It deacetylates many protein substrates, including histones and transcription factors, thereby controlling many physiological and pathological processes. Several synthetic inhibitors and activators of SIRT1 have been developed, and some therapeutic applications have been explored. The indole EX-527 and its derivatives are among the most potent and selective SIRT1 inhibitors. EX-527 has been often used as a pharmacological tool to explore the effect of SIRT1 inhibition in various cell types. Its therapeutic potential has, therefore, been evaluated in animal models for several pathologies, including cancer. It has also been tested in phase II clinical trial for the treatment of Huntington’s disease (HD). In this review, we will provide an overview of the literature on EX-527, including its mechanism of inhibition and biological studies.The human sirtuin silent information regulator 1 (SIRT1) is a NAD It is the most studied of the seven human sirtuins known to date. It is a NADin vitro, its effect on various cell types , biological studies in animal models, and results of a clinical trial. This review primarily describes studies in which EX-527 is the main compound of interest, but we also included selected studies using EX-527 as a control and/or pharmacological tool to explore SIRT1 related pathways. To complete this overview, we also included some examples in which the inhibitor EX-527 was used to counteract the effects of other molecules, such as SIRT1 activators.In addition to these genetic manipulation studies, pharmacological modulation of SIRT1 has been the subject of intense research. SIRT1 modulators in general and their roles in cancer in particular have been often reviewed, usually giving an overview of several inhibitors and activators, but limited information on each one2.2.1.EX-527 was identified in 2005 by high throughput screening of libraries of compounds on the enzyme SIRT1 15. It haα to the ketone, affording a bromo keto ester, which was heated in the second step with an aniline, affording the tetrahydrocarbazole ester. The ester was then converted to the primary amide under pressure. In case enantiomerically pure material was needed, separation by chiral column chromatography was achievedA typical synthesis of this family of compounds is depicted in 50 values as low as 38 nM, depending on assay conditionsS), whereas the other (R) isomer (designated EX-242) is inactive. IC50 values for sirtuin inhibition by EX-527 have been measured in several studies, using a variety of assay methods and peptide substrates . Very stringent structure/activity relationships were described in the original article35 (50 of the (S) isomer is 60 nM, and the IC50 of the racemic mixture is 124 nM. It is selective for SIRT1, with an IC50 for SIRT2 of 2.77 µMEX-527 is a potent and selective SIRT1 inhibitor, with ICbstrates . They rarticle35  is an anN-((dimethylamino)methylene)acetamide derivative (a dimethylformamide adduct), which was rapidly hydrolysed in aqueous solution to form EX-527 and dimethylformamide  and SIRT135 (rac-35) have been tested in vitro on other isolated enzyme and receptor targets. Overall, they displayed very little to no activity. They did not inhibit class I and II HDACs and NAD+ glycohydrolase at 100 µM+ as cosubstrate for ADP-ribosyl transfer, producing nicotinamide, like sirtuins. Therefore, inhibitors targeting the nicotinamide binding pocket like EX-527 could have an inhibitory effect on PARP enzymes. No inhibition was observed on PARP1 and PARP10,Kr), EX-527 had an IC50 of 43 µM, with 0% inhibition at 10 µMrac-35 displayed only 10% inhibition at 10 µMrac-35. IC50 values determined for EX-527 were higher than 100 µM for all cytochromes P450 except 2C9 (62.4 µM), 2C19 (72.2 µM), and A2 (8.7 µM),In addition to sirtuins, EX-527 and racemic 2.2.+ cofactor, releasing nicotinamide. The 1′-O-alkylimidate intermediate formed reacts intramolecularly to generate a bicyclic intermediate. This intermediate is subsequently hydrolysed to form the deacetylated product and the 2′-O-AcADPr coproduct.A simplified mechanism of deacetylation of a substrate catalysed by sirtuins is represented in Figure 2(A)Thermotoga maritima) demonstrated in all three cases that the inhibition by EX-527 was non-competitive with substrate and uncompetitive with NAD+. Therefore, the inhibition potency depends on the NAD+ concentration. Binding parameters are summarised in d values for EX-527 measured for the apo enzymes and in the presence of NAD+ confirmed the uncompetitive nature of the inhibition. Indeed, EX-527 does not bind to the apo enzyme, but binds with low micromolar affinity in the presence of NAD+.The mechanism of SIRT1 inhibition by EX-527 is represented in Another interesting aspect of these mechanistic studies concerns the specificity of EX-527 for sirtuin isoforms. The authors propose that the difference between EX-527-sensitive enzymes (like SIRT1 and Sir2Tm) and less sensitive ones (like SIRT2 and SIRT3) comes from differences in their kinetics of catalysis, and not from differences in the binding pockets, which are very similarS)-35 and EX-243, have been described. The first one was between SIRT1, NAD+, and compound (S)-35 (PDB 4I5I)+/EX-243 (4BV3), SIRT3/ADPr/EX-243 (4BVB), SIRT3/2′-O-AcADPr/EX-243 (4BVH), Sir2Tm soaking , and Sir2Tm/2′-O-AcADPr/deacetyl p53 peptide/EX-243 (4BV2)Six crystal structures of sirtuins in complex with the active enantiomers of the inhibitors, compound  of the sirtuin, and one of the following molecules is also co-crystallised, forming a ternary complex: NAD or ADPr . This obO-AcADPr. Finally, from the comparison of crystals structures with and without the inhibitor, it appears that a flexible cofactor-binding loop moves towards the inhibitor and the coproduct during inhibition, resulting in a “closed” conformation preventing product releaseMoreover, the mechanistic studies showed that sirtuin inhibition with EX-527 allows the formation of one molecule of product per molecule of enzyme, indicating that the inhibitor binds most efficiently after bicyclic intermediate formation and allows coproduct formation3.EX-527 has been tested on several cell lines, either as the main molecule of interest for potential therapeutic applications, or as a control experiment for comparison with other sirtuin modulators (inhibitors or activators). Often, it has been used as a pharmacological tool to demonstrate the involvement of SIRT1 in a biological response. An overview of literature data is summarised in ,,,,,,,,increased cell proliferation of cancer cell lines,On tumour cell lines, several reports demonstrated the ability of EX-527 to increase p53 acetylation from 1 to 25 µM concentrations, when used either alone or in combination with cytotoxic moleculesIn summary, our results suggest that both activators and inhibitors of SirT1 have therapeutic potential as anti-tumor agents. A simple scenario is that SirT1 activators may impart cancer prevention effects by enhancing the growth-inhibitory effect of SirT1 in benign tumors. Its effect on advanced stage tumors may be heterogeneous, depending on whether a tumor has evolved to rely on SirT1 for survival. However, when tumors are being treated with chemotherapy, SirT1 inhibitors may be useful for enhancing apoptotic response,Ten years after this report, the list of EX-527 studies has grown to reinforce this view . For exa2O2 damage,in vivo results)On non-cancer cell lines, fewer studies were published than on cancer-cell lines. For example on HUVEC, EX-527 was shown to protect from Hin vitro studies show that the extent of its specificity, in particular towards SIRT2, depends on the assay types (nature of the substrate and concentration of NAD+ for example) and may not be so high under certain conditions  and in humans in exploratory clinical trials The fraction of unchanged EX-527 excreted in the urine was very low for all doses in male subjects (<0.02% up to 24 h post-dose). The compound was transformed Pharmacogenomics studies suggested that EX-527 treatment in human was associated with a specific transcriptional signature in blood cells, with genes involved in mechanisms of signal transduction and transmembrane transport, as well as metabolic and redox processesThe conclusion of the safety study in healthy volunteers indicated that EX-527 was safe and well tolerated by female and male subjects after single doses up to 600 mg and multiple doses up to 300 00/d for 7 d. Moreover, no meaningful cardiovascular effects were observed in beagle dogs up to 100 mg/kgIn vivo, numerous studies have been carried out to explore the effect of EX-527 under physiological or pathological conditions  clinical trial with EX-527 (Selisistat) will enrol around 30 women with unexplained failure after embryo transfer with euploid embryos. This phase II trial will start on 1 January 2021, and finish on 31 December 2022. The drug will be administered daily for 5 d, beginning with the start of progesterone therapy, and ending 24 h before embryo transfer. Pregnancy rates and pregnancy outcome will be monitored .New derivatives of EX-527 with greater activity and selectivity for SIRT1, as well as improved pharmacokinetic and pharmacodynamic properties, may lead to results that are even more promising, and reach further advanced clinical trials.


Real-time monitoring of fruit ripeness in storage and during logistics allows traders to minimize the chances of financial losses and maximize the quality of the fruit during storage through accurate prediction of the present condition of fruits. In Pakistan, banana production faces different difficulties from production, post-harvest management, and trade marketing due to atmosphere and mismanagement in storage containers. In recent research development, Wireless Sensor Networks (WSNs) are progressively under investigation in the field of fruit ripening due to their remote monitoring capability. Focused on fruit ripening monitoring, this paper demonstrates an Xbee-based wireless sensor nodes network. The role of the network architecture of the Xbee sensor node and sink end-node is discussed in detail regarding their ability to monitor the condition of all the required diagnosis parameters and stages of banana ripening. Furthermore, different features are extracted using the gas sensor, which is based on diverse values. These features are utilized for training in the Artificial Neural Network (ANN) through the Back Propagation (BP) algorithm for further data validation. The experimental results demonstrate that the projected WSN architecture can identify the banana condition in the storage area. The proposed Neural Network (NN) architectural design works well with selecting the feature data sets. It seems that the experimental and simulation outcomes and accuracy in banana ripening condition monitoring in the given feature vectors is attained and acceptable, through the classification performance, to make a better decision for effective monitoring of current fruit condition. Fresh produce, especially fruits and vegetables, is considered an important part of our day to day diet because it is a major source of vitamins, minerals, organic acids, dietary fibers, and also antioxidants. According to the food guide pyramid, a balanced diet should include at least 2–4 servings of fruit every day . The conBanana is a major harvesting fruit yield in Pakistan and is grown in the large area of the province Sindh with an approximate production of 155 K tons in the farming season because of the favorable climatic and soil conditions for its successful farming. Major farming areas are Badin, Tando Allahyar, Naushero Feroz, Hyderabad, Nawabshah, Sangar, Thatta, and Tando Muhammad Khan, and farming has been extended to some other northern areas of the province of Sindh. These areas of production amount to 87–90% of total production in Pakistan : Banana Normal Condition,: Banana Rotten Condition,: Banana Unknown Condition.In support of the required target output, classified vector classes are prepared and given by:2, and humidity) were stored in text files and allocated values with banana health. Matlab scripts were simulated to combine all the feature sets and produce the range of training data for the testing process and its validation in both healthy and ripening cases. All feature values , 4.89 ppm (sample 2), 8.05 ppm (sample 3), and 10 ppm (sample 4) at high accuracy rate 0.01 ppm. All the data were captured through Xbee nodes and analyzed at the coordinator level. The experimental results demonstrated a dramatic increment of temperature values of fruit when ethylene volume values were high, as shown in For the measurement of the sensor values, the ethylene dissolves the electrolyte that counts the electrodes by oxidization at a sampling rate of 50 Hz. A small amount of current is produced by the oxidization reaction. Ethylene gas is measured in ppm under the parched condition of the experimental room and container. Four samples are taken at different time frames according to the banana ripening process. The ethylene sensor measures the gas concentration from 0 to 10 ppm. The practical flow ratio of ethylene gas was measured at 0.4 L/minThe following stage is to classify the uncertainty management in Xbee sensor values in diverse frames using multi-layer FFNN from the ripening process. It can be observed in The next step is to measure the data validation coming from the Xbee motes. It can be easily being observed from 2) are selected from sample data sets and extracted at different time frames for analysis and training. For classification and training purposes, a supervised ANN architecture is presented to show the efficiency of the network in the diagnosis of the current condition of banana . The simulated results showed the precise and general behavior of the ripening process of different parameters, especially ethylene gas, on fruit condition. To improve the mean squared error rate, three types of ANN architecture were tested and [4 × 12 × 3] demonstrated a reasonable quantity of hidden layers with a high accuracy rate in the classification of features vector.The feasibility of Xbee-based motes was experimentally demonstrated for monitoring the banana ripening process while in storage. The role of the network architecture of the Xbee sensor node and sink end-node was discussed in detail regarding their ability to monitor the condition of all the required diagnosis parameters and stages of banana ripening. Different significant features (temperature, humidity, ethylene, and COFuture development of this research would be extended toward the utilization of multiple fruit and vegetables for the diagnosis of their type and existing condition in a normal atmosphere environment and cold storage refrigeration. A multilayered structure in which the outer layer is an effective barrier to 1-MCP can be used to prevent loss of 1-MCP gas molecules to the general environment. A comparison of different Xbee motes would be an interesting area with another artificial intelligence technique for better communication between nodes and to precisely predict when different parameter measurements can be taken in parallel with fruits and vegetables to create the complexity.


Wnt/β-Catenin signaling is involved in embryonic development, regeneration, and cellular differentiation and is responsible for cancer stemness maintenance. The HSP90 molecular chaperone TRAP1 is upregulated in 60–70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/β-Catenin pathway and preventing β-Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/β-Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/β-Catenin signaling was highlighted by a TCF/LEF Cignal Reporter Assay in Wnt-off HEK293T and CRC HCT116 cell lines. Of note, this regulation occurs through the modulation of Wnt ligand receptors LRP5 and LRP6 that are both downregulated in TRAP1-silenced cell lines. However, while LRP5 mRNA is significantly downregulated upon TRAP1 silencing, LRP6 mRNA is unchanged, suggesting independent mechanisms of regulation by TRAP1. Indeed, LRP5 is regulated upon promoter methylation in CRC cell lines and human CRCs, whereas LRP6 is controlled at post-translational level by protein ubiquitination/degradation. Consistently, human CRCs with high TRAP1 expression are characterized by the co-upregulation of active β-Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/β-Catenin signaling is modulated at multiple levels by TRAP1. Wnt pathway is an evolutionarily conserved signaling broadly involved in regulating embryonic development, regeneration, and cellular differentiation . Indeed,Wnt signaling is classified into canonical and non-canonical pathways, based on the dependency of its activity on β-Catenin. The canonical pathway (β-Catenin-dependent) controls self-renewal/differentiation and cellular proliferation , whereasc-Myc, cyclin D1, and CD44 [In the canonical pathway, Wnt proteins act as ligands, interacting with two major classes of cell membrane receptors: Frizzled (FZD) and Lipoprotein-related proteins 5/6 (LRP5/6) . Upon Wnand CD44 . In the and CD44 .Given its relevance in cellular homeostasis, the aberrant activation of Wnt pathway is a key mechanism in human colorectal carcinogenesis . AberranWe previously reported that TRAP1 favors stemness maintenance in CRC cells, and that this occurs through Wnt/β-Catenin pathway and the regulation of β-Catenin ubiquitination/phosphorylation process . In ordeThis was achieved upon cell transfection with a Cignal TCF/LEF Reporter and subsequent stimulation with Wnt3A recombinant protein. Immunoblot analysis of active β-Catenin expression and Wnt receptor LRP6 phosphorylation were used as markers of pathway activation/inactivation. TRAP1 silencing resulted in the downregulation of total and active β-Catenin levels in both cell lines, as previously reported by Lettini et al. . A signiIn subsequent experiments, we questioned whether TRAP1 modulates the spheroid-forming ability of CRC HCT116 cells and SA41 and SA54 patient-derived spheroids. TRAP1-silenced and relative parental control cells were seeded as single-cell suspensions and compared for their sphere-forming ability. Noteworthy, TRAP1 knocking down A, insertAs TRAP1 modulates β-Catenin phosphorylation/degradation without being part of its degradation complex , the hypTo prove the specificity of TRAP1 regulation on LRP5/6 receptors, the molecular chaperone was silenced in HCT116 cells by two independent siRNAs, and this resulted in a significant downregulation of LRP5 and LRP6 . ConsistIn order to further evaluate the molecular mechanism used by TRAP1 to modulate the expression of LRP5/LRP6 receptors, we questioned whether this regulation occurs at transcriptional level. Thus, mRNA levels of LRP5 and LRP6 were analyzed by RT PCR in CRC HCT116 cells and SA54 spheroids upon TRAP1 interference. LRP6 mRNA was unchanged after TRAP1 silencing in HCT116 and SA54 cells, whereas LRP5 mRNA was significantly downregulated C, even iThus, we hypothesized that TRAP1 regulation on LRP6 may occur at post-translational level. This hypothesis is consistent with the current knowledge about TRAP1 regulation of its protein network, as it is widely accepted that TRAP1 regulates the quality of its interactors on the endoplasmic reticulum, preventing their degradation . This TRIn subsequent experiments, TRAP1 was immunoprecipitated from total lysates of HCT116 cells and TRAP1 immunoprecipitates were evaluated by LRP6 immunoblot analysis. Interestingly, a positive co-immunoprecipitation was observed between the two proteins B, suggesAs LRP5 mRNA level was significantly downregulated in parallel with its protein level, we questioned whether the downregulation of LRP5 was due to a transcriptional control by TRAP1 protein network. To address this issue, we took advantage from literature data, which suggest that LRP5 expression is controlled by DNA methylation mechanisms in in vitro cell models and in human malignancies ,27. ThusTo address the relevance of TRAP1 regulation on Wnt/β-Catenin pathway through LRP5/LRP6 in vivo, an in-house cohort of 41 human CRCs  was evalp-value: 0.005; LRP5 Sperman r: 0.6, p-value: <0.0001), confirming the dependence of active β-Catenin expression on Wnt LRP5/6 receptors   C. These Finally, to further support the role of epigenetic mechanisms in LPR5 regulation by TRAP1 in vivo, LRP5 mRNA expression and promoter methylation were assessed in a few CRC specimens, selected based on the availability of DNA, RNA and protein extracts from the same sample. Two representative cases are reported in Several molecular chaperones are determinants of cancer cell stemness and potential targets for therapeutic approaches directed against CSCs ,29. TheyTRAP1 regulation of CSCs occurs through the modulation of Wnt/β-Catenin signaling, by preventing β-Catenin phosphorylation and degradation . HoweverThe mechanism of TRAP1 regulation of LRP5 and LRP6 receptors is quite complex. LRP6 is controlled at post-translational level by preventing its ubiquitination and degradation. Indeed, LRP6 interacts with TRAP1 and its ubiquitination is enhanced in conditions of low TRAP1 expression. This observation is consistent with TRAP1 role in proteostasis, as this molecular chaperone plays a co-translational quality control on selective client proteins, most of them key genes involved in cancer progression . This reMore complex is the mechanism of TRAP1 regulation of LRP5. Indeed, no interaction was observed between the TRAP1 and LRP5 and this correlated with lack of ubiquitination of LRP5 in TRAP1 silencing conditions (data not shown). Noteworthy, LRP5 transcript was downregulated upon TRAP1 knocking down, this suggesting a transcriptional control by TRAP1 protein network. Interestingly, our data suggest that LRP5 gene promoter is hypermethylated in condition of low TRAP1 expression in CRC cell lines, patient-derived spheroids and human CRC samples and this correlates with mRNA and protein downregulation. This observation is consistent with the evidence that LRP5 expression is regulated by DNA methylation mechanisms in in vitro cell models and in human malignancies ,27 and wIn conclusions, this study suggests that Wnt/β-Catenin signaling is modulated at multiple levels by TRAP1 protein network and that the modulation of LRP5 and LRP6 receptors represents an additional mechanism responsible for TRAP1 regulation of stemness maintenance in human CRC.v/v) FBS, 1.5 mM glutamine, and 100 U/mL penicillin–streptomycin, and HCT116 cells were cultured in McCoy’s medium supplemented with 10% (v/v) FBS, 0.75 mM glutamine, and 10 U/mL penicillin–streptomycin. Unless otherwise specified, reagents were purchased from Thermo Fisher Scientific . ShTRAP1 HCT116 cells were cultured as previously reported [Human CRC k-ras G13D HCT116 and embryonic kidney HEK293T cells were purchased from American Type Culture Collection . Cell lines were routinely monitored in our laboratory by microscopic morphology, whereas cell line authentication was verified before starting this study by STR profiling, according to ATCC product description. HEK293T cells were cultured in DMEM supplemented with 10% . Experiments were performed according to the Ethics Approval obtained by Prof. De Maria from the Ethics Committee of the Istituto Superiore di Sanità  and a Material Transfer Agreement was signed between the Legal Representatives of the two Research Institutes. CSCs were cultured as spheroids in ultra-low attachment tissue culture flasks  in humidified atmosphere at 37 °C and 5% COTRAP1 transient silencing were performed with two independent siRNAs purchased from Qiagen . TRAP1 siRNA #1 was used for the vast majority of the experiments, unless otherwise specified. TBP7 transient silencing were performed with siRNAs SI00301469 . For control experiments, cells were transfected with a similar amount of control siRNA . Transient transfections of siRNAs were performed using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s protocol. The Wnt pathway activation was evaluated by the Cignal TCF/LEF Reporter Assay Kit (GFP) (CCS-018G Qiagen) according to the manufacturer’s protocol. TRAP1 cDNA was transfected as previously reported [v/v) glycerol, 2 mM EDTA, 1% (v/v) Triton X-100, 1 mM PMSF, 2 mg/mL aprotinin, 2 mg/mL leupetin, and 0.2% (w/v) deoxycholate) for 30 min on ice. Immunoblot analysis was performed as previously reported [Total cell lysates were obtained by homogenization of cell pellets and tissue samples in a cold lysis buffer  were used: mouse monoclonal anti-HSP75 (sc-73604), mouse monoclonal anti-PSMC4 (TBP7) (sc-166003), mouse polyclonal anti-MCT4 (sc-50329), mouse monoclonal anti-Ubiquitin (sc-8017), mouse monoclonal anti-PKM2 (sc-365684), mouse monoclonal anti-LDH (sc-137243), rabbit polyclonal anti-SOX2 (sc-20088), mouse monoclonal anti-c-Myc (sc-40), mouse monoclonal anti-GAPDH (sc-47724), mouse monoclonal anti-Tubulin (sc-8035) and mouse monoclonal anti-β-Actin (sc-47778). The following antibodies from Cell Signaling Technology  were used: rabbit monoclonal anti-active-β-Catenin (#8814), rabbit polyclonal anti-total-β-Catenin (#9562), rabbit monoclonal anti-LRP6 (#3395), rabbit polyclonal anti-phosphoLRP6 (#2568), rabbit monoclonal anti-LRP5 (#5731), rabbit monoclonal anti-TCF1 (#5731), and rabbit monoclonal anti-Survivin (#2808). The following antibody was also used; rabbit monoclonal anti-Axin2 .Protein levels were quantified by densitometric analysis using the Quantity One 4.5 software  and normalized according to the expression of the housekeeping gene.Total RNA was extracted from cell pellets and CRC specimens using the TRIzol reagent . RNA concentrations were determined spectrophotometrically at 260 using the NanoDrop™ 2000/2000c Spectrophotometer (Thermo Scientific). Each RNA was transcribed to cDNA using a Transcriptor First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. For real-time PCR analysis, 0.5 ng of cDNA samples was amplified using the LightCycler 480 SYBR Green I Master (Roche) in a LightCycler 480 (Roche) according to the manufacturer’s instructions. The following primers were used; TRAP1 forward 5′-CGCAGCATCTTCTACGTGC-3′, reverse 5′-CTGATGAGTGCGCTCTCC-3′ (PCR product 200 bp); LRP6 forward 5′-ACAGAGCTGCAATGGATG-3′, reverse 5′-TGAGCCCAAGCATATTTG-3′ (PCR product 150 bp); LRP5 forward 5′-AGACCAATAACAACGACGTG-3′, reverse 5′-TTCTTGCCCATCCAGTC-3′ (PCR product 211 bp); β-Actin forward 5′-CGCAAAGACCTGTACGC-3′, reverse 5′-CACACGGAGTACTTGCGC-3′ (PCR product 152 bp). Reaction conditions were as follows; pre-incubation at 95 °C for 5 min, followed by 45 cycles of 10 s at 95 °C, 10 s at 59 °C, and 10 s at 72 °C. β-Actin was chosen as an internal control.Genomic DNA (gDNA) from cell pellets and CRC specimens was extracted by using the QIAamp DNA Mini kit (Qiagen). DNA concentration and purity were assessed by NanoDrop technology (Thermo Scientific) using, respectively, the absorbance at 260 nm and the 260/280 nm absorbance ratio. Five-hundred nanograms of total gDNA was treated with sodium bisulfite using the Zymo EZ DNA Methylation Kit (Zymo Research D5002), according to the manufacturer’s protocol. The presence of methylation in bisulfite converted gDNA was detected by MS-PCR ,39. Prim5 cells/mL in PBS containing 0.1% (w/v) NaN3 and 5% (v/v) FBS. As a preliminary step, intrinsic cell fluorescence was analyzed, using unlabeled samples. For specific staining, 100 μL of each cell suspension were incubated with 10 μL of mouse monoclonal anti-CD44 (APC) (559942) from Becton Dickinson  for 15 min at room temperature. Cells were finally analyzed by flow cytometry with Navios  and data were processed by Kaluza 2.1 software. Cell suspensions were reconstituted to a final concentration of 1.0 × 104) were suspended in a medium containing Advanced DMEM F12 supplemented as described above, seeded in ultra-low attachment tissue culture flasks, and left growing for 3 days in humidified atmosphere at 37 °C and 5% CO2. Density of transformation colonies were compared by cell counts and represented as percentage of colonies in TRAP1-silencing conditions respect to the respective siNeg control (means ± S.D).Cells (1.25 × 10Specimens from 41 human CRCs and corresponding normal, non-infiltrated, peritumoral mucosa were obtained from the IRCCS-CROB Tissue Biobank. Patients’ baseline characteristics are reported in Expression and methylation data of TCGA samples were downloaded from XENA data repository . To evalThe two-sided paired t-test was used to establish the statistical significance between different variable in silenced/transfected/treated cells and related controls. The Spearman test was used to establish the statistical significance of the correlation between TRAP1, β-Catenin, LRP5, and LRP6 levels in human CRCs. Statistically significant values are reported in figure legends. All experiments were independently performed at least three times and three technical replicates were used for statistical analysis. Data represent means + S.D.


The use of renewable resources for plastic production is an imperious need for the reduction of the carbon footprint and the transition towards a circular economy. With that goal in mind, fully biobased epoxy resins have been designed and prepared by combining epoxidized linseed oil, lignin, and a biobased diamine derived from fatty acid dimers. The aromatic structures in lignin provide hardness and strength to an otherwise flexible and breakable epoxy resin. The curing of the system was investigated by infrared spectroscopy and differential scanning calorimetry (DSC). The influence of the different components on the thermo-mechanical properties of the epoxy resins was analyzed by DSC, thermal gravimetric analysis (TGA), and tensile tests. As the content of lignin in the resin increases, so does the glass transition, the Young’s modulus, and the onset of thermal degradation. This correlation is non-linear, and the higher the percentage of lignin, the more pronounced the effect. All the components of the epoxy resin being commodity chemicals, the present system provides a realistic opportunity for the preparation of fully biorenewable resins at an industrial scale. We live surrounded by plastics: from packaging to clothing, including construction materials, components of vehicles, furniture, electronics, and an endless list of commodities. In these days of banishment of plastics, their benefits should be kept in mind. For example, the use of plastics for automotive parts results in lighter vehicles that consume less fuel, therefore reducing the greenhouse emissions . HoweverHere, we report a fully biobased epoxy resin from biorenewable bulk chemicals: lignin and vegetable oils . Current® from Croda, derived from dimerized fatty acids , molecular weight (Mw), and glass transition (Tg), was chosen for the biopolymer development. Initial efforts were directed towards using lignin as a curing agent for the epoxy group. The quest for a lignin-epoxy resin goes decades back [®) was considered and successfully tested. The miscibility of the non-polar oils with polar lignin was not always optimal but could be addressed by transesterifying the EVO with methanol (me and ELOme) led to samples with higher Tg than those prepared with the EVO. Although the transesterification process was carried out in the lab, it is worth noting that there are commercial suppliers of ESOme and ELOme.The methanol extracted fraction, KLdes back ,16. Therdes back ,19,20,21methanol . Apart f1H NMR. It showed that the leaching corresponded to the diamine and not to lignin  was exponentially correlated  was also tested but resulted in a brittle sample. Besides the mechanical properties, the effect of lignin was also noticeable in the color of the samples, which ranged from dark orange to dark brown  had a greater impact than that from not having lignin at all to having a 12.5% content.Likewise, the presence of KLceptions , the incceptions . TGA expceptions . By contBreak) or elongation at break (ΕBreak). The Tg of the epoxy resins made from different Kraft lignins had a remarkably similar value, and only that of the acetone extracted fraction was slightly higher than the rest. A similar effect had been noted before for lignin-based epoxy resins [After the observed strong effect of the amount of lignin on the thermomechanical properties of the resins, we wondered how these would be influenced by the characteristics of lignin . Differey resins .Young lower than KLAcetone, which had a Mw of 2540. Despite its relatively low Young’s modulus, the epoxy resin made from West Fraser KL showed the highest ΣBreak and ΕBreak of the series.One of the features of lignin is the variability and heterogenicity among different samples, and therefore we were interested in comparing a KL from another source. West Fraser KL led to epoxy resins with not striking but noticeable differences. The resin containing West Fraser KL had a slightly lower Young’s modulus than Lignoboost KL, which points again to a relation between the Mw and the Young’s modulus. However, this seems to not be a linear relation, because West Fraser KL had a Mw of 4080 but a Eg of 9 °C, significantly higher than the samples prepared with KL. Moreover, the structural differences of lignin had a great impact on the mechanical properties, leading to harder epoxy resins  was kindly provided by BASF; Epoxidized linseed oil (Lankroflex™) was kindly provided by Valtris. Dimer diamine (Priamine 1074®) was kindly provided by Croda (AHEW 138g/equiv.). Lignoboost® Kraft lignin was provided by Innventia Institute. West Fraser Kraft lignin was kindly provided by West Fraser. Base-catalyzed depolymerized (BCD) solvent-extracted eucalyptus lignin oil was kindly provided by Fraunhofer Institute. Tetrahydrofuran was purchased from Fisher Scientific, methanol and ethanol from Acros Organics, and 1-methylimidazol from Alfa Aesar. Sodium hydroxide was purchased from Sigma-Aldrich.Epoxidized soybean oil (EfkaThe formation of methyl esters of epoxidized soybean and linseed oil was carried out following a modified literature procedure : 2 g of 31P NMR according to a literature procedure [Solvent extraction was performed by dissolving 10 g of lignin in 150 mL of a solvent (methanol or acetone), stirring overnight, filtering through a Büchner funnel, followed by concentrating the filtrate and finally drying the lignin at 50 °C under vacuum for 16 h. The lignin hydroxyl content was determined by rocedure .An amount of lignin (LignoBoost Kraft or the solvent-extracted fractions) was dissolved in 2 mL of tetrahydrofuran and mixed with 1 g of epoxidized vegetable oil . Alternatively, ethanol can be used as a solvent. The mixture was stirred at 70 °C for 1 h in an open flask. Afterwards, an amount of Priamine 1074 was added, and the mixture was stirred for 1 min and poured in a Teflon mold. The Teflon mold was placed in a vacuum oven (850 mbar) at 50 °C for 1 h to allow the solvent to evaporate. Then, the Teflon mold was placed in an oven and cured at 120 °C for 4 h and at 150 °C for 16 h. The curing could also be done at 120 °C for 20 h in the presence of 1-methylimidazole as a catalyst without an influence on the outcome. When BCD Eucalyptus lignin was used, no solvent was required, and thus the required amount of lignin was mixed with 1 g of epoxidized vegetable oil and stirred at 70 °C for 30 min in an open flask. The rest of the protocol was as described above.31P NMR spectroscopy, as described in the literature [Gel permeation chromatography was performed on a Styragel HR1 column eluting with tetrahydrofuran with RID detection. The hydroxyl content was quantitatively determined using terature . The epoTg) was defined as the half value of the heat capacity change (ΔCp/2). Thermal gravimetric analyses (TGA) were performed to gain insight into the degradation temperature of the resins. TGA measurements were carried out in a nitrogen environment (flow rate 20 mL min−1) using a Netzsch STA 449c machine at a 10 °C min−1 heating rate from 25 to 800 °C.Possible thermal phase transitions in all of the cured resin specimens were investigated using differential scanning calorimetry (DSC). The DSC measurements were conducted with TA Discovery DSC 250 by subjecting the cured epoxy resins to a heat–cool–heat cycle from −80 to 200 °C at a 20 °C/min rate under a nitrogen atmosphere. Normalized heat flows were plotted as a function of the temperature, and the plots were analyzed. The glass transition temperature  analyses of the epoxy resins were performed using a Nicolet iS10 spectrometer  operating at laboratory atmospheric conditions. The measurements were performed in the attenuated reflectance (ATR) mode using a diamond crystal. The spectral range was 4000 to 500 cmThe tensile tests of the cured epoxy resins were conducted according to ASTM D638, using an Instron machine model 2412 . Three replicates were measured for each sample, with the dimensions of ASTM D638 Type V. The tests were performed at 22 ± 2 °C and at 50–60% relative humidity. The crosshead speed was 2.5 mm/min. Young’s modulus, ultimate tensile strength, and percentage elongation at break were calculated from the stress-strain curves on the basis of the initial sample dimensions.A series of fully biobased epoxy resins made from renewable resources were prepared, and their thermo-mechanical properties were analyzed. Lignin, rich in aromatic structures, gives stiffness and consistency to the epoxy resins, which are otherwise soft, flexible, and breakable due to long aliphatic chains of the fatty acid derivatives. Solvent extracted Kraft lignin was used due to its more defined profile. At a low lignin content, the effects were barely noticeable, but at a 12.5 weight% of lignin both the glass transition temperature and the Young’s modulus increased, and the rise was even more pronounced at a 22 weight%. It is thus not a linear but an exponential correlation. Higher amounts of lignin could not be incorporated if solvent-extracted Kraft lignin was used. Depolymerized and subsequently solvent-extracted lignin, liquid and with low Mw, could be used in higher amounts (36.4%), leading to harder epoxy resins. The fact that all the components of the resin are bulk chemicals opens the door to the production of fully biobased resins on an industrial level.Ortiz, P.; Vendamme, R. Bio Based Epoxy Resin. PCT/EP2019/075067.


Inflammatory bowel disease (IBD) is a group of chronic intestinal inflammation that is a risk factor for many gastrointestinal cancers. Exosomes are gradually gaining attention as an emerging treatment method for IBD due to their important biological characteristics. NF‐κB is an important pro‐inflammatory transcription factor kept inactive by IκB protein in the cytoplasm by masking the nuclear localization signal of NF‐κB. The deterioration of IκB is mainly ubiquitination, and this depends on neddylation.In this study, we established a dextran sulfate sodium (DSS)‐induced IBD model in BABL/C mice to evaluate the effect of human umbilical cord mesenchymal stem cell‐derived exosomes  on the repair of IBD. At the same time, human colorectal mucosa cells (FHC) were stimulated by LPS (lipopolysaccharide) in vitro to activate the inflammatory environment to study the mechanism of hucMSC‐Ex regulating neddylation. The microRNA (miRNA) obtained by sequencing and transfection with hucMSC‐Ex was used to verify the role of miR‐326/neddylation/IκB/NF‐κB signaling pathway in IBD repair.HucMSC‐Ex inhibited the process of neddylation in relieving DSS‐induced IBD in mice. The binding of NEDD8  to cullin 1 and the activation of NF‐κB signaling pathway were suppressed along with reduced expression levels of neddylation‐related enzyme molecules. The same phenomenon was observed in FHC cells. The miRNA comparison results showed that miR‐326 was highly expressed in hucMSC‐Ex and played an important role in inhibiting the neddylation process. The therapeutic effect of hucMSC‐Ex with high expression of miR‐326 on IBD mice was significantly stronger than that of ordinary hucMSC‐Ex.HucMSC‐Ex relieves DSS‐induced IBD in a mouse model by inhibiting neddylation through miR‐326. HucMSC‐Ex carrying miR‐326 relieves intestinal inflammatory damage by inhibiting neddylation within the intestinal mucosal cells. A, HucMSC‐Ex contains multiple active substances, of which miR‐326 is highly expressed. B, miR‐326 targets NEDD8 and inhibits the binding of cullin 1 and NEDD8 (the neddylation of cullin 1), thereby preventing the degradation of IκB; NF‐κB is masked, and the activation of its signaling pathway is suppressed to alleviate inflammation. CDCrohn's diseaseCo‐IPcoimmunoprecipitationCRLcullin‐RING ligaseDAIdisease activity indexDCdendritic cellDSSdextran sulfate sodium saltEvsextracellular vesiclesFBSfetal bovine serumFHChuman colorectal mucosa cellH&Ehematoxylin and eosin stainingHucMSChuman umbilical cord mesenchymal stem cellHucMSC‐Exhuman umbilical cord mesenchymal stem cell‐derived exosomesIBDinflammatory bowel diseaseIECintestinal epithelial cellIFimmunofluorescenceIFN‐γinterferon‐γIHCimmunohistochemistryIκBinhibitor of NF‐κBLPSlipopolysaccharideMSCmesenchymal stem cellNEDD8neural precursor cell expressed developmentally down‐regulated 8NF‐κBnuclear factor‐κBNKnatural killer cellPCNAcell proliferation nuclear antigenqRT‐PCRreal‐time fluorescence quantification PCRTGF‐βtransforming growth factor‐βTNBS2,4,6‐trinitrobenzene sulfonic acidTNF‐αtumor necrosis factor‐αTregregulatory T cellUCulcerative colitis1Inflammatory bowel disease (IBD) is a group of inflammatory disorders caused by environmental, genetic, infection and immune factors. These factors interact to affect the intestinal mucosal immune system. IBD mainly includes ulcerative colitis (UC) and Crohn's disease (CD). The incidence of IBD has increased rapidly in recent years.Mesenchymal stem cells (MSCs) isolated from various tissues have the ability of inducing regeneration, maintaining general tissue dynamic balance, and home to target site.Neddylation is a post‐translational protein modification in which a ubiquitin‐like protein NEDD8  is covalently tagged to a targeted protein. In this process, NEDD8 is activated by NEDD8 enzyme E1 (heterodimer of NAe1 and Uba3), NEDD8 conjugated enzyme E2 (UBC12), and NEDD8 E3 ligase, after which it binds to a specific substrate protein, thereby forming a unique complex structure. NEDD8 combined with substrate does not degrade the protein, but rather regulates the conformation, stability, localization, and function of the substrate protein.Current research has shown that neddylation plays an important regulatory role in IBD, but the mechanism by which human umbilical cord mesenchymal stem cell‐derived exosomes (hucMSC‐Ex) specifically regulate neddylation needs further exploration.2The study was approved by the Ethical Committee of Jiangsu University (2012258).2.12.MSCs were isolated and cultured in α‐MEM medium (Invitrogen) as previously described.2.2The MSCs were normally cultured in α‐MEM medium (containing 10% FBS). After 24 h, the supernatant was discarded, the cells were washed with PBS, followed by the addition of a new α‐MEM medium . The supernatant was collected in sterile tubes after 48 h, and organelles were removed by centrifugation; during which cell debris was also removed. The resultant supernatant was poured into a 100 KD ultrafiltration centrifuge tube and repeatedly centrifuged until the supernatant was concentrated to 250 μL. Exosome extraction reagent (SBI) was added (supernatant:reagent = 5:1), and allowed to precipitate at 4°C overnight. The precipitated supernatant was centrifuged and washed to obtain hucMSC‐Ex. It was then resuspended in PBS, filtered through a 0.22 μm filter, and finally stored at −80°C.2.3HucMSC‐Ex was diluted with PBS, and 1 mL of suspension analyzed by NanoSight Nano Analyzer .2.4The diluted solution of hucMSC‐Ex in PBS was pipetted onto the copper net and made to stand at room temperature for 2‐3 min, after which it was counterstained with 3% phosphotungstic acid for 1‐2 min. The observation was done using an electron microscope (Philips).2.5Male BABL/C mice  were purchased from the Animal Research Center of Jiangsu University , and related research was carried out after the approval by the Ethics Committee of Jiangsu University and the Experimental Animal Management and Use Committee.Mice were randomly divided into three groups (n = 6/group): negative control group (Neg), DSS‐induced mouse IBD model group (DSS), and hucMSC‐Ex remission mouse IBD experimental group (hucMSC‐Ex). Mice in the Neg group drank autoclaved purified water, while both DSS group and hucMSC‐Ex group drank 3% DSS water in autoclaved purified water. In the hucMSC‐Ex group, 1 mg hucMSC‐Ex was injected into the tail vein of mice on the third, sixth, and ninth day after drinking 3% DSS, the other mice were injected with PBS. The body weights of the mice were recorded at the same time point every day, in addition to observation of the fecal characteristics and the presence of bloody stools. All the mice were sacrificed on the 10th day. The disease activity index (DAI) of the mice was analyzed , FHC + LPS group (LPS), FHC + LPS + hucMSC‐Ex group (hucMSC‐Ex), and FHC + LPS + MLN4924 group (MLN4924). 2 μg of LPS , 200 μg of hucMSC‐Ex, and 0.2 μL of MLN4924 (MedChemExpress) were added to the respective groups. After co‐cultivation for 12 h, cells were collected for subsequent experiments.FHC concentration was adjusted to 1 × 102.86 FHC was added to each well of the six‐well plate. After 60% cellular adherence was obtained, the original culture solution was discarded, followed by the addition of blank 1640 without FBS (including transfection reagent). The cells were cultured in the dark for 6 h, after which the medium was replaced with a normal 1640 nutrient solution (containing 10% FBS) for an additional 48 h; the cells were then collected for subsequent analysis. HucMSC‐Ex was transfected with Exo‐Fect™ Exosome Transfection Reagent (SBI).The transfection reagent (Gamma gene) was configured, 1 × 102.9A portion of the colorectal tissue (about 4 mm) was cut out and fixed in 4% paraformaldehyde. Following subsequent tissue processing, they were finally embedded in paraffin and sectioned with microtome. The sections were fixed on slides, dewaxed, and stained with H&E, followed by mounting and scanning using pathological section scanner.2.10RIPA lysate (Pierce) was added to the tissues/cells to obtain the protein that was mixed with the loading buffer (Life Technologies) at a ratio of 3:1, and boiled at 100°C for 8‐10 min. Protein samples were added to the gel well and the target protein was separated by electrophoresis. The protein was transferred to a PVDF membrane (Millipore), and non‐specific antigens blocked with 5% skim milk. The membranes were incubated with corresponding primary antibodies: anti‐CD9 , anti‐CD63 , anti‐CD81 , anti‐NEDD8 , anti‐cullin 1 , anti‐IκB , anti‐NF‐κB , anti‐P‐NF‐κB , anti‐PCNA , and anti‐β‐actin  overnight at 4°C. After washing with 1× TBS/T buffer, the membranes were incubated with the secondary antibody for 30 min at 37°C. Pictures were taken with a chemical gel imaging system (GE).2.11The Pierce TM Co‐Immunoprecipitation Kit (Thermo Fisher Scientific) was used to immobilize the antibodies in the resin, followed by Coimmunoprecipitation (Co‐IP). The eluted target protein was pretreated and then detected and analyzed by Western blot.2.12Trizol (Gibco) was added to the tissues/cells, and RNA was obtained by chloroform extraction. cDNA was obtained through a corresponding reverse transcription kit (Vazyme), real‐time fluorescence quantification PCR (qRT‐PCR) was carried out in a Step One Plus Real‐Time PCR System (ABI) to detect gene expression. The sequences of primers used are listed in Tables 2.13The tissue embedded in paraffin were fixed and dewaxed, followed by endogenous peroxidase blocking with 3% hydrogen peroxide solution at room temperature for 30 min. Tissues were steamed for 30 min in citrate buffer to repair antigens and incubated with 5% BSA solution for 30 min to block non‐specific antigens. The tissues were then incubated with antibodies: anti‐NEDD8 , and anti‐cullin 1  at 4°C for overnight, and with the secondary antibody at 37°C for 30 min. Subsequently, tissue color rendering by DAB (Biorad) was used, resin sealing applied, and pathological section scanner used to obtain pictures.2.14Cells obtained from culture were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 30 min. Triton X‐100 (0.1%) was added, gently mixed for 30 min, and non‐specific antigens blocked by the addition of 5% BSA solution. The cells were incubated with the antibodies: anti‐NEDD8 , and anti‐cullin 1  overnight at 4°C, then incubated with fluorescent secondary antibody at 37°C for 60 min. They were finally incubated with honchest (Biorad) for 10 min at room temperature, and anti‐quenching agent, after which pictures were taken by scanning with a confocal laser microscope (Nikon). The light was avoided in the entire process.2.15A total of 100 μL of FHC suspension was added to a 96‐well plate. When the cell density reached 60%, the nutrient solution was replaced and CCK8 solution (Vazyme) added at 12, 24, 36, and 48 h to the culture wells. The resultant cell suspensions were incubated for 30 min in the dark, and absorbance measured at 450 nm with microplate reader (Thermo Fisher Scientific).2.16The NEDD8 mRNA 3′‐UTR region containing the miR‐326 binding site, WT/Mut (GGCCCAGAG mutated to gcaagcggc), was cloned into a dual‐luciferase miRNA target expression vector . The two vectors were co‐transfected into FHC with miR‐326 mimics, mimics NC, inhibitor, and inhibitor NC, and then cultured for 12 h. Dual‐luciferase reporter gene kit (Promega) was used to detect related expression.2.17P < .05 considered statistically significant.All data were shown as mean ± SEM. Statistical analysis was performed by using GraphPad Prism software for analysis of variance (ANOVA), with 33.1The results of NanoSight and TEM showed that the extracted hucMSC‐Ex was 30‐200 nm in diameter, vesicle‐like, and highly concentrated Figure .3.2To determine the effects of hucMSC‐Ex in damaged colon tissue, we labeled hucMSC‐Ex with DIR fluorescent dye and injected it into IBD mice. 12 h post‐injection, treated mice (with hucMSC‐Ex‐DIR) showed a large area of fluorescence in vivo Figure . The micThe weight of the mice in the Neg group increased with time, but the weight of the mice in the DSS group decreased on the fifth day with blood also appearing in stool. The body weight of the mice in the hucMSC‐Ex group basically remained unchanged in the first 6 days, but slightly decreased after day 6 though not obvious Figure . Althoug3.3P < .05). For a more intuitive observation of the expression of NEDD8 and cullin1 in the colon tissue, IHC was performed, and results showed increased levels of NEDD8 and cullin1 in DSS group but decreased levels in hucMSC‐Ex treated group , but the expression level in cells of LPS and hucMSC‐Ex groups increased. On the other hand, PCNA expression in cells of MLN4924 group significantly decreased  experiment indicated that cell proliferation did not change after 12, 24, 36, and 48 h when FHC was induced by LPS. However, after co‐culture with MLN4924, although the cell proliferation did not change at 12 and 24 h, the cell viability began to decline after 36 h, and at 48 h, viability had significantly decreased Figure , P < .05From the background that hucMSC‐Ex and MLN4924 had no effect on FHC proliferation at 12 h, few other investigations were performed. Western blot analysis showed overexpression of NEDD8 but decreased IκB in FHC of the LPS group. Again, NEDD8 expression was decreased in the MLN4924 group cells, and IκB degradation was inhibited. At the same time, P‐NF‐κB expression was upregulated in the LPS group and decreased in the MLN4924 group. The expression changes of NEDD8, IκB, and P‐NF‐κB in cells of HucMSC‐Ex group were consistent with those of MLN4924 group Figure . QRT‐PCR3.5http://www.targetscan.org website. Alignment results singled out four miRNAs: miR‐27b, miR‐326, miR‐411‐5p, and miR‐665. To verify the cellular expression of selected miRNAs, FHC was treated with different batches of hucMSC‐Ex. QRT‐PCR results showed that only miR‐326 was stably and significantly expressed in cells under hucMSC‐Ex treatment, and less expressed in Neg and LPS groups . There was no significant difference in the ratio of luciferase activity  to sequence hucMSC‐Ex and HFL1‐Ex separately for comparison  superfamily. The TM4SF family is a group of scaffold membrane proteins including CD63, CD81, and CD9. They are located on the surface of exosomes and serve as detection markers.NF‐κB is a ubiquitous pro‐inflammatory transcription factor in mammalian cells and plays a key role in regulating the production of pro‐inflammatory factors .As a selective NAE inhibitor, MLN4924 regulates multiple signaling pathways by inhibiting the neddylation of target molecules.miRNAs are a class of endogenous noncoding single‐stranded small RNAs that are involved in regulating the expression of the gene of interest at the post‐transcriptional level. The expression levels of miRNAs in different tissues and different developmental stages are significantly different, further indicating that miRNAs play an important role in regulating gene expression. miRNAs regulate many physiological processes, including early development,To verify the reparative effect of miR‐326 in IBD mice, the study transfected hucMSC‐Ex with miR‐326 mimics and inhibitor to establish miR‐326 high/low expression. Since miR‐326 cannot be injected directly into mice, hucMSC‐Ex acted as corresponding carriers, with results showing that hucMSC‐Ex carrying miR‐326 relieves IBD by inhibiting neddylation of cullin 1.5HucMSC‐Ex homes to damaged colorectal tissues to relieve IBD in mice. In the process of alleviating inflammation, it inhibits the binding of NEDD8 to the substrate protein cullin 1, the expression of E1, E2, and E3 enzymes in the neddylation process, as well as the activation of NF‐κB signaling pathway. HucMSC‐Ex is rich in miR‐326 which targets the expression of NEDD8 to inhibit the neddylation process and achieve the effect of relieving IBD.6Findings in this research add up to knowledge on the regulatory effect of hucMSC‐Ex in intestinal mucosal inflammation, as it modulates cullin1 neddylation. miR‐326, an active cargo in hucMSC‐Ex, targets NEDD8 and prevents it from binding to cullin 1. CRL/NF‐κB signaling pathway is a classic pathway in neddylation, and loss of its transduction signal inhibits the synthesis and production of related inflammatory factors. MLN4924 is currently used as a clinical antitumor drug and has not been applied in the treatment of inflammatory damage. HucMSC‐Ex has no detrimental effect on target cells, avoids unwanted immune response caused by immunogenicity, hence providing a new target and strategy for future treatment of IBD.The authors declare no conflict of interest.Figure S1. Related parameters of NanoSight Nanoparticle Tracking AnalyzerClick here for additional data file.Figure S2. (A) Gray‐scale scanning analysis of P‐NF‐κB in Figure 3A; (B) Gray‐scale scanning analysis of IκB, P‐IκB, NEDD8 in Figure 3A, *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA.Click here for additional data file.Figure S3. (A) Gray‐scale scanning analysis of PCNA in Figure 4B; (B) Gray‐scale scanning analysis of P‐NF‐κB in Figure 4C; (C) Gray‐scale scanning analysis of IκB, P‐IκB, NEDD8 in Figure 4C, *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA.Click here for additional data file.Figure S4. (A) Gray‐scale scanning analysis of P‐NF‐κB in Figure 6A; (B) Gray‐scale scanning analysis of IκB, P‐IκB, NEDD8 in Figure 6A, *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA.Click here for additional data file.Figure S5. (A) Gray‐scale scanning analysis of P‐NF‐κB in Figure 8A; (B) Gray‐scale scanning analysis of IκB, P‐IκB, NEDD8 in Figure 8A, *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA.Click here for additional data file.


Pulpal inflammation is known to be mediated by multiple signaling pathways. However, whether melatonin plays regulatory roles in pulpal inflammation remains unclear. This study aimed at elucidating an in situ expression of melatonin and its receptors in human pulpal tissues, and the contribution of melatonin on the antagonism of lipopolysaccharide (LPS)-infected pulpal fibroblasts.Melatonin expression in pulpal tissues harvested from healthy teeth was investigated by immunohistochemical staining. Its receptors, melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2), were also immunostained in pulpal tissues isolated from healthy teeth and inflamed teeth diagnosed with irreversible pulpitis. Morphometric analysis was subsequently performed. After LPS infection of cultured pulpal fibroblasts, cyclo-oxygenase (COX) and interleukin-1 β (IL-1 β) transcripts were examined by using reverse transcription-polymerase chain reaction (RT-PCR). Analysis of mRNA expression was performed to investigate an antagonism of LPS stimulation by melatonin via COX and IL-1 β induction. Mann-Whitney U test and One-way ANOVA were used for statistical analysis to determine a significance level.P-value< 0.001), but not MT1 . Co-culturing of melatonin with LPS resulted in the reduction of COX-2 and IL-1 β expression in primary pulpal fibroblasts, indicating that melatonin may play an antagonistic role to LPS infection in pulpal fibroblasts.Melatonin was expressed in healthy pulpal tissue within the odontoblastic zone, cell-rich zone, and in the pulpal connective tissue. Furthermore, in health, strong MT1 and MT2 expression was distributed similarly in all 3 pulpal zones. In contrast, during disease, expression of MT2 was reduced in inflamed pulpal tissues (Human dental pulp abundantly expressed melatonin and its receptors MT1 and MT2 in the odontoblastic layers and pulpal connective tissue layers. Melatonin exerted antagonistic activity against LPS-mediated COX-2 and IL-1 β induction in pulpal fibroblasts, suggesting its therapeutic potential for pulpal inflammation and a possible role of pulpal melatonin in an immunomodulation via functional melatonin receptors expressed in dental pulp. Pulpal inflammation is an outcome of complex host-bacteria interactions that involves multiple intracellular machineries in tooth pulp. Early stage of pulpal injury caused by bacterial stimuli is mediated via innate immune sensors i.e. Toll-like receptor (TLR), particularly TLR4 which recognizes lipopolysaccharide (LPS), a potent immunostimulatory component derived from gram-negative bacterial cell walls , 2. ReceIn deep dental caries, the chance of chronic exposure of pulp cells to polymicrobial infections is dramatically increased due to the abundance of cariogenic bacterial species found in carious dentin . Such miPrevious studies have elucidated the contribution of melatonin on the modulation of host tissue homeostasis in a number of tissues. Melatonin is produced from the mammal pineal gland by melanocytes and is known for its distinct versatility on the variety of tissues such as salivary glands and the oral epithelium –12. In bIt is important to note that immunomodulatory properties of melatonin have been extensively investigated. Melatonin potentiates an inflammatory immune response in human myeloid cells involved in both innate and adaptive immunity . Remarkan = 3) without carious lesions from dental patients who underwent a simple extraction procedure were collected immediately in cold sterile Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum , 2 mM L-glutamine , 100 U/mL penicillin, and 100 μg/mL streptomycin. Each tooth was washed thoroughly with 1X phosphate buffer saline (PBS) and the periodontal tissue was removed. A carborundum disc was used to cut around the cemento-enamel junction and the cervical portion between crown and root was subsequently split. Pulpal tissue was removed by using sterile forceps and immediately submerged in a 35-mm culture dish containing DMEM. Extirpated pulpal tissue was cut into 1-mm piece using scalpel blade no.15 and was incubated at 37 °C under 5% CO2 environment and 95% relative humidity. Culture medium was replenished every 2 days until 100% cell confluence of pulpal fibroblasts was achieved at which the grown cells were passaged.The need for consent was waived by Khon Kaen University Ethics Committee for human research based on the declaration of Helsinki (Reference number: HE592265). Freshly extracted intact third molars  was performed at 20 μg/mL final concentration in growth medium. Dose of LPS stimulation was tested from our preliminary experiments to be effective in the transcript induction of inflammatory mediators. Purified melatonin  was used in a wide dose range for stimulation , predetermined for their safety to pulpal fibroblasts by cell viability assays. Cultures of pulpal fibroblasts were stimulated for 3 h or 24 h, as indicated in Figs. Third to sixth passage of isolated pulpal fibroblasts was used in the experiments when cultured cells reached 80% confluence. Stimulation of cultured pulpal fibroblasts with Total RNA from cultured pulpal fibroblasts was extracted and purified using RNeasy mini kit  according to the manufacturer’s protocol and subsequently tested for its purity. Reverse transcription and polymerase-chain reactions (RT-PCR) were performed separately by using a two-step procedure. First, cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit  with 150 ng of total RNA. Control sample without reverse transcriptase enzyme as well as control without RNA template were prepared similarly and served as negative controls. Next, each PCR reaction was setup using TopTaq Master Mix kit  to a total volume of 50 μL, containing 15 ng cDNA and 200 nM of each primer sequence. List of primer pairs used in this study was shown in Table Pulpal tissues from freshly extracted third molars, 12 sound third molars and 6 third molars diagnosed with irreversible pulpitis without apical periodontitis or abscess, were collected by splitting off the roots and immediately placed the pulpal tissues in 10% formalin for 24 h prior to paraffin embedding, similar to a previously published study . Embedde+ System-HRP labeled polymer, Carpinteria, CA, USA). Diaminobenzidine detection system was employed by the addition of DAB tetrahydrochloride solution  onto HRP-conjugated tissue sections. Sections were then counterstained with hematoxylin , dehydrated and mounted. Tissue biopsy specimens from human pineal glands were stained similarly and used as a positive control, while omission of primary antibodies served as a negative control.Immunohistochemistry analysis was performed in paraffin-embedded pulpal tissues. Briefly, Peroxo-Block™  was used to inhibit endogenous peroxidase activity. Specimens were subsequently blocked for non-specific antibody binding with Protein Block Serum-free . Immunohistochemistry staining was performed by using antibodies raised against human proteins as follows: rabbit anti-human melatonin , rabbit anti-human MT1 , rabbit anti-human MT2 . Concentrations of primary antibodies used in the study were optimized for their optimal immunostaining signals in our preliminary experiment. Each primary antibody were incubated with tissue sections separately for 24 h at 4 °C overnight. Tissue slides were thoroughly washed with PBS supplemented with 0.5% Tween (PBS-T) and incubated with anti-rabbit secondary antibody  under 10X eyepiece lens coupled with 20X objective lens for the staining profile of melatonin. Descriptive analysis was used to qualitatively evaluate the signal intensities emanated from each layer of pulpal tissues. For MT1 and MT2 analysis, approximation of stained cells was enumerated from 3 tissue sections obtained from each pulp sample. Total number of 12 healthy pulps and 6 inflamed pulps were fixed and stained for human MT1 and MT2. Each slide was blindly scored for the percentage of stained cells under a light microscope at 200X magnification. Semi-quantitative rating system was utilized to assess the distribution of stained cells and number of positively stained cells. Level of MT1 and MT2 expression within pulpal tissues was scored according to the relative intensity of the staining signals on a scale of 0 to 4 . Histological assessment and rating of staining intensity were performed twice more time with one-week time gap to minimize recall bias.P-value of less than 0.05 was considered statistically different.All experiments conducted in this study were summarized in Fig. We first sought to determine whether melatonin protein was expressed in dental pulp. Histological structure of normal pulpal anatomy was shown by H&E staining Fig. b. LocaliP-value< 0.001) between normal and inflamed dental pulpal tissue  compared to its expression in health. Our results demonstrated a striking contrast between MT2 and MT1 expression, with a significant decrease in MT2, but not MT1, in inflamed pulpal tissues.Next, we investigated the expression of MT1 and MT2 in pulpal tissues in relation to the health of the pulp. Our H&E staining demonstrated that, in healthy pulps, dental pulp periphery was comprised of an intact palisade of odontoblast linings Fig. a, while sue Fig. . MT1 expMT1 and MT2 mRNAs  is able to promote vascular permeability , and indE. coli LPS-stimulated human pulpal fibroblasts with melatonin reduced COX-2 and IL-1 β transcripts. Antagonism mediated by melatonin is thought to occur in part at TLR4 because LPS derived from E. coli exerts host hyperresponsiveness specifically at human TLR4 [A recent study has shown that systemic administration of melatonin during LPS-induced pulpal inflammation dampened TLR4-mediated inflammatory response in dental pulp cells . Our resman TLR4 , 39. Altman TLR4  and LPS man TLR4 , indicatman TLR4 . This suman TLR4 . In addiman TLR4 . In agreman TLR4 . HoweverIn summary, we have reported the ubiquity of melatonin and its receptors i.e. MT1 and MT2 in human dental pulp and a possible implication of melatonin in pulpal tissue homeostasis via the antagonism of LPS-induced COX-2 and IL-1 β signaling. Abundance of extra-pineal melatonin and its hormone receptors MT1 and MT2 in pulpal tissue suggests that melatonin might license dental pulp an immune regulation both via a direct melatonin-receptor engagement and its cross interactions with pulpal microbial sensors in the vicinity, which has yet to be elucidated. Interesting to note is the increased MT1, but not MT2 expression in inflamed pulps, indicating a distinct utilization of melatonin receptors during pulpal pathogenesis. Melatonin in orchestration with its receptors might thus provide a first line defense against microbial insult in dental pulp. Perhaps, melatonin may prove beneficial for the treatment of inflamed dental pulpal tissues. Nonetheless, induction of melatonin expression by polymicrobial infection especially in pulpal disease still needs further elucidation.Additional file 1 Fig. S1 Semi-quantitative analysis of COX-1 mRNA expression by pulpal fibroblasts cultured with LPS or various dose of exogenous melatonin alone for 3 h.Additional file 2 Fig. S2 Semi-quantitative analysis of COX-1 mRNA expression by pulpal fibroblasts stimulated with LPS alone or LPS with the presence of exogenous melatonin in the culture for 24 h.


Cardiovascular disease (CVD) is globally known as the number one cause of death with hyperlipidemia as a strong risk factor for CVD. The initiation of drug treatment will be recommended if lifestyle modification fails. However, medicines currently used for improving cholesterol and low-density lipoprotein cholesterols (LDL-C) levels have been associated with various side effects. Thus, alternative treatment with fewer or no side effects needs to be explored. A potential agent, oil palm phenolics (OPP) recovered from the aqueous waste of oil palm milling process contains numerous water-soluble phenolic compounds. It has been postulated that OPP has shown cardioprotective effects via several mechanisms such as cholesterol biosynthesis pathway, antioxidant and anti-inflammatory properties. This review aims to summarize the current evidence explicating the actions of OPP in cardiovascular health and the mechanisms that maybe involved for the cardioprotective effects.  Cardiovascular disease (CVD) is globally known as the number one cause of death with increasing numbers of people dying annually due to CVD compared to other diseases. An estimation of 18 million death per year from CVD has been made, which represents 31% of all death caused by disease globally . Primula veris L. extract that contains various phenolics such as aglycons and glycosides has demonstrated increased myocardial contractility with a good safety profile in a heart failure experimental study [Hyperlipidemia, which has been well established as a potent risk factor for CVD, can be defined as an isolated elevation of fasting total cholesterol (TC) concentration, which may or may not be related to isolated elevation of triglyceride (TG) concentration . A stronal study . Additioal study  and coroal study , respectElaeis guineensis Jacq.) is mainly done for palm oil production [The cultivation of an agricultural plant known as African oil palm  as the main component and other phenolic acids including caffeic acid, protocatechuic acid (PCA), as well as p-hydroxybenzoic acid (PHBA) and hydroxytyrosol (HT) . Their cThis review elucidates the effects of OPP and its postulated cardioprotective mechanism in preventing and treating CVD. This information will be beneficial and relevant for future research on CVD and the development of a comprehensive natural medicine approach to CVD. The direct effects of OPP in cardiovascular health have been previously investigated, as tabulated in Abeywardena et al. (2014)  conductep < 0.05). For ventricular fibrillation (VF) incidence, the OPP-treated group had a lower rate of 52% compared to the control group with 90% incidence. The supplementation of OPP also demonstrated a lower mortality rate of 20% in comparison with a control group of 40% [Abeywardena et al. (2014)  also perp of 40% . The redThe potential of OPP fractions in lowering vascular resistance has been tested using noradrenaline pre-contracted aortic ring and mesenteric vascular assays . This moIn a molecular study using microarray gene expression profiling on mice fed a 6-week normal diet, Leow et al.  reportedp = 0.025) and LDL-C (p = 0.04) compared to the control treatment. However, the significant result was only obtained when using the Wilcoxon signed rank test. Following Bonferroni correction, the reduction in TC and LDL-C was insignificant. In addition, the supplementation of OPP showed insignificant levels of triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) when compared to control subjects. Nevertheless, it should be noted that the subjects were healthy with normal range of lipid profiles [The positive results of OPP related to CVD in the preclinical studies further suggested its possible application in humans. A phase 1 clinical trial has been performed in healthy subjects to assess the effects of OPP on physiologic condition. The subjects were supplemented with OPP at 450 mg GAE/day for 60 days, which eventually demonstrated significantly lower plasma TC  de novo biosynthesis from acetyl-CoA by the multi-enzyme pathway or (ii) certain types of food ingestion via extracellular transport by low LDL-mediated endocytosis ,63. CholIn a physiological state, reactive oxygen species (ROS) may act as carriers in signaling pathways. During homeostatic balance, fluctuation of ROS concentrations occurs under a controlled environment with the presence of antioxidants and enzymes. However, perturbation to the balanced state of ROS may cause oxidative stress to become apparent . High ROThe feasible connection between atherosclerosis and oxidative stress might suggest that nutritional antioxidants may inhibit atherosclerosis development ,76. MoreIt has been reported that gut microbiota, which are the microorganisms living in the gut can improve the bioavailability of polyphenols. Following oral ingestion of polyphenols, the molecules must be absorbed into the body and carried through the bloodstream from the absorption site to target tissues and organs. The gut microbiota may be involved in the metabolism of polyphenols by converting dietary polyphenols to low-molecular-weight phenolic compounds (such as phenolic acids), which are more readily absorbed by intestinal epithelial cells . In addiIn addition, an absorption characterization of phytochemicals extracted from palm fruit biomass has been performed in a recent study by bio-matching the plasma pharmacokinetic plasma peak (Tmax) with the onset of oxidative stress and inflammation (OSI). Through bio-matching of Tmax and OSI, the Phytochemical Absorption Prediction (PCAP) model and methodology were able to be predicted. From the study, it was shown that the post-consumption absorption of plasma peak (Tmax) ranged between 0.5 and 12 h and 2 and 6 h for intake in liquid and solid forms, respectively, and generally showed high antioxidant activity of the extracts. Thus, this might indicate that the phytochemicals of the palm fruit biomass have broad potential to be used for human health as dietary antioxidants in foods and nutraceutical products . To dateInflammatory events are crucial in the pathogenesis of CVD. The role of cytokines is progressively apparent as studies have proven the association of inflammatory/immune mechanisms in atherosclerosis and heart failure . In the Leow and colleagues demonstrated that the OPP has modulated Th1/Th2 axis of the immune system towards the Th2 cytokines that possess anti-inflammatory actions. This modulation might in turn contribute to the reduction of atherosclerosis development. Moreover, in comparison to the control mice, OPP supplementation to atherogenic-diet fed mice had significantly reduced pro-inflammatory IL-12 cytokine while significantly increased anti-inflammatory IL-13 cytokine . The chaFatty acids are part of the lipid macronutrient class commonly found in the nature, food and organisms. They act as an important constituent of the cell membrane and possess significant biological, structural and functional roles. They have a vital role to generate a source of energy. Their metabolism yields a large quantity of adenosine triphosphate (ATP), in which the major pathway is mitochondrial fatty acid β-oxidation (FAO) ,96. ThisA microarray data from the liver of BALB/c mice supplemented with OPP showed the upregulation of FAO genes, which indicated increased hepatic lipid catabolism . These uTricarboxylic Acid (TCA) cycle or Kreb’s cycle is the principal metabolic pathway that produces energy in cells. In order to produce the energy by means of adenosine triphosphate (ATP), oxidation of the reduced coenzymes by the electron transport chain (ETC) may takes place . TCA relOPP has been demonstrated to prevent metabolic shift towards glycolysis by upregulating a key enzyme, Pdk4 . Pdk4 inAlterations in proteins have been identified to be associated with heart failure development. For example, the downregulation of uncoupling proteins (UCPs) may interfere with the regulation of mitochondrial membrane potential, energy metabolism and may promote ROS generation . OPP canElectron transport chain (ETC), which occurs in the inner membrane of mitochondria is important for oxidative metabolism. ETC consists of several substantial protein complexes (Complex I–V). While electrons flow through the ETC, active proton pumping occurs across the membrane which subsequently creates an electrical potential and pH gradient across the membrane. The free energies of this pump may cumulatively contain proton motive force. The discharge of force through the final complex of the chain, complex V, triggers the conversion of ADP and Pi to ATP . Leow etIn a previous study, OPP provided at antioxidant value of 1500 ppm GAE as ad libitum drinking fluids to mice showed no toxicity effects, as indicated by insignificant alteration to the organ histology, hematology and clinical biochemistry analyses. OPP had upregulated three genes namely Cxcl12, Gjb1 and Ar . These gSambanthamurthi and colleagues performed a teratological study by monitoring three generations of Sprague Dawley rats supplemented with 1500 or 2400 mg/L GAE OPP as the only drinking fluid for 21–22 days . For both doses of OPP, the offspring of rats showed the absence of developmental birth defects or congenital anomalies. Moreover, the OPP supplementation to the rats did not affect their well-being. The macroscopic observation on all major organs showed no signs of OPP-induced toxicity . SeveralThe safety aspects of OPP administration is not limited only to pre-clinical data, as it has been evaluated in phase I single-blind trial in healthy volunteers. Following the daily administration of 450 mg GAE/day OPP for 60 days, there was no major or serious adverse effect detected. Normal range of clinical biochemistry profiles implied that OPP supplementation was not toxic. None of them reported any hypersensitivity reactions throughout the treatment period . Thus, OThis review summarized the current evidence of OPP for cardiovascular health. In the future, studies from in vitro systems should be well-integrated with in vivo studies, as well as clinical trials to be conducted to evaluate the applicability of these proposed mechanisms of OPP in human cardiovascular health. Therefore, in elucidating the OPP safety profile and potential as an alternative cardioprotective agent, further in-depth studies are warranted to explore a conclusive mechanism for its therapeutic action. Currently, there are several mechanisms proposed for its cardioprotective effects. The understanding of the proposed mechanism that leads to cardiovascular health will provide more insight into the development of potent cardioprotective agents that specifically target the pathways. The proposed pathways include the downregulated genes of cholesterol biosynthesis pathway that could mimic the action of a well-known anti-hyperlipidemic agent, for example, statin, by targeting the HMG-CoA reductase. Other mechanisms involved are the antioxidative and anti-inflammatory properties as well as modulation of vital clinical biochemistry pathway including fatty acid β-oxidation, TCA and ETC. In conclusion, the health benefits of OPP for CVD have been clearly elucidated by literature and it is important to conserve this valuable nutraceutical derived from the oil palm fruit.


Prunus persica L. Batsch) trees grow vigorously and are subject to intense pruning during orchard cultivation. Reducing the levels of endogenous gibberellins (GAs) represents an effective method for controlling branch growth. Gibberellin 2-oxidases (GA2oxs) deactivate bioactive GAs, but little is known about the GA2ox gene family in peach. In this study, we identified seven PpGA2ox genes in the peach genome, which were clustered into three subgroups: C19-GA2ox-I, C19-GA2ox-II, and C20-GA2ox-I. Overexpressing representative genes from the three subgroups, PpGA2ox-1, PpGA2ox-5, and PpGA2ox-2, in tobacco resulted in dwarf plants with shorter stems and smaller leaves than the wild type. An analysis of the GA metabolic profiles of the transgenic plants showed that PpGA2ox-5 (a member of subgroup C19-GA2ox-II) is simultaneously active against both C19-GAs and C20-GAs,which implied that C19-GA2ox-II enzymes represent intermediates of C19-GA2oxs and C20-GA2oxs. Exogenous GA3 treatment of shoot tips activated the expression of all seven PpGA2ox genes, with different response times: the C19-GA2ox genes were transcriptionally activated more rapidly than the 20-GA2oxC genes. GA metabolic profile analysis suggested that C20-GA2ox depletes GA levels more broadly than C19-GA2ox. These results suggest that the PpGA2ox gene family is responsible for fine-tuning endogenous GA levels in peach. Our findings provide a theoretical basis for appropriately controlling the vigorous growth of peach trees.Peach ( Prunus persica L.) is one of the most widely cultivated deciduous fruit trees worldwide. According to FAOSTAT data, the global cultivation area of peach and nectarine reached 1712.4 ha in 2018. Peach trees must be pruned to keep the tree size manageable and to facilitate harvesting, but most cultivated peach varieties grow vigorously, increasing the labor costs required for orchard management. One effective way to inhibit the rapid growth of peach branches is to treat the trees with paclobutrazol (PBZ), an inhibitor of gibberellin (GA) biosynthesis, indicating that endogenous GA is at least one of the phytohormones that promote the vigorous growth of peach branches.Peach , ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 13-oxidase (GA13ox), GA 20-oxidase (GA20ox), and GA 3-oxidase (GA3ox)  . Because (GA3ox) . The mai (GA3ox) .Arabidopsis thaliana (GA2ox genes are expressed throughout plant development and in different tissues (GA2oxs belong to the 2OG-Fe (II) oxygenase superfamily . GA2oxs thaliana , rice  in addition to bioactive GAs (GA1 and GA4). GAs are classified into different groups based on their number of carbon (C) atoms, including C20-GAs  and C19-GAs . GA2ox proteins are classified as C20-GA2ox or C19-GA2ox based on their preference for C20-GA or C19-GA substrates, respectively. Phylogenetic analysis of GA2ox proteins further divided the C19-GA2ox group into two subgroups  and PpGA2ox4-PpGA2ox7 (52% identity) share the highest sequence identity among the GA2ox genes  oxygenase superfamily. All seven PpGA2oxs contain conserved amino acid residues that are thought to bind Felutarate .19-GAs  or C20-GAs (such as GA12 and GA53), placing them into the C19-GA2ox or C20-GA2ox class , the bryophyte Sphagnum fallax, and the lycophyte Selaginella moellendorffii and GA2ox genes from the flowering plants rice (Oryza sativa), peach (Prunus persica), and Arabidopsis thaliana , which is highly conserved among C20-GA2ox-I proteins. This observation suggests that the C19-GA2ox-II subgroup is intermediate between the C19-GA2ox-I and C20-GA2ox-I subgroups.Motif analysis of GA2ox, GA20ox, and GA3ox proteins uncovered an amino acid sequence in the N-termini of these proteins that is the signature motif of GA2oxs , pointinubgroups . The mosPpGA2ox genes in different tissues, we analyzed their transcript levels in six tissues   ; PpTEF2 GA2ox gene from each of the three subgroups , we generated the 35S:PpGA2ox1, 35S:PpGA2ox5, and 35S:PpGA2ox2 constructs and introduced them into tobacco by Agrobacterium-mediated transformation. For each construct, more than three independent transgenic tobacco lines were obtained, which were confirmed by PCR and qRT-PCR . These results strongly suggest that PpGA2ox1 is a C19-GA2ox and that PpGA2ox2 is a C20-GA2ox. Interestingly, plants overexpressing PpGA2ox5 showed an intermediate phenotype compared to the PpGA2ox1-OE and PpGA2ox2-OE lines. These results suggest that PpGA2ox5 is active against GA1. However, PpGA2ox5 is less efficient than PpGA2ox1, suggesting that PpGA2ox5 might not be a classical type of C19-GA2ox.Based on our phylogenetic analysis , we preda C19-GA . GA3, wh and C-3 , was usePpGA2ox1, PpGA2ox2, or PpGA2ox5  and seven C19-GAs  using HPLC-MS/MS. The levels of the four C20-GAs  were higher in the PpGA2ox1-OE line and lower in the PpGA2ox5-OE and PpGA2ox2-OE lines vs. the wild type. GA24 and GA44 levels were significantly higher in the PpGA2ox1-OE line, whereas the levels of all four C20-GAs were significantly lower in the PpGA2ox5-OE and PpGA2ox2-OE lines compared to the wild type. Only PpGA2ox2-OE plants showed significantly lower levels of both C19-GAs (GA9 and GA20) compared to the wild type. GA8 and GA29 levels were significantly higher in all transgenic plants compared to the wild type. GA5 levels were significantly higher in all three transgenic lines vs. the wild type as well. Moreover, GA5 contents were 30.3-fold higher in PpGA2ox5-OE than in the wild type. GA12, GA110, GA97, GA51, and GA34 were not detected in any of the plants. These results suggest that PpGA2ox5 is active not only against C19-GAs, but also against C20-GAs.To determine how PpGA2ox5 may differ from PpGA2ox1 and PpGA2ox2, we analyzed the GA metabolic profiles in shoot tips with young leaves from wild-type tobacco plants and transgenic lines overexpressing PpGA2ox5 . We meas8 content among PpGA2ox1-OE, PpGA2ox5-OE, and PpGA2ox2-OE plants, but PpGA2ox1-OE plants showed higher activity against GA1 compared to PpGA2ox5-OE and PpGA2ox2-OE. The main bioactive GA in tobacco is GA4, whereas GA1 levels are low in this plant (1 (endogenous vs. exogenous GA1) in the plants.Surprisingly, there were no differences in GAis plant . This paGA2oxs in Arabidopsis  than the wild type, which was similar to the results for PpGA2ox2-OE (a predicted C20-GA2ox) but opposite to those for of PpGA2ox1-OE (a predicted C19-GA2ox). These changes in C20-GA levels in the PpGA2ox5-OE lines were consistent with those in Arabidopsis 19-ga2oxc and 20-ga2oxc mutants  (PpGA2ox1-OE showed higher levels of GAand GA1) .19-GA2oxs, C20-GA2oxs, and GA biosynthetic oxidases (GA20ox and GA3ox) are all members of the 2OG-Fe (II) oxygenase superfamily , GA20ox, and GA3ox were absent in this moss  is highly conserved in C19-GA2ox-I proteins. This tryptophan is also highly conserved in four clades of the 2OG-Fe (II) oxygenase superfamily   in the signature motif of GA2ox is highly conserved in both C GAox-C) . We specGA2ox1  illustrate how these enzymes fine-tune GA levels. Three PpGA2ox genes, one each from the C19-GA2ox-I, C19-GA2ox-II, and C20-GA2ox-I subgroups, were significantly upregulated after GA3 treatment at similar time points, whereas one gene from C19-GA2ox-II and one from C20-GA2ox-I were significantly upregulated more slowly after GA3 treatment. After GA3 treatment, the C19-GA2oxs were activated first, followed by the C20-GA2oxs. Six C20-GAs and four C19-GAs are intermediates in the GA biosynthesis pathway. The C20-GA2oxs significantly depleted the C20-GAs and some of the C19-GAs, while the C19-GA2oxs significantly depleted the C19-GAs but increased the levels of some of the C20-GAs  and the maximum width of the leaf (width).The peach cultivar used in this study, “QiuMiHong” (QMH), was provided by the Fruit Tree Germplasm Repository of Henan Agricultural University . For RNA extraction, young fruits, flower buds, flowers, sepals, petals, young leaves, mature leaves, and shoot tips were collected from 3 year old “QMH” trees in the spring, immediately frozen in liquid nitrogen, and stored at −80°C for subsequent experiments. Tobacco variety “K326” seedlings were grown GA2ox genes in the peach genome, a BlastP with default parameters of the Phytozome database1 was performed based on the conserved domain of genes in the 2-oxoglutarate-dependent oxygenase family using the amino acid sequences of seven Arabidopsis GA2oxs as queries. The search results were aligned by ClustalW algorithm with default parameters to reduce duplicate and redundant sequences . PCR products that produced a single band on the gel were purified and sent to Sangon Biotech (Shanghai5) for sequencing. All primers used in this study are listed in The full-length sequences of the PpGA2oxs were amplified from the pEASY® -Blunt Vector (TransGen Biotech) using specific primers containing restriction sites and inserted into the pSAK277 vector containing the CaMV35S promoter. The plant overexpression vectors 35S:PpGA2ox1, 35S:PpGA2ox5, and 35S:PpGA2ox2 were introduced into Agrobacterium tumefaciens strain GV3101 and used to transform tobacco leaf discs as described previously (A. tumefaciens (OD600 = 0.6) harboring the chosen construct for 5 min, and transferred to MS medium. After 2 days of co-culture in the dark, the leaf discs were transferred to selection medium . When shoots appeared, individual shoots were cut and transferred to root-inducing medium . After rooting, the plantlets were grown in pots in a greenhouse and used for phenotypic analysis and further experiments. A pSAK277-GFP vector was transformed into tobacco in our lab and the transgenic lines showed no dwarf phenotype and smaller leaves.The full-length sequences of the eviously . BrieflyTEF2 (Peach EST database accession number: TC3544) and β-tubulin (accession number: U91564) were used for data normalization . Purified total RNA (0.5–2 μg) was reverse transcribed into first-strand cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser . The cDNA product was used for semiquantitative RT-PCR and qRT-PCR analysis. qRT-PCR analysis was conducted using SYBR Select Master Mix  according to the manufacturer’s protocol on an ABI PRISM 7500 FAST Sequence Detection System . All experiments were conducted in triplicate. To quantify relative transcript level, lization .3 (100 mg/L) or with distilled water (as a control). Three biological replicates were performed for all treatments. Shoot tips were collected at 0.5, 2, and 4 h after treatment for RNA extraction to analyze GA2ox transcript levels.One year old branches of “QMH” peach trees were sprayed with GATo measure endogenous phytohormone contents, approximately 1.0 g of young leaf tissue was collected from transgenic and wild-type tobacco plants and frozen in liquid nitrogen. The concentrations of different GAs were measured by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) as described previously . Three b1 or 0.4 mg/L GA3. Changes in growth were observed 10 days after treatment. The lengths of 8–10 leaves were measured and averaged. The experiments were performed three times.The shoots of transgenic tobacco were cultivated in MS medium containing 0.4 mg/L GAThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/JF, XZ, and JC conceived and designed the experiments. JM, HL, and MZ performed the experiments. JF and JC wrote the manuscript. BT, XY, and WW performed the GA metabolic analysis. LZ, ZL, and JL revised the manuscript. All authors contributed to the article and approved the submitted version.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.


P ˂ .05). The control slices showed greater increase in browning index (BI) and greater decrease in lightness (L*) than pretreated dried samples. Ascorbic acid and QSM treatments can be effective in the control of the enzymatic browning along with maintaining the quality properties of banana chips. Therefore, using of immersion pretreatment with 0.25% QSM and 0.05% ascorbic acid is recommended to prevent enzymatic browning as well as maintain the quality of banana chips before the drying process.The objective of this investigation was to illustrate the effects of quince seed mucilage (QSM) and ascorbic acid pretreatments to prevent the quality of freeze‐dried banana slices. The studied parameters were moisture content, antioxidant activity, total phenol, color properties, structural properties, and sensory evaluation. Both treatments were effective in protecting total phenolic content and antioxidant activity in dried banana slices  than pre‐treated dried samples. Ascorbic acid and QSM treatments can be effective in the control of the enzymatic browning along with preserving the quality properties of banana slices. Therefore, using of immersion pretreatment with 0.25% QSM and 0.05% ascorbic acid is recommended to prevent enzymatic browning as well as maintain the quality of banana slices prior to the drying process. It destroys the color and quality of the cut fruit and effects adversely on its quality  is a set of a cellulosic fractions with a more hydrolyzed polysaccharides Schmidt, . As we pP ˂ .05). As stated by Sikora and Świeca, . Bananas and quince seed were prepared from a local market in Tehran (Iran).2.2A sequential process was employed to extract mucilage from quince seeds using the technique explained by Jouki, Yazdi, Mortazavi, and Koocheki .2.3Bananas (cv. Cavendish) with uniform size and color were selected, peeled, and cut into 30 equal slices (3 mm thickness) using a sharp steel knife. The fresh‐cut banana slices were immersed in solutions Table  for 1 mi2.4The banana slices were dried using an Armfield vacuum freeze‐dryer  according to the method of Gębczyński, Skoczeń‐Słupska, and Kur . Briefly2.5b is the moisture content of dried banana slices before drying in oven and Ma is the moisture content after oven.The moisture content of freeze‐dried banana slices was determined in a vacuum oven at 70°C for 48 hr, according to AOAC . The dig2.6g for 15 min at 5°C by an Ultra Centrifuge (Sigma 8K) and 0.1 ml of the supernatant was added to 3.9 ml of methanolic DPPH solution (at a concentration of 0.1 mM). Then, the obtained mixture was shaken and placed in a dark place for half an hour at room temperature. Absorption of the samples against a blank of methanol without the DPPH reagent was determined at 517 nm (using a PerkinElmer Spectrophotometer). The antioxidant activity of dried banana slices was expressed as µmol TE / g based on dry weight.The antioxidant activity of the dried banana chips was evaluated by method of the free radical scavenging effect on 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical described by Brand‐Williams, Cuvelier, and Berset . Briefly2.7g for 15 min and 0.1 ml of the supernatant was reacted with 0.2 ml FCR, comprehensively vortexed, and 0.8 ml (700 mmol/L) Na2CO3 was added to the assay mixture. Then, the mixture was placed in a dark place for an hour at room temperature. After 1‐hr incubation, UV–Visible Spectrophotometer at 765 nm was used to measure the absorbance of banana extracts. Gallic acid was applied as the standard, and the results were obtained based on a standard curve equation of gallic acid (0.05–0.75 mM). Experiment was performed in four replications, and the mean of replicates was expressed as mg of gallic acid equivalent (GAE) per gram based on dry weight.Total phenolic content (TPC) was determined by Folin–Ciocalteu reagent (FCR) method as cited by Lee et al. . Briefly2.8After freeze‐drying, color values of banana slices were measured by using Konica Minolta Colorimeter. Color was measured using the CIE L (lightness), a (redness), and b (yellowness) coordinates. For each sample, color values  were read 6 times at different locations on the banana slice surface. As described by Perez‐Gago et al. , the bro2.9Scanning electron microscopy, SEM (AIS‐2100), was applied to analyze the internal structure of freeze‐dried banana slices after dipping pretreatment according to the method of Jouki et al. . The sam2.10Sensorial tests were performed by comparison with 15 semitrained panelists  in standardized individual cabins possessing adequate illumination. The panelists were trained to evaluate sensorial parameters of dried banana slices. The scores  were given by the panelists  and were presented as mean values with standard deviations. All experiments were performed in four replications, and Duncan's multiple range tests were used to compare the differences among means.33.1The moisture content of the untreated dried banana slices was 7.67 ± 0.18% after freeze‐drying process Figure  and PanaThe results showed that the amount of ascorbic acid was not a critical parameter in the levels of 0 to 0.25%. It can be observed that after drying process, the ascorbic acid pretreated slices showed the minimum amount of moisture content (7.38% w.b), followed by the untreated banana samples (7.67% w.b) and the mucilage dip treated banana slices (7.94% w.b).In similar research, Abano et al.  showed t3.2−1 of dry basis, respectively. A decrease in TPC of dried banana chips was seen as compared to the TPC value of the fresh banana slices. This reduction in total phenols of freeze‐dried banana slices was 5.12, 19.23, and 24.35% for control (untreated), QSM pretreated, and pretreated by ascorbic acid, respectively. As shown in Figure The antioxidant activity and total phenolic content of fresh‐cut banana slices before dying stage were 40.32 µmol/g and 7.88 mg GAE gThe lower effect of freeze‐drying on the reduction of phenolic compounds compared to other methods of drying fruit slices has been reported in many studies. However, total phenolic contents of mango, hawthorn, starfruit, papaya, and sour cherry watermelon have decreased by freeze‐drying . A similar trend has been reported by Pan et al. . As stated by Askari, Emam‐Djomeh, and Mousavi , immersi3.5p < .05) than untreated and ascorbic acid treated samples . The results were in accordance with the results of the browning index and microstructure, which showed that the samples pretreated with both mucilage and ascorbic acid solutions at medium concentrations had lower browning index and higher porosity.As shown in Figure s Figure . Panelis4The application of both QSM and ascorbic acid treatments inhibited the enzymatic browning, thereby prevented the conversion of phenols to quinones and reduced enzymatic browning, while increased the overall acceptance of dried banana slices. The banana slices treated with both sodium chloride and gum had crisper texture and golden color than the regular freeze‐dried slices. According to the results of physicochemical, microstructural, and sensory evaluation, application of immersion treatment with 0.05% ascorbic acid + 0.25% QSM solution to inhibit enzymatic browning with preservation of qualitative properties of banana chips before drying process is suggested.The authors have declared no conflicts of interest for this article.We declare no ethical issue related with this article.