GHQ-28), Profile of Mood State Short Form (POMS-SF), and Satisfaction with Life Scale (SWLS).The survey included an introductory page describing the background and the aims of the survey and the ethics information for participants. A dedicated, self-report questionnaire was set up to collect demographic and epidemiological variables of interest , information on lockdown conditions  and encouraged participants to answer carefully through questionnaire explanations. Moreover, questionnaires that were completed less than 1 min or more than 20 min would be excluded from the analysis.General Health Questionnaire-28 (GHQ-28) has been developed by The SWLS was designed as a measure of individuals’ global judgment of their life . The SWLThe POMS-SF is a reliable and valid measure of subjective mood states that has been used extensively in a wide variety of studies . This qun) and percentage (%).The data were analyzed using the Statistical Package for the Social Sciences . Data were checked for missing, outliers, and normal distribution. The Kolmogorov–Smirnov normality test showed that most variables were not normally distributed. Values were computed and reported for illustrating purposes as mean ± SD (standard deviation) and frequency  was analyzed using Cochran’s Q test for comparison of three conditions and McNemar Tests were utilized as post hoc comparisons. Furthermore, Kruskal–Wallis and Mann–Whitney U tests were utilized for the comparison of psychological variables in gender, age categories, types of sport, and competitive levels in each phase of this study. Pearson correlation tests were used to assess possible relationships in the difference scores (Δ) of three phases of study between the assessed psychological parameters . Statistical significance was evaluated as p < 0.05 for all tests.To test the effect of COVID-19 pandemic condition on the self-reported measure (training during COVID-19 pandemic), a repeated measure analysis of variance (ANOVA) with a within-subject factor was conducted for the three conditions . Moreover, we applied Bonferroni correction for multiple comparisons, when the ANOVA outcomes were significant. For those variables without normal distribution and ordinal variables, we utilized Friedman tests for comparison of the three conditions  and subsequently, Wilcoxon Signed Ranks tests as n = 464). In the third phase (semi-lockdown condition), 36  people did not participate compared with the second phase (reopening condition) (n = 428). Descriptive statistics presenting the demographic information of the participants, including age, gender distributions, educational level, marital status, life form, sports type, and competitive levels, are summarized in For the first phase (home confinement condition), 525 (100%) valid responses were received. Our sample decreased by 61 (11.6%) people in the second phase  and team  sports. Most respondents competed at the national level , followed by at the international level  and super league .The mean age of participants was 27.85 ± 9.09 years at the home confinement condition, 27.53 ± 8.74 years at the reopening condition, and 27.43 ± 8.56 years at the semi-lockdown condition. Of the total sample, 205 (39%) were men and 320 (61%) were women at the first phase of this study. In the second phase, 39.4% of the sample were men and 60.6% were women. In the third phase, we observed that 169 (39.5%) of the sample were men and 259 (60.5%) were women; 69.9% of our sample were single and 26.7% were married and 3.4% were divorced or widow/widower at the first phase. In the second phase, 70.3% were single, 26.1% were married, and 3.7% were divorced or widow/widower. At the final phase, 70.1% were single, 26.9% were married, and 4% were divorced or widow/widower. Only 7.4% of the elite athletes lived alone and 92.6% were with family. The same results were found at the second and third phases. The elite athletes were active in individual  = 217.11, p = 0.001]. Furthermore, Wilcoxon Signed Ranks tests as post hoc comparison showed that economic damage by COVID-19 was significantly different for the home confinement condition compared with the reopening condition  and home confinement condition compared with the semi-lockdown condition . However, no significant difference was found between the reopening condition and semi-lockdown condition .As shown in 2(2) = 27.20, p = 0.001]. Furthermore, post hoc comparison by Wilcoxon Signed Ranks tests indicated that this variable was significantly different for the home confinement condition compared with the reopening condition  and home confinement condition compared with the semi-lockdown condition . There was a significant difference between the reopening condition and the semi-lockdown .Related to the acceptance of the rules of quarantine and COVID-19 instruction, our findings revealed a significant difference in the COVID-19 condition . In this regard, Wilcoxon Signed Ranks tests as post hoc comparison confirmed this finding .The coaches follow-up variable did not show any significant change between the three conditions . Subsequently, Wilcoxon Signed Ranks tests as post hoc comparison showed that the need for psychosocial services was significantly different for the home confinement condition compared with the semi-lockdown condition  and the reopening condition compared with the semi-lockdown condition . However, no significant difference was observed between the home confinement condition and the reopening condition .The analyses yielded significant differences in need for psychosocial services variable , somatic symptoms , social impairment , depression , and total score .As shown in Post hoc comparison analysis with Wilcoxon Signed Ranks tests revealed that in home confinement condition, GHQ-28 subscales and total score were higher , whereas in the reopening condition  and in the semi-lockdown condition , those parameters were lower. Moreover, there was a significant difference between the reopening condition and the semi-lockdown condition .2(4) = 2.79, p = 0.593; somatic symptoms: χ2(4) = 4.42, p = 0.328; social impairment: χ2(4) = 1.49, p = 0.828; depression: χ2(4) = 6.38, p = 0.172; total score: χ2(4) = 3.89, p = 0.441] and competitive levels . According to the Mann–Whitney U test, no significant difference was found in gender  and types of sport .At the home confinement condition, our results using Kruskal–Wallis test indicated that there is no significant difference between age categories  and competitive levels . According to the Mann–Whitney U test, no significant difference was found in gender  and types of sport .Under the reopening condition, our results using Kruskal–Wallis test indicated that there is no significant difference between age categories , competitive levels , gender , and type of sport .The same results were found for semi-lockdown condition in age categories . Furthermore, Wilcoxon Signed Ranks tests as post hoc comparison showed that life satisfaction was significantly different for the home confinement condition compared with the reopening condition  and the home confinement condition compared with the semi-lockdown condition . Moreover, a significant difference was found between the reopening condition and the semi-lockdown condition .Our results for life satisfaction scores indicated a significant difference between the studied phases , reopening condition , and semi-lockdown condition . The same results were found for competitive levels . No significant difference (based on Mann–Whitney U test) was found by gender  and sport type .The Kruskal–Wallis test indicated that there is no significant difference between age categories in life satisfaction scores in the home confinement condition , anger , depression , vigor , fatigue , and tension  between the studied conditions. In this regard, Wilcoxon Signed Ranks tests as post hoc comparison revealed that mood states scores were significantly different for the home confinement condition compared with the reopening condition  and for the home confinement condition compared with the semi-lockdown condition . Moreover, there was a significant difference between the reopening condition compared with the semi-lockdown condition   .2(4) = 4.75, p = 0.313; anger: χ2(4) = 3.47, p = 0.482; depression: χ2(4) = 5.57, p = 0.234; vigor: χ2(4) = 3.31, p = 0.381; fatigue: χ2(4) = 4.18, p = 0.381; tension: χ2(4) = 1.48, p = 0.830] and competitive levels . According to the Mann–Whitney U test, no significant difference was found by gender  and types of sport .At the home confinement condition, our results using Kruskal–Wallis test indicated that there is no significant difference between age categories , competitive levels , gender , competitive levels , gender , and types of sport .Similar to the previous findings, no significant differences were found in the semi-lockdown condition between age categories  and new restrictions posed during this pandemic. Besides, comparing results in different countries with different languages and cultures, per capita income levels, diverse support, and diverse constraints over a single period or at different times can help to integrate conclusions. Future studies can also be conducted experimentally and a variety of psycho-behavioral interventions (under an ICT setting or face-to-face and even group therapy) and training plans for elite athletes can be applied to investigate if any improvement would show up for mental health factors and physical fitness.Findings from this explorative study suggest that mental health, life satisfaction, and mood states were influenced during the different phases of the COVID-19 pandemic. Reopening and semi-lockdown phases were found to be associated with higher mental health, mood state, and life satisfaction. Home confinement created problems with trainings and plans and economic damage. Overall, this study provided the first evidence on dynamic changes of the COVID-19 phases on several psychological factors in elite athletes. According to the results, specific programs to support elite athletes at a psychological level could be recommended, especially to improve training rate, mental health, mood states, and life satisfaction. Accordingly, safe conditions and a rigorous monitoring system must be established by federations and institutions to protect the integrity of elite athletes. Learning from the past crises caused by the previous pandemics, planning for the current situation, and eventual future strategies seem crucial.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Tehran University of Medical Sciences (number: 1399-335) and was conducted according to the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.JCJS conceived the original study in Spain. AM, AZ, PG, MN, and MT conceived the study and collected the data. AM, MT, MA, and AZ designed the questionnaire and analyzed the data. All authors wrote the manuscript. AM, AZ, PG, JCJS, MN, and MA provided critical revisions on the successive drafts. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A pivotal role of thyroid hormones and their nuclear receptors in intestinal development and homeostasis have been described, whereas their involvement in intestinal carcinogenesis is still controversial. In this perspective article we briefly summarize the recent advances in this field and present new data regarding their functional interaction with one of the most important signaling pathway, such as WNT, regulating intestinal development and carcinogenesis. These complex interactions unveil new concepts and will surely be of importance for translational research. Indeed, during this postnatal developmental process the gastrointestinal tract is dramatically remodeled, with a first phase of apoptosis followed by a strong increase of cell proliferation correlating with an increase of circulating THs (The role of the Thyroid Hormones (THs) in intestinal development have been established since the beginning of the 20ting THs .The intestinal epithelium is a tissue which combines the absorptive properties of a single layered/high-surface epithelium with the protective advantages of a constantly renewing barrier. It comprises six different mature cell types, which are separated into absorptive (enterocytes and M cells) and secretory  lineages. The different cell types allow functions such as the nutrient uptake, metabolic control and immune regulation. Thus, the intestinal epithelium is a multifunctional dynamic tissue highly dependent on continuous supply of all cell types in appropriate ratios . The epiCdx genes) and several pathways  play crucial roles in diverse developmental and homeostatic processes  still need to be better defined.Homeodomain transcription factors  show a strong impairment in epithelial renewal and the ISC compartment is entirely absent  context, blocks the functionality of the β-catenin and thus the activity of the pathway (AXIN2/Axin2 (LOF) as well as CTNNB1/Ctnnb1 gain-of-function (GOF), the last encoding for the β-catenin protein. Specifically, CTNNB1/Ctnnb1 mutations are responsible for a stabilized oncogenic form of the protein  can be included in this list  results in crypt hyperplasia and enhanced proliferation up to adenoma development  . During elopment .i) mutational status of oncogenes  when hypo- or hyper-thyroidism appears, (ii) combination of mutations and/or expression of key genes and/or activation or repression of signalling pathways in different cell types influencing the biology of the cancer cells, (iii) local availability of the hormone , Dio3 (i.e. low T3) and Shh , which is another component of the WNT pathway (in vivo and in primary cells (0/0 and TRβ-/- mice (vil-TRα1 mice) not only confirmed their regulation but showed an increased cell proliferation and adenoma development even though it was not able per se to induce tumorigenesis    while TRApc mice . ImportaApc mice .Ccnd1 and c-Myc  compared to the simple vil-TRα1 mice (in vitro using the respective luciferase-driven reporters DR4/TRE (in vivo chromatin immunoprecipitation (ChIP) approach to analyze the TREs and WREs of the specific target genes of TRα1 and β-catenin/Tcf4, respectively. As shown in Sfrp2 and Ctnnb1 genes containing TRE elements  it stimulates its target genes including β-catenin, resulting in the activation of the WNT pathway and, by consequence, increased crypt proliferation, through the mechanisms we have already described (vil-TRα1/Apc), the stronger increase of the β-catenin/Tcf4 complex not only induces the WNT pathway activity but may also act to “displace” TRα1 from its own targets to the WNT targets. We may speculate that the “displacement” of TRα1 can be considered the molecular counterpart of the WNT hyperactivation resulting from the interaction of this two signalling pathways. Indeed, in the intestinal tumors of vil-TRα1/Apc mice the transcriptional activity of TRα1 on its own target is similar to that observed in WT condition, despite the nuclear receptor is expressed at augmented levels comparable with those of vil-TRα1/Apc mice. It is worth noting that TRβ or other nuclear receptors and β-catenin/Tcf  have been shown to form protein complexes .MS participated in the design of the study, performed all the experiment, carried out the data analysis and drafted the manuscript. LC helped in design the manuscript, figure preparation and revised the manuscript. MP conceived and supervised the study, carried out the data analysis drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.MP lab is supported by the FRM , by the Inca (PLBIO19-289) and by the Ligue Contre le Cancer, Département Grand Est (01X.2020). LC received support from the FRM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.