Edwardsiella tarda is a severe fish pathogen, featured by its capacity to live inside host phagocytes. For intracellular survival, it is crucial for E. tarda to neutralize the deleterious effect of host reactive oxygen species (ROS). Accumulating evidence suggests that bacterial metabolism is closely connected to oxidative resistance. However, the roles of E. tarda metabolic proteins in antioxidative adaptation and intracellular proliferation remain elusive. In this study, we performed a proteomic analysis on E. tarda and identified 111 proteins responsive to H2O2-mediated oxidative stress. Based on this data, we further obtained eight crucial proteins, including seven metabolic proteins, for E. tarda antioxidation and intracellular infection. Among them, two C4-dicarboxylate transporters were found necessary for E. tarda to disseminate in fish tissues. Furthermore, the substrate of the two transporters was identified as L-aspartate, which was proven to be essential for the full antioxidative capacity of E. tarda. Our results indicate that reprogramming the metabolic flux to the production of pyruvate, a ketoacid capable of neutralizing ROS, was likely a pivotal strategy of E. tarda to survive the oxidative environments inside host cells. Together, the findings of this study highlight the significance of metabolic reprogramming for bacterial redox homeostasis and intracellular infection.Edwardsiella tarda is a significant fish pathogen that can live in challenging environments of reactive oxygen species (ROS), such as inside the phagocytes. Metabolic reconfiguration has been increasingly associated with bacterial oxidative tolerance and virulence. However, the metabolic proteins of E. tarda involved in such processes remain elusive. By proteomic analysis and functional characterization of protein null mutants, the present study identified eight crucial proteins for bacterial oxidative resistance and intracellular infection. Seven of them are metabolic proteins dictating the metabolic flux toward the generation of pyruvate, a key metabolite capable of scavenging ROS molecules. Furthermore, L-aspartate uptake, which can fuel the pyruvate generation, was found essential for the full antioxidative capacity of E. tarda. These findings identified seven metabolic proteins involved in bacterial oxidative adaptation and indicate that metabolic reprogramming toward pyruvate was likely a pivotal strategy of bacteria for antioxidative adaptation and intracellular survival. Edwardsiella tarda is a Gram-negative facultatively anaerobic bacterium, belonging to the Enterobacteriaceae family , which can be converted into hydroxyl radicals by the Fe2+-dependent Fenton reaction with superoxide dismutase (SOD) or participate in the generation of hypochlorous acid and chloramines (Reactive oxygen species (ROS) production, termed respiratory burst, is a common programmed response of phagocytes upon bacterial infection . NADPH o p67phox . Upon imoramines . These horamines –14.2·− to H2O2. The latter can be further detoxified by conversion to O2 and H2O with bacterial catalases . A total of 1,588 proteins were identified in the H2O2 treatment group  and the control group , of which 1,505 were shared in common  were identified in the comparison of H2O2 group vs control group, 67 of which were upregulated and 44 were downregulated , molecular function (MF), and cellular component (CC) (Gene ontology (GO) analysis showed that the DAPs of Hent (CC) . The sigE. tarda . The DAPs were categorized into the top 10 KEGG pathways of pentose and glucuronate interconversions, arginine biosynthesis, pantothenate and CoA biosynthesis, tryptophan metabolism, valine, leucine and isoleucine biosynthesis, biosynthesis of vancomycin group antibiotics, one carbon pool by folate, polyketide sugar unit biosynthesis, acarbose and validamycin biosynthesis, and two-component system (Table S6).Kyoto Encyclopedia of Gene and Genome (KEGG) analysis was conducted to reveal the signaling pathways contributing to the oxidative defense of 2O2 group (ranked based on the proteomic abundance) were analyzed for their relative mRNA levels in the H2O2 group vs the control group  and the top 25 DAPs that were detected only in the Hol group . The resol group . These eE. tarda, the isogenic mutants of wild-type strain TX01, namely TX01Δ0310, TX01Δ0790, TX01Δ1753, TX01Δ1821, TX01Δ2289, TX01Δ2291, TX01Δ2977, and TX01Δ3323, were created by markerless gene deletion. The essentiality of KPOD proteins for the resistance of E. tarda to oxidative stress was assessed by H2O2 pulsing assay. The result showed that, under H2O2 stress, the survival rates of the mutants were all significantly reduced, in comparison with that of the wild-type TX01 , C4-dicarboxylate transporter, DcuC1 (ETAE_0790), and putative dicarboxylate-binding periplasmic protein (ETAE_2977), are involved in C4-dicarboxylate transportation. To investigate the potential role of CDTs in the infectivity of nfection , indicatIn order to determine the possible substrate(s) transported by CDTs, growth curves of TX01Δ0310 and TX01Δ0790 were, respectively, compared with those of the wild-type TX01 in a chemically defined medium, with or without the potential substrates of Dcu family, namely L-aspartate, malate, or fumarate. The results indicated that in the absence of L-aspartate both TX01Δ0310 and TX01Δ0790 showed similar growth curves with TX01, while in the presence of L-aspartate both TX01Δ0310 and TX01Δ0790 exhibited notably delayed growth compared to that of TX01 . In contE. tarda, we conducted H2O2 pulsing assay to evaluate the resistance of TX01, TX01Δ0310, and TX01Δ0790 to oxidative stress in the medium without or with L-aspartate. The results showed that in the absence of L-aspartate, the survival rates of TX01, TX01Δ0310, and TX01Δ0790 were similar , mannonate dehydratase (ETAE_2289), glycerate 2 kinase (ETAE_3323), C4-dicarboxylate transporter DcuA (ETAE_0310), C4-dicarboxylate transporter DcuC1 (ETAE_0790), putative dicarboxylate-binding periplasmic protein (ETAE_2977), and dihydromonapterin reductase (ETAE_1753), and one non-metabolic protein, i.e., starvation-inducible DNA-binding protein, Dps (ETAE_1821).thriving , 5, 23. Escherichia coli, fructuronate reductase and mannonate dehydratase are important for glucuronate assimilation  cycle through oxaloacetate or fumarate  medium at 28°C  were cultured at 37°C in Dulbecco’s modified Eagle’s medium  supplemented with 10% (vol/vol) fetal bovine serum , 100 units/mL penicillin, and 100 µg/mL streptomycin  in a humidified atmosphere containing 5% COScophthalmus maximus), averaging 20 g, were purchased from Hai-Shuo-Jia-Yuan aquaculture company in Qingdao city, Shandong province, China. The fish were maintained at ~20°C in aerated seawater and fed with commercial feed daily. Turbot were acclimatized in the laboratory conditions for 2 weeks before the experiments.Healthy turbot  or 10 mM hydrogen peroxide (H2O2). After 3 h, the bacteria were centrifuged and washed with phosphate buffered saline (PBS) at 4°C, and frozen in liquid nitrogen for subsequent experiments.2O2 treatment group) were lysed with SDT  buffer. The protein of each sample was extracted and quantified with the BCA Protein Assay Kit . Subsequently, trypsin digestion of protein was performed according to the method of filter-aided sample preparation procedure described by Matthias Mann  . In brieEdwardsiella tarda EIB202_6916_20201010 database using the MaxQuant software (1.5.3.17), as previously described . The GO annotation and the KEGG pathway of DAPs were annotated using the software program Blast2GO and KEGG Automatic Annotation Server. After Fisher’s exact test, GO terms and pathways with P-value < 0.05 were considered to be significantly enriched.The statistical significance was determined with an unpaired Student’s -ΔΔCT) method with topA as an internal control . RNA was extracted using Bacterial RNA Kit , with an optional on-membrane DNase I treatment step included to remove the residual genomic DNA. cDNA was obtained by reverse transcription using the RevertAid First Strand cDNA Synthesis Kit . Quantitative real-time reverse transcription PCR (qRT-PCR) was performed in technical duplicates  using the ChamQ Universal SYBR qPCR Master Mix  in a QuantStudio 3 Real-Time PCR System. Relative transcription was quantified by the comparative Ct . The resulting plasmids were introduced into E. coli S17-1 λpir and then E. tarda TX01 by conjugative transfer. The mutant strains were generated by two-step homologous recombination and verified by PCR and sequencing (Table S1).In-frame gene deletion was carried out as reported previously . In brieEcoRV site by homologous recombination. The resulting plasmid was introduced into the mutant strain TX01Δ0310 or TX01Δ0790 by electroporation, yielding the complementary strain TX01Δ0310c and TX01Δ0790c.For the construction of the ETAE_0310 or ETAE_0790 complementary strain, the entire coding sequence of ETAE_0310 or ETAE_0790 was amplified by PCR and then inserted into pBT3  at the EAll primers used in this study are listed in Table S2.E. tarda strains were grown in LB medium at 28°C until OD600 ≈ 0.5. The bacteria were collected by centrifugation, washed with PBS, and suspended in PBS with 0 or 1 mM H2O2 to a final concentration of 1 × 105 CFU/mL. After 2-h incubation at 28°C, the cells were diluted in PBS and plated on LB agar plates. The plates were incubated at 28°C for 24 h; the number of colony-forming units was counted and bacterial survival was determined as follows: (number of survived cells after H2O2 treatment/number of recovered cells without H2O2 treatment) × 100%.E. tarda to H2O2 pulsing, TX01, TX01Δ0310, and TX01Δ0790 were grown in FMCM medium at 28°C until OD600 ≈ 0.2, and then 5 mM or 0 mM (as control) of L-aspartate was added to the medium. The bacteria were grown for another 2 h at 28°C, then subjected to H2O2 pulsing and plate count as described above.To examine the effects of exogenous metabolite on the resistance of E. tarda wild-type TX01 and derivates as described previously with slight modification  to a final concentration of 1 × 107 CFU/mL. Bacteria were added to RAW264.7 cells in a 24-well plate at a multiplicity of infection of 10:1. The plates were centrifuged at 800 g for 10 min and incubated at 30°C for 1.5 h. Then, the culture medium was replaced by fresh Opti-MEM containing 400 µg/mL gentamicin. The plates were incubated at 30°C for 40 min for the antibiotics to kill the extracellular bacteria. The cells were then washed three times with PBS and cultured in Opti-MEM containing 20 µg/mL gentamicin for 0, 2, 4, and 6 h to allow intracellular proliferation of the bacteria. At each time point, 600 µL 1% (vol/vol) Triton X-100 was added to the plate to lyse the cells; the lysate was diluted and plated onto LB agar. The plates were incubated at 28°C for 24 h, and the number of colonies was counted.RAW264.7 cells were infected with fication . BrieflyIn vivo infection was performed as described previously with slight modification (7 CFU/mL. Turbot were randomly divided into five groups (nine fish/group) and infected via intramuscular injection with 100 µL TX01, TX01Δ0310, TX01Δ0310c, TX01Δ0790, or TX01Δ0790c. At 12-, 24-, and 48-h post-infection, fish were euthanized with an overdose of tricaine methanesulfonate . The spleen and kidney were collected aseptically, homogenized, and serially diluted in PBS. The diluted homogenates were plated onto LB plates. The plates were incubated at 28°C for 24 h, and the number of colonies was counted.fication . Brieflyt-test. Statistical significance was defined as P < 0.05.All experiments were performed three times. Statistical analyses were carried out with GraphPad Prism version 7.00 . Data were analyzed with Student’s


Apolyaromatic receptor with a virtually isolated, hydrophobic cavityexclusively binds one molecule of phenylalanine dipeptide from a mixturewith its amino acid and tripeptide in water via multiple CH−πand hydrogen-bonding interactions in the complementary cavity. Thebinding selectivity persists even in the presence of other dipeptides,such as leucine–leucine, leucine–phenylalanine, tyrosine–phenylalanine,tryptophan–tryptophan, and aspartame, revealed by NMR/MS-basedcompetitive binding experiments. ITC studies reveal that the selectivebinding of the phenylalanine dipeptide is relatively strong (Ka = 1.1 × 105 M–1) and an enthalpically and entropically favorable process (ΔH = −11.7 kJ mol–1 and TΔS = 17.0 kJ mol–1). In addition, the present receptor can be used for the emissiondetection of the dipeptide through a combination with a fluorescentdye in water.Precise recognitionof peptides is a daunting task owing to thesubstantial number of available amino acids and their combinationinto various oligo/polymeric structures in addition to the high hydrationof their flexible frameworks. Here, we report the selective recognitionof a dipeptide through a  Furthermore, (iii) selectiveemission detection of the dipeptide can be accomplished using 1 and a fluorescent coumarin dye in water.Here, we propose a newstrategy for the selective recognition ofoligopeptides using d cavity 1c. Synthc panels 1d.10,11 1 is a spherical supramolecularcapsule, composed of two metal ions and four bent polyaromatic ligands,usable under ambient aqueous conditions.10 Besides typical hydrophobic guest molecules ,11 its virtually closed cavity (∼1.3 nm in diameter and ∼580Å3 in volume) displays efficient binding abilitiestoward hydrophilic oligomers,12 such assynthetic oligo(ethylene glycol)s and biorelated oligo(lactic acid)s,through the small yet flexible gaps (∼0.3 nm windows) in waterat room temperature, without the ligand dissociation. However, thelength-selective recognition of such oligomers by the receptor hasbeen unachieved so far, owing to their flexibility and none/smallsubstituents. We posited that oligopeptides, bearing similar flexibleframeworks yet characteristic side chains , would be promising new targets for receptor 1 with high binding selectivity. Importantly, this study is focusedon unprotected natural oligopeptides rather than N- and C-termini-protected ones because the bindingproperties are largely affected and changed by the protecting groups.9Syntheticreceptor 1 toward two molecules of benzene derivatives  in water at room temperature,13 phenylalanine (F) and its dipeptide FF and tripeptide FFF were selected as the first set ofa mixture among a huge number of amino acid/oligopeptide combinations.Prior theoretical estimations by DFT calculations predicted that 1:1host–guest structure 1•FF shouldbe the most stable product among the possible four products 2, 1•FF, and 1•FFF; Table S1), dueto the size and shape matching.15Inspiredby the selective binding ability of polyaromatic receptor 1, F, FF, and FFF (0.13 μmol each) was stirred in water (0.5 mL) at room temperaturefor 30 min, 1:1 host–guest structure 1•FF was formed in a selective and quantitative fashion .14 The large upfield shifts of the aliphaticsignals (Δδmax = approximately −4 ppm)are derived from the effective aromatic shielding effect by 1. The splitting of the aliphatic signals from 6 to 12 uponbinding indicates tight host–guest interactions in the desymmetrizedpolyaromatic cavity,18 through efficientcarbonyl-based hydrogen-bonding interactions, as discussed later.The ESI-TOF MS spectrum of the resultant solution displayed molecularion peaks, corresponding to 1•FF n and 1•FFF were hardly detected in the spectrum. To our surprise,neither F nor FFF was bound by 1 under the same conditions, separately and thus, even from a complexmixture of F, FF, and FFF ina 5:1:5 ratio, receptor 1 bound FF in anexclusive manner .14 The quantitative binding of FF bythe receptor was found even in an acetic acid buffer solution . To support the weak NMR signalsof bound FF, C-terminal methyl-protectedphenylalanine dipeptide FF-Me was used as a referenceguest  and thermodynamic parameters(ΔH and TΔS) of 1 toward FF were estimated by ITCanalysis.14 The titration study in waterat 25 °C revealedthat the host–guest interactions are relatively high . Weak host–guest π–πinteractions were supported by the UV–visible study of 1•FF, where the anthracene-based absorptionbands (λ = 300–420 nm) remain virtually intact .14 The entropic energy gain indicates the dehydrationof the host cavity and the guest surface upon inclusion. As we expected,ITC studies revealed that C-terminal and C,N-terminal protections on FF with methylgroups enhance the host–guest interactions (2.5-fold for FF-Me and 4.4-fold for FF-3Me based on Ka) with 1 under the same conditions21 and four hydrogen-bonding interactions (2.1–2.5Å) was indicated between 1 and FF inthe isolated cavity and leucine–leucine (LL), bearing hydrophobicisopropyl groups, potentially interacting with the receptor cavitythrough multiple CH−π interactions. Stirring a mixtureof dipeptides FF, LF, and LL (1.0 equiv. each) with receptor 1 in water (0.5 mL)for 30 min at room temperature led to the selective and quantitativeformation of 1•FF .14 Host–guest interactions between 1 and LF or LL were scarcely observed even when addingexcess amount of LF or LL (5.0 equiv) to 1 in water .Next,the ability of  of 1•FF 4a. The pFF was demonstratedby 1, even from a mixture of FF and aspartame,known as a representative dipeptide-based artificial sugar, underthe same conditions .14 The binding selectivity persisted in the presence of long aliphaticoligopeptides, such as alanine trimer AAA and tetramer AAAA, with multiple methyl groups .Notably, the exclusivebinding of nditions S10.14 Tharomatic and aromatic side chains on the dipeptides was further examined. Tryptophan (W) provides a heterocyclic indole ring capable of efficientlyinteracting with aromatic and aliphatic surfaces so that W and its oligomers have been preferentially bound by the majorityof previous synthetic receptors so far.22 In contrast, the closed polyaromatic cavity of 1 couldcapture FF with 75% selectivity from a mixture of FF, WF, and WW as well as with 100%selectivity from a mixture of FF and WW.When a mixture of 1, FF, WF, and WW (1.0 equiv. each) was stirred in water in thepresence of NaNO3 (0.2 equiv. based on 1)at room temperature for 30 min, the quantitative formation of host–gueststructures 1•FF and 1•WF was confirmed by 1H NMR and ESI-TOFMS analyses . Thesespectra indicated the absence of both empty 1 and host–gueststructure 1•WW. Observed aliphaticproton signals in the range of 0.7 to −1.0 ppm were derivedfrom bound FF and WF in the cavity of 1 (1 toward FF and WF was estimated to be 75:25 on the basis of theintegration of proton Hg (at ∼9.2ppm) on the receptor.14 It is worth notingthat the simple addition of the nitrate salt improved the selectivityby 1.4-fold, most probably due to the slight shrinking of the polyaromaticshell at high ionic strength. From a 1:1 mixture of FF and sequential isomer FW, receptor 1 bound FF in 79% selectivity , even without NaNO3 addition.14 Neither 1•WW nor 1•W was generated even by the treatment of 1 with WW or W under various aqueousconditions. These results revealed the detailed recognition orderof 1 to be FF > WF > FW ≫ WW ≈ W. On thebasis of ITC-based binding constants, host–guest interactionsof 1 toward WF (Ka = 2.6 × 104 M–1) are 4.1-foldweaker than those toward FF even without NaNO3 .14The distinction between 1 in anexclusive way. From a 1:1 mixture of FF and tyrosine–phenylalanine(YF) with 1 in water at room temperature.14 The selectivity stems fromthe steric bulkiness of the para-substituted OH groupon the phenyl ring, which is unfit to the closed and rigid, sphericalcavity.23 The exclusive binding of FF by 1 was also observed from a 1:1 mixtureof FF and phenylalanine–tyrosine (FY) under the same conditions .Furthermore, among phenyl-based dipeptides, theabsence and presenceof a single hydroxy group were recognized by perature5b, the 1 of 1•FF S21.14 Th1, the facileyet sensitive detection of dipeptide FF was performedby the use of a fluorescent coumarin dye. Although hydrophobic coumarin C153 employed here is water-insoluble under ambient conditions,aqueous host–guest structure 1•C153 emitted green fluorescence (ΦF = 20%) in waterupon light irradiation at 423 nm . A similar strong emission was observed from an aqueous solutionof 1, F, and FFF (in a 1:1:1ratio) after 5 min, upon addition of C153 (2.0 equivbased on 1), due to the formation of 1•C153  in water,with the small exception of dipeptides with a tryptophan and phenylalanineresidue. The quantitative experimental data such as the binding constantand thermodynamic parameters were obtained by ITC analysis. The observedunprecedented high selectivity is derived from size and shape host–guestcomplementarity as well as multiple host–guest interactions, enhancedby the closed polyaromatic cavity. In addition, the highly selectiveand sensitive detection of the dipeptide was achieved by the simpleaddition of the present receptor and a fluorescent dye.closed cavity strategy can be widely applied for not onlysimilar capsules, bearing other polyaromatic panels 24 and spacers 25 but alsovarious capsules with closed large cavities, reported by other groups.26 Further investigations of the present cavity-directedhost–guest system will lead to the development of made-to-orderrecognition tools for complex biomolecules with high specificity inwater.Fordifferent selectivities, we hope that the coordination-based 3 was used as an external standard forhost–guest studies in D2O), ESI-TOF MS: Bruker micrOTOFII, UV–visible: JASCO V-670DS, FT-IR: SHIMADZU IRSprit, ITC:MicroCal system, VP-ITC model. Peptide synthesizer: Gyros ProteinTechnologies, PurePep Chorus. PM6 and DFT calculations: Gaussian 16program (Rev. C.01) package. Molecular mechanics calculation: Forcitemodule, BIOVIA Materials Studio 2020, version 20.1.0.5 (Dassault SystèmesCo.).The following analyticalinstruments and software were used inthis study. NMR: Bruker AVANCE III HD 500  in CDCl


Lansium domesticum Corr. cv. kokosan against estrogen receptor alpha (ERα) using in silico techniques. Utilizing molecular docking, Lipinski’s rule of five, in silico ADMET, and molecular dynamics simulations, we assessed the potency of five onoceranoid triterpenes against ERα. Molecular docking indicated competitive binding energies for these triterpenes relative to the active form of tamoxifen (4OHT) and estradiol, an ERα native ligand. Three triterpenes met drug-likeness criteria with favorable ADMET profiles. Notably, 2 demonstrated superior binding affinity in molecular dynamics simulations, outperforming estradiol, closely followed by 3 and 4. Hierarchical clustering on principal components (HCPC) and the spatial distribution of contact surface area (CSA) analyses suggest that these triterpenes, especially 2, may act as antagonist ligands akin to 4OHT. These findings highlight the potential of onoceranoid triterpenes in treating ERα-related breast cancer.Breast cancer is a significant global concern, with tamoxifen, the standard treatment, raising long-term safety issues due to side effects. In this study, we evaluated the potential of five onoceranoid triterpenes from  Breast cancer is the most prevalent cancer in women around the world ,2. It isα, ERβ, and membrane receptors such as G protein-coupled estrogen receptor 1 (GPER1) [Breast cancer has various subtypes characterized by different criteria, including pathological type, lymph node involvement, tumor size, and molecular subtypes . The mos (GPER1) .α and ERβ, are activated by estrogens such as estradiol, forming homodimers or heterodimers with other ER-ligand complexes [α is the predominant subtype in breast cancer, accounting for approximately two-thirds of cases, and can be modulated by agents like tamoxifen [ERs, including ERomplexes . These domplexes . ERα is amoxifen .Tamoxifen, a selective ER modulator, is commonly used to treat ER-positive breast cancer by blocking the ER pathway . HoweverLansium domesticum Corr. cv. kokosan exhibit cytotoxic activities against MCF-7 breast cancer cells, which overexpress ERα [L. domesticum Corr. cv. kokosan. They include lansiolic acid (1) [2) [3) [4) [5) [Reinwardtiodendron cinereum [L. domesticum Corr. [L. domesticum Corr. cv. kokosan may also inhibit breast cancer.Natural products have gained attention as potential alternative agents for combating breast cancer ,23,24,25ress ERα ,29,30. Sacid (1) , 8,14-se (1) [2) , 8,14-se [2) [3) , kokosan [3) [4) , and 3-h [4) [5)   to predict the drug-likeness properties of the onoceranoid triterpenes. Additionally, we assessed the pharmacokinetics and toxicity of the screened onoceranoid triterpenes using in silico ADMET . Ultimately, we subjected the onoceranoid triterpenes to 500 ns molecular dynamics simulations to better understand the inhibition of onoceranoid triterpenes against ERα. In this study, we evaluated the anti-breast cancer activities of five onoceranoid triterpenes from L. domesticum Corr. cv. kokosan against ERα. Before subjecting all onoceranoid triterpenes to molecular docking, we performed a redocking procedure using the complex of ERα and 4-hydroxy tamoxifen (4OHT) with a PDB code, 3ERT. Such a procedure warrants the finding of global minimum using the stochastic search method during molecular docking, which eventually minimizes errors [−1 (−1)   possesse[−1 (−1) . These rα facilitates cancer cell proliferation through complex molecular signaling pathways [α shows the best binding affinity score of −9.63 kcal mol−1  to predict the drug-likeness properties of the onoceranoid triterpenes. This approach foresees whether the compounds are orally active drugs in humans but does not indicate their pharmacological properties . The rul2, 3, and 4 satisfy MW, HBA, and HBD criteria but not logP. Their molecular weight values are below 500 g mol−1. Therefore, they may have a good permeability across the intestine and membrane lipid bilayer. According to Ro5, compounds should possess hydrogen bond donors of less than five and hydrogen bond acceptors of less than ten to be permeable to the lipid bilayer membrane, which is fulfilled by the three onoceranoid triterpenes  fulfill drug-likeness criteria [The prediction results  show thaterpenes . For the above 5 , suggestcriteria .Subsequently, we subjected the three onoceranoid triterpenes to pharmacokinetic prediction, including absorption, distribution, metabolism, and excretion properties. These pharmacokinetic criteria are essential in drug development, as they provide insights into the behavior of compounds in the body and their potential efficacy . Additio−1 , fraction unbound, blood–brain barrier (BBB) permeability, and central nervous system (CNS) permeability . VDss prs > 0.45 , while 3and 0.45 . Thus, tes are 0 , lowerinan −2.00 . http://biotransformer.ca (accessed on 23 July 2023), suggests that they may not metabolized by CYP450, but 4 may undergo glucurodination. In terms of metabolism, the three onoceranoid triterpenes are not predicted as substrates or inhibitors for the cytochrome P450 (CYP450) family, except as CYP3A4 substrates . These xFor the excretion criterion, the three onoceranoid triterpenes are predicted to have low total clearance values , reflect2 and 3 are predicted to be hERG II inhibitors according to the pkCSM web server [α inhibitor candidates.The assessment of toxicity is crucial in drug development to mitigate the risk of candidate failures. Thus, toxicity assessment using in silico approaches is beneficial for new drug candidates. In the case of the three onoceranoid triterpenes, they are predicted to be free from AMES toxicity, indicating a potential absence of carcinogenicity. Additionally, they may not cause hepatoxicity. However, it is worth noting that b server , raisingb server , these cL. domesticum Corr. cv. kokosan. However, molecular docking uses a rigid receptor, which does not account for the flexibility of the protein–ligand complex [2, 3, and 4 to molecular dynamics (MD) simulations, which accommodate receptor and ligand flexibility [Molecular docking is extensively employed in structural-based virtual screening because of its rapid evaluation . Our stu complex ,44. This complex . Hence, xibility . α, may affect the conformational changes in both ligand and protein [α in its apo form is relatively stable after 10 ns. Its RMSD values remain between 3.00 and 4.00 Å, with median and interquartile range (IQR) values of 3.28 and 0.32 Å, respectively  and IQR (0.54 Å) values of protein RMSD. Moreover, estradiol-bound ERα shows some conformational changes in different clusters at the beginning and the end of the simulation  than that of the apo form (0.32 Å)  . HCPC su(0.32 Å) . Four diy of ERα . During ng poses B. 3- and 4-bound ERα proteins have higher flexibilities than the others, but at the end of this coil, the highest flexibility is shown by the apo form of ERα. Interestingly, both tamoxifen- and estradiol-bound ERα proteins are the least fluctuated at the whole part of coil2-3. At the coil of H12, all ligand-bound and apo forms of ERα display comparable flexibility, but for the end of H12, 4-bound ERα has the highest flexibility. H12 plays a role in regulating the activity of ERα by switching between active and inactive conformations [While RMSD describes conformational changes in the entire ERα structure over time, RMSF indicates the flexibility of specific residue during MD simulations. As depicted in rmations . This se2-bound ERα complexes are in the same cluster, suggesting their RMSF profile similarity. Interestingly, both complexes share the same parent branch with the estradiol-bound ERα, with a dissimilarity height of 36.5. They have strikingly different RMSF values at H1 and slightly different ones at H2, Coild2-3, H9, H10, and H12 , while estradiol has the lowest fluctuated values . From the obtained 500 ns MD trajectories, we calculated MMGBSA binding energy values every 10 ns window. As shown in 2 , 3 , and 4  are stronger than estradiol  in binding to ERα  have close median values of MMGBSA binding energy. These results are consistent with our in vitro experiment that 2 and 4OHT exhibited inhibition activities to MCF-7, with IC50 29.73 µg mL−1 (unpublished result) and 20.5 µg mL−1, respectively. Therefore, 2 is the most potent ERα inhibitor.Nonetheless, MMGBSA binding energy values overlap between ligands, confusing further analysis. Thus, we performed a Kruskal–Wallis rank-sum test, followed by the Games–Howell test. The results suggest that g to ERα . Compounr than 2 . Additioα  is more similar to that of 4OHT, compared with estradiol, supporting our notion that these compounds will likely be the antagonist ligands similar to 4OHT. Studies showed that the majority (over 60%) of effective anti-cancer medications currently utilized in clinical practice are sourced from natural products found in plants, marine life, and microorganisms [L. domesticum Corr. cv. kokosan would provide new insight into the discovery and development of cancer drugs.The same high CSA values pattern was also found in residues Leu346 and Leu387, although their contribution to the binding energy is not as notably high as the key residues. In addition, the pattern in the CSA map of the three onoceranoid triterpenes  was utilized to draw the 2D structures of onoceranoid triterpenes manually. MarvinView 18.21.0  was employed to predict the protonation states of the onoceranoid triterpenes under physiological pH conditions (pH 7.4). BIOVIA Discovery Studio 2021 Visualizer v21.1.0.20298  was utilized to transform 2D into 3D structures in a pdb format.α binding site. The procedure was deemed successful if the root-mean-square deviation (RMSD) value for the docked 4OHT was less than 2.00 Å [α binding 4OHT (3ERT) [α and 4OHT structures were separately saved as individual pdb files, with water and ion molecules excluded. Next, each file was subjected to molecular docking preparation in AutoDockTools 1.5.6 [α and 4OHT, respectively; nonpolar hydrogen removal; and conversion to pdbqt format. The active torsions in the 4OHT structure were configured based on recommendations from AutoDockTools 1.5.6. Grid parameters were initiated from the center of 4OHT, located at the ERα binding site. The grid box size was 40 × 40 × 40 with center coordinates of 30.010, −1.913, 24.207 , and 0.375 Å spacing. Grid maps for the ERα within the grid box were computed using AutoGrid4.2 [Molecular docking was validated by redocking the 4OHT structure into the ERn 2.00 Å . BrieflyT (3ERT)  was obtals 1.5.6  , and number of hydrogen bond acceptors (HBA) and donors (HBD).The SwissADME web server  was usedIn silico absorption, distribution, metabolism, excretion, and toxicity assessments were carried out using the pkCSM web server . The criα structure was prepared using the pdb4amber program, AmberTools21, to adjust histidine residues according to their local chemical environments. We assigned ff19SB [α structure. The tleap program, AmberTools21, was used to prepare each ERα and ligand complex. The TIP3P explicit water model was employed to solvate every ERα–ligand complex, with a minimal boundary box of 10 Å. Moreover, we introduced a few Na+ and Cl− ions to achieve a physiological salt concentration of 0.15 M.Initially, we prepared partial charges of onoceranoid triterpenes and 4OHT structures using Austin Model 1—Bond Charge Corrections (AM1-BCC), as implemented in the antechamber program, AmberTools21   on AMBER20, employing GPU acceleration [−1 Å−2 harmonic force. Subsequently, 5000 steps of unimpeded conjugate gradient minimization rectified spatial overlaps. The system temperature ramped to 300 K in 20 ps increments  over 60 ps. Equilibration ensured density, pressure, and gradual force release during 1000 ps. The production runs for 500 ns with a 2 fs time step. Molecular dynamics simulation of every ERleration . Initialccptraj program in AmberTools21 was employed for MD trajectory analyses. The analyses include root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and conventional H-bond conservation. Molecular dynamics simulation trajectories were assessed using VMD. The ante-MMPBSA.py in AmberTools21 was used to calculate MMGBSA binding energy and decomposition analysis. To compute contact surface analysis (CSA), we used the dr_sasa program [The  program . Jupyter program  was used program . Statist program . Hierarc program , while f program  and fact program  were use program , ggplot2 program , and ggp program . Inkscap program  was usedLansium domesticum Corr. cv. kokosan against estrogen receptor alpha (ERα) using a range of in silico techniques, including molecular docking, Lipinski’s rule of five, in silico ADMET, and molecular dynamics simulations. The potential onoceranoid triterpenes based on molecular docking—namely 8,14-secogammacera-7,14-dien-3,21-dione (2); 8,14-secogammacera-7,14(27)-dien-3,21-dione (3); and kokosanolide B (4)—fulfill drug-likeness criteria and potential ADMET profiles. Molecular dynamics simulations revealed 2 as a highly promising contender, exhibiting the most favorable MMGBSA binding energy of −56.10 kcal mol−1, closely followed by 3 and 4, with MMGBSA binding energy values of −52.27 and −52.17 kcal mol−1, respectively. These computational findings align well with our experimental data, where 2 demonstrated notable inhibition activity against MCF-7 cells, with an IC50 of 29.73 µg mL−1, while tamoxifen exhibited an IC50 of 20.5 µg mL−1. Consequently, 2 emerges as the most potential inhibitor of ERα. HCPC analysis on RMSF suggests that 2 and 4OHT are in the same cluster. Moreover, the spatial distribution of CSA among the three onoceranoid triterpenes  bears greater similarities to that of 4OHT than estradiol. This observation suggests a hypothesis that these compounds may potentially function as antagonist ligands akin to 4OHT. These findings collectively provide valuable insights into the potential therapeutic significance of these triterpenes in targeting ERα-associated breast cancer. In the present study, we evaluated the anti-breast cancer potential of five onoceranoid triterpenes found in


Trauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3–7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less.Three potential assays were examined—assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2–72 h.Suppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h.Takeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2–6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h. Frontiers Immunology 2023  are at the forefront of the cellular therapy revolution –3 whetheogy 2023 .MSCs are adult, stem-like cells commonly isolated from bone marrow, adipose, umbilical cord, or cord blood . While tThe most important mechanism of action of MSCs is thought to be immunomodulation , 24 for In vitro immunomodulation assays are routinely based on the ability of MSCs or MSC-produced products to block the effects of T-cell activation , as well as PBMC production of tumor necrosis factor alpha (TNFα). These assays use either flow cytometry or rapid enzyme-linked immunoassay (ELISA) methods and cryopreserved pooled donor human PBMCs in co-culture with MSCs to determine suppressive ability. Results are compared to current standard methods—either a 72-h proliferation assay by ATP measurement or a 96-h assay of proliferation by CFSE label dilution. Both PS externalization and TNFα release prove to be robust assays of MSC potency.Human MSCs were purchased from RoosterBio, Inc. , expanded in the recommended medium, and cryopreserved at the initial passage following receipt. Population doublings were limited to less than 15–20.7 per mL in CryoStor10, frozen at 1°C/min cooling rate in a Nalgene™ Mr Frosty and stored in liquid nitrogen until use. Viability upon thaw was 94 ± 2.4% (n = 9) by 1:1 dilution with acridine orange propidium iodide (AOPI) stain and cell counting in the Cellaca MX High-Throughput Automated Cell Counter .Whole blood was collected from healthy donors in citrate tubes according to an approved institutional standard operating procedure. PBMCs were purified using gradient centrifugation through Ficoll-Paque Premium  in Greiner Bio-One Leucosep tubes  as described by the manufacturer . Briefly6 PBMCs per mL in PBMC media. In 96-well plate assays , 150,000 PBMCs were loaded per well with or without MSC co-culture in a volume of 200 µL. Proliferation was stimulated by the addition of 50 µL of stimulus as indicated in the text. Stimuli included phytohemagglutinin P  and phytohemagglutinin-L . After 2, 4, 6, 24, 48, 72, or 96 h incubation at 37°C, 5% CO2, cells in the media were resuspended and aliquots were taken for analysis. In designated experiments, a monocyte-depleted population of PBMCs was obtained by incubating the thawed, washed, resuspended PBMCs at 1.5 × 106 in culture media in a T-75 tissue culture flask overnight. After incubation, monocyte-depleted PBMCs were removed, washed in culture media, and resuspended at 1.5 × 106 cells per mL for use in experiments. After overnight incubation, monocyte-depleted PBMCs were generally 85% viable.Cryopreserved PBMCs were rapidly thawed; washed twice in PBMC media  containing 10% MSC qualified FBS , 1 mM Glutamax , and 10 mM HEPES, pH 7.4 ; and resuspended at 1.5 × 10For routine experiments, MSCs were serially diluted starting at 50–60,000 cells/well and cultured in 96-well plates for 24 h . On Day g for 6 min, resuspended in 50 µL of Hanks’ Buffered Saline Solution (HBSS) containing 2% bovine serum albumin (BSA) and then incubated with 20 µL of APC-CD3 antibody  for 20 min at room temperature. After using the wash-only setting on the BD FACS Lyse Wash assistant, 5 µL of 7AAD was added and the cells were incubated for an additional 5 min before analysis on a BD FACS Canto II flow cytometer  using an expanded FSC-A/SSC-A gate to capture the larger proliferating population (The classic carboxyfluorescein succinimidyl ester (CFSE) assay was performed in 96-well plates as previously described , 45. MSCpulation . A totalpulation , 53. TheProliferation of both MSCs and PBMCs was assessed by using ATP levels as a surrogate for cell number and quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo 2.0  as described previously , 54. At 6 PBMC per mL of PBMC media as above. In a 6-well plate, 4.5 × 106 PBMCs in 2.5 mL were placed per well and treated as control (no addition), mitogen stimulated (15 µg/mL PHA-P with the addition of 7.5 µL of 5 mg/mL PHA-P) or apoptosis-induced . At 24 and 48 h, PBMCs were resuspended and analyzed for viability, phosphatidylserine externalization, and caspase 3/7 activity.Cryopreserved hPD-PBMCs were rapidly thawed, washed twice in PBMC media, and resuspended at 1.5 × 106 PBMC per tube were centrifuged at 400 × g for 6 min and the pellet was resuspended in 100 µL of Annexin V staining buffer  or 100 µL of HBSS (for lactadherin only samples) containing 5 µL of Human TruStain FcX™ (BioLegend Cat # 422302). After a 5-min room temperature incubation in the dark, the cells were then stained with 2.5 µL of Pacific Blue™ anti-human CD45 Antibody  and 5 µL of Alexa Fluor® 647 Annexin V  with and without 1 µL of FITC-bovine lactadherin  for 15 min at room temperature in the dark. The addition of 5 µL of 7AAD for a further 5 min in the dark was used to discriminate live from dead cells. Prior to analysis, the samples were diluted with 100 µL of HBSS (Lactadherin only) or 100 µL of Annexin V buffer (Annexin V ± lactadherin samples) and then analyzed on a BD FACS Canto II flow cytometer . Alternatively, doubly labeled cells were analyzed on an AMNIS imaging flow cytometer . Data are expressed as percent of parent.After treatment as above, 0.6 × 106 cells were centrifuged at 600 × g for 6 min, resuspended in 100 µL of HBSS containing 5 µL of Human TruStain FcX™, and incubated for 5 min at room temperature. PBMCs were then stained with the following markers: 2.5 µL of APC/Cyanine7 anti-human CD3 Antibody  and 2 µL of bovine lactadherin  with or without 2.5 µL of Brilliant Violet 421™ anti-human CD4 antibody . Cells were stained for 15 min at room temperature in the dark followed by a 5-min incubation using 5 µL of 7AAD . HBSS (100 µL) was added to the samples before analysis on a BD FACS Canto II flow cytometer . Data are expressed as % positive cells of the live population. Suppression was calculated as above.PBMCs with or without mitogen treatment and MSC co-culture were harvested and pooled from the 96-well plates at the times specified; 1 × 10TM−Cell Fitness Panel (Nexcelom Cat # CSK-V0024-1) for a further 30-min incubation at 37°C before transfer to a Cellaca MX plate and collection of brightfield and fluorescent images on a Cellaca MX automated cell counter using the caspase 3/7 apoptosis assay. Caspase active cells are expressed as percent of total cell number determined by brightfield analysis.Caspase 3/7 activity was determined by automated cell counting. After incubation at 37°C for 2, 4, 6, 24, 48, or 72 h, control, mitogen-stimulated, and apoptosis-induced samples were resuspended, aliquoted into 0.6 mL of polypropylene micro-centrifuge tubes and mixed with 12.5 µL of a 1/50 dilution of NucView™ caspase 3/7 reagent in the ViaStainActivity of caspases 3/7, 8, and 9 were determined from singly cultured or co-cultured PBMCs and MSCs incubated in the presence or absence of mitogen as described above. At either 24 or 72 h, PBMCs in each well were triturated to suspend the PBMCs in media and 25-µL aliquots were diluted into 25 µL of dPBS in a white LUMITRAC assay plate; 50 µL of the appropriate caspase assay reagent was added . After 30–60 min, luminescent readings were determined in a dedicated luminometer. Data are expressed as relative luminescent readings (RLU).g for 5 min before analysis. Samples were diluted threefold in ELISA kit assay buffer; 60 µL from each sample was added to the dedicated ELISA plate and the plate loaded onto the automated ELISA. Cytokine levels were determined after 70 min. Alternatively, a rapid, no-wash ELISA  was performed, which relies on proximity interactions of bound antibody to reconstitute a light-generating enzyme that can produce luminescence signals. Samples were either from frozen, thawed, centrifuged media from mitogen-stimulated assays or on samples taken directly from mitogen-stimulated samples without centrifugation and were assayed according to the manufacturer’s recommendations. For thawed frozen samples, 50 µL each of diluted standards or centrifuged media was added to a 96-well Lumitrac plate followed by the addition of 50 µL of a 2× antibody mix and then 60-min room temperature incubation, per the manufacturer’s recommendations. For freshly harvested samples, 80 µL of diluted standards or resuspended PBMCs was transferred to a 96-well Lumitrac plate with 20 µL of 5× antibody mix, per the manufacturer’s recommendations. After incubation, 25 µL of room temperature equilibrated, diluted detection substrate in buffer was added to each well. After brief mixing, and a 3- to 5-min incubation, the luminescence was read on a luminometer. The concentration of TNFα was determined from the linear standard curve. Suppression of the PBMC signal in the presence of MSCs was calculated as described above.Tumor necrosis factor-alpha, interleukin 6, and interleukin 10  were quantitated by multiplexed ELISA using the Ella Simple Plex platform from Bio-Techne  according to manufacturer’s recommendations. Conditioned media from stimulated or unstimulated PBMSC-MSC cocultures treated as described above in 96-well plates were frozen, thawed on the date of assay, and centrifuged at 20,000 × RT-PCR was performed as described previously . mRNA wawww.graphpad.com). TNFα standards were calculated internally (Simple Ella platform) or were determined by linear regression using GraphPad Prism, version 8.1.2 for Windows. Significant differences were determined with one- and two-way ANOVA using JMP version 15.2.0, from SAS Institute, Inc., Cary, NC, USA (www.jmp.com). Pairwise comparisons of means were made using Tukey HSD. Significance was set at p < 0.5. Unless otherwise noted, data are presented as average ±standard deviation.ATP standards were fit to a four-parameter dose–response logistic curve using GraphPad Prism version 8.1.2 for Windows, GraphPad Software, Inc., San Diego, California USA , although there was no significant difference with MSC co-culture at these times. There was significant immunosuppression of PBMC caspase activity by MSC co-culture only at the 72-h time points . Cells with caspase activity cleave the DEVD peptide, allowing the dye to bind DNA and display bright green fluorescence. In validating the assay, cells were monitored at 24, 48, and 72 h using an automated cell counter , but hBM-MSCs in co-culture show little suppression of PBMC caspase activity (p < 0.001 for both caspase 8 and 9). While this suggests that monitoring caspase activity could be used in potency assays, the 72-h time frame does not markedly improve other existing potency assays following PBMC stimulation.Caspase 3/7 is only one family of caspases. Therefore, luminescent-based assays were used to detect activity by other caspases. These assays also rely on the cleavage of caspase specific tetrapeptides . In these assays, aminoluciferin becomes the substrate for a proprietary thermostable luciferase upon cleavage of the respective tetrapeptide, generating light that is detected with a luminometer. Using these luminescent assays for caspase activity, caspase activity of stimulated PBMCs (15 µg/mL PHA-P) was examined. Triplicate determinations for caspase 3/7, caspase 8, and caspase 9 were run on each sample. Data from three experiments are shown. These experiments were done in co-culture in the presence or absence of three different concentrations of one hBM-MSC preparation that had been allowed to attach for 24 h prior to co-culture with PBMCs to examine the ability of MSC co-culture to suppress caspase activation. Controls included PBMCs in the absence of stimulus and PHA-treated MSCs without PBMCs. Caspase activity was assayed on PBMC samples at 24 and 72 h Figure 2Phosphatidylserine (PS) externalization is commonly referred to as a marker of early apoptosis –63. HoweThe flow cytometry gating strategy for PS externalization is shown in PBMCs respond to mitogens with multiple cytokine responses , 72–75. In p < 0.0001), no difference exists between assay type (p = 0.2369). A correlation of r2= 0.74 is found  and “late stage” apoptosis markers (caspase activity) were used. The apoptotic response generally completes within 6–24 h of stimulation, so even the late-stage apoptosis becomes a possible candidate for a rapid immunomodulation assay. While caspase 3/7 activity was evidence of late-stage apoptosis and was seen in mitogen-activated PBMCs, reliable suppression of this activity by MSC co-culture only occurred at 72 h. Thus, this assay did not allow for earlier assessment of MSC suppression activity.in situ luminescent assays. In this assay, there was no caspase 3/7 activity detected with MSC co-culture that could be attributed to PBMC activation; all caspase 3/7 activity in this assay appeared to be due to the plated MSCs. This contrasts with the significant caspase 3/7 activity detected at 24 h on PBMCs alone treated with PHA and staurosporine in the previous assay. The reason for this discrepancy is unclear. Possibly the release of caspase 3/7 by MSCs alone masked PBMC activity. Caspase 8 and 9 activity in mitogen-stimulated PBMCs was detected at 72 h and that activity was suppressed by MSC co-culture. However, while suppression of caspase 8 and 9 activation in mitogen-stimulated PBMCs might be a surrogate for immunomodulation by MSCs, the time frame is such that it provides no improvement over current assays. Whether this activity is due to apoptosis is in question; there are reports of non-apoptotic caspase 8 and caspase 9 activity , PS externalization (2–6 h), and TNFα secretion (2–6 h), immunosuppression by MSC co-culture could not be detected at these times for any of the methods. Immunosuppression was only reliably found at 24 h.The promises of cellular therapies are beginning to be realized. Therapies focused on preventing secondary damage post injury and suppressing a dysregulated immune response will serve to improve patient outcomes. Although treating coagulopathic patients with pro-coagulant MSCs must be approached with caution , 20, 103The views expressed in this article are those of the author(s) and do not reflect the official policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. This study was conducted under a protocol reviewed and approved as exempt from IRB review by the US Army Institute of Surgical Research Regulatory Compliance Division and in accordance with the approved protocol.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study used commercial mesenchymal stem cells and prepared PBMCs from an institutional blood bank. The US Army Institute of Surgical Research (USAISR) Research Regulatory Compliance Division (RRCD) did not require the study to be reviewed or approved by an ethics committee because it does not involve living individuals about whom an investigator conducting research obtains information or biospecimens through intervention or interaction with the individual, and uses studies or analyzes the information or biospecimens or obtains, uses, studies, analyzes, or generates identifiable private information or identifiable biospecimens.MH, BC, JB and AC contributed to conception and design of the study. MH, RM, CC-G, GB, JL, NM, JT, and IA performed assays related to the study. MH performed statistical analysis and wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.


This study was performed on a population of 300 graphene field effect transistors. The output characteristic of the transistors revealed that a C60 thin film adsorbate increased the graphene hole density by (1.65 ± 0.36) × 1012 cm−2, whereas a Pentacene thin film increased the graphene electron density by (0.55 ± 0.54) × 1012 cm−2. Hence, C60 induced a graphene Fermi energy downshift of about 100 meV, while Pentacene induced a Fermi energy upshift of about 120 meV. In both cases, the increase in charge carriers was accompanied by a reduced charge mobility, which resulted in a larger graphene sheet resistance of about 3 kΩ at the Dirac point. Interestingly, the contact resistance, which varied in the range 200 Ω–1 kΩ, was not significantly affected by the deposition of the organic molecules.Graphene is an excellent 2D material for vertical organic transistors electrodes due to its weak electrostatic screening and field-tunable work function, in addition to its high conductivity, flexibility and optical transparency. Nevertheless, the interaction between graphene and other carbon-based materials, including small organic molecules, can affect the graphene electrical properties and therefore, the device performances. This work investigates the effects of thermally evaporated C60 ( In fact, the van der Waals interactions of carbon materials, including small organic molecules, e.g., C60-Fullerene and Pentacene, have an important influence on the electrical properties of graphene and therefore, on the device performances. While the growth mechanisms and orientation of C60 and Pentacene molecules on graphene have been widely studied [Hybrid van der Waals heterostructures made of Graphene (Gr) and Organic Semiconductors (OSC) are being widely investigated for their potential applications in sensors ,2, solar studied ,15,16,17n- and p-type organic semiconductors [This work investigates the effect of C60 and Pentacene adsorbates on the electrical properties of large-area graphene grown by Chemical Vapor Deposition (CVD). C60 and Pentacene molecules are commonly used as nductors ,19,20 thW of 5 μm and five different channel lengths L of 5, 10, 20, 50 and 100 μm. The two sets include 30 GFETs per channel length and were fabricated on two separate chips (3). The remaining graphene sheet was transferred on the pre-patterned Ti/Au electrodes (5 nm/50 nm) on a Si/SiO2 (525 μm/300 nm) substrate and patterned into geometrically defined channels by e-beam lithography. Then, the molecules were thermally evaporated, without using any physical mask nor photolithographic patterning, resulting in a uniform coverage of the whole chip. L = 50 μm and W = 5 μm. The output and transfer characteristics of the GFETs were measured in vacuum before and after depositing the molecules, after 24 h of vacuum exposure to assure complete desorption of the moisture and water. 2O and/or resist residues [This study was performed on two sets of 150 Graphene Field Effect Transistors (GFETs) with a fixed channel width te chips . The detresidues ,27,28,292 substrates were about 10 nm and 5 nm, as shown in the 2 and graphene. The AFM profile of 2 and graphene, as was previously reported [2 [2 and on the graphene channel. The presence of the graphene channel under the organic thin films was further confirmed using Raman spectroscopy. 2 substrate) showed the characteristic G (1587 cm−1) and 2D (2681 cm−1) peaks, as well as the D (1340 cm−1) peak [−1) and second-order scattering band (940–980 cm−1) of the Si substrate [1g at 264 cm−1, H2g at 430 cm−1, A1g at 491 cm−1, H3g at 707 cm−1, H4g at 772 cm−1, H5g at 1099 cm−1, H6g at 1242 cm−1, A2g at 1462 cm−1 and H8g at 1568 cm−1, as well as the characteristic Raman peaks of CVD graphene (G at 1587 cm−1 and 2D at 2681 cm−1) observed before the deposition of C60. −1 were assigned to the Ag bands, while the peak at 1595 cm−1 was assigned to the B3g band. The 2D peak (2687 cm−1) of the CVD graphene could be clearly distinguished, whereas the G peak overlapped with the B3g band of Pentacene. The AFM surface morphology combined with Raman chemical analysis confirmed the presence of the geometrically well-defined Gr/C60 and Gr/Pentacene hybrid heterostructures.reported . The avereported . The disreported . Here, treported ). The AForted [2 , whereasorted [2 ,16. Figu−1) peak , with a s the D 10 cm−1 peubstrate , i.e., HDSI vs. GSV) of pristine GFETs and coated GFETs measured under vacuum and at room temperature. The standard deviations are indicated by the shaded areas. Refer to the Experimental Methods section for details on the data filtering and population selection process and to the DSI vs. DSV) and transfer characteristic (DSI vs. GSV) of graphene field effect transistors [GSV range  × 1012 cm−2, against the residual doping of pristine graphene with n0 = (0.20 ± 0.65) × 1012 cm−2 (Chip 1). For the Pentacene molecules, the graphene was electron-doped with an electron density of n0 = (0.55 ± 0.54) × 1012 cm−2, against the residual hole doping of graphene before the deposition with n0 = (0.09 ± 0.48) × 1012 cm−2 (Chip 2). Such a charge transfer could not be anticipated a priori from the HOMO and LUMO energy levels of the individual molecules relative to the graphene Fermi level only, as illustrated for C60 and Pentacene in DSI vs. GSV traces. n0 was accompanied by a decrease, from n0 which was accompanied by a decrease, from GSV range.nsistors . The traGS range , while ang level ,25,26,27acitance ,38, is ge ϵr=3.9  and t=30graphene , i.e., Δurements . The fiety reads ,(2)μ=LgDS=50 mV . The holSR increased by about 200 Ω and shifted to a more positive CR shifted with SR and remained close to the Dirac point, suggesting that the charge carrier injection mechanism at the Au/Gr interface was not significantly affected by the molecules. Assuming that the charge injection mostly occurred at the edge between the metal electrodes and Gr (W = 5 μm), the gate dependent contact resistivity n- and p-type organic semiconductors [ne GFETs a,b showegraphene ,45, weregraphene . On the mobility , assuminon model ,13. Thisnductors ,10,11,1212 cm−2, whereas the deposition of Pentacene results in an increased residual graphene electron density of (0.55 ± 0.54) × 1012 cm−2. In both cases, the increase in the residual charge carriers was accompanied by a reduced charge mobility, possibly due to an increased charge-impurity scattering in graphene. The Fermi energy of graphene shifted to (−150 ± 15) meV after the deposition of C60, and it shifted to (+86 ± 35) meV after the deposition of Pentacene, while the charge carrier injection at the Au/Gr did not seem to be significantly affected. Overall, this work provides useful insight into the graphene in-plane charge transport and on the energetic of the Gr/C60 and Gr/Pentacene hybrid heterostructures, which could be exploited in more complex organic electronic devices. For instance, the energy barriers forming at the Gr/C60 and Gr/Pentacene interfaces and the weak electrostatic screening of graphene could be beneficial for forming n- and p-type channels in graphene-based vertical organic transistors.In summary, CVD graphene field effect transistors (GFETs) with a fixed channel width (5 µm) and different channel lengths  were fabricated and electrically characterized under vacuum. Subsequently, thin films of C60 and Pentacene were deposited on two distinct GFET chips by thermal evaporation and electrically characterized again under vacuum. The GFETs’ transfer characteristic revealed that the deposition of C60 on graphene results in an increased residual graphene hole density of (1.65 ± 0.36) × 102. Then, the foils were placed in Acetic Acid (CH3COOH) for 30 min, rinsed in de-ionized water (DIW) and Ethanol and dried with N2. The cleaned foils were pre-annealed at 1000 °C for 1 h in a H2 (20 sccm) and Ar (200 sccm) atmosphere in the CVD oven (approximately 1 mbar). Then, graphene grew for 35 min in a CH4 (0.05 sccm), H2 (20 sccm) and Ar (200 sccm) atmosphere under a pressure of about 120 mbar. After the CH4 flow was stopped, the CVD oven was left to cool down to room temperature (at approximately 1 mbar).Graphene was grown in-house by chemical vapor deposition (CVD) on copper foils  with a fully automated setup. The foils were ultrasonicated in Acetone, rinsed with Isopropyl Alcohol (IPA) and dried with N2 flow (30 sccm) for 2 min. The Cu substrate was etched away in a Ferric Chloride  bath for 1 h, and then transferred to de-ionized water (DIW). Subsequently, the remaining graphene/PMMA sheet was placed in hydrochloric acid (HCl 10%) for 5 min and transferred back to DIW. Finally, graphene could be transferred onto the target substrate and dried overnight in vacuum oven .The CVD graphene was transferred as previously reported ,46. To p2 (300 nm) substrates. The Au bottom contacts architecture . The remaining PMMA, protecting the graphene channels, was removed in Acetone (55 °C) for 1 h and IPA (55 °C) for 1 h.Two chips including 150 Graphene Field Effect Transistors (GFETs) were fabricated. Graphene sheets grown by CVD on Cu foils were transferred onto photolithography pre-patterned Ti (5 nm)/Au (50 nm) electrodes on Si (525 μm)/SiOitecture b was cho−6 mbar) without further treatments. The C60 thin film was deposited by thermal evaporation at about 0.2 Å/s from a molybdenum evaporation boat (Umicore). The Pentacene thin film was thermally evaporated at about 0.05 Å/s using a low-temperature-controlled source (about 120 °C) with an Al2O3 crucible (Creaphys). The evaporation rate was maintained by monitoring the quartz crystal microbalance of the evaporator. The temperature of the target substrate was not actively controlled during the evaporation.Fullerene-C60  powder was purchased from Sigma-Aldrich , while Pentacene powder  was purchased from Tokyo Chemical Industry . The two types of molecules were thermally evaporated under vacuum (approximately 10−6 mbar) and at room temperature. The samples were shortly exposed to ambient conditions during the transport from the evaporation chamber to the electrical prober vacuum chamber. They were kept in vacuum conditions (approximately 10−6 mbar) for at least 24 h before being characterized. The electronics comprised an AdWin Gold II ADC-DAC unit, a low-noise current-to-voltage converter  to measure the drain-to-source current (DSI), and a high-voltage amplifier  to provide the gate-to-source voltage (GSV). The drain-to-source voltage (DSV) was directly provided by the ADC-DAC. The automated probe station and the ADC-DAC were controlled via LabVIEW and Matlab scripts. In the drain-to-source voltage (DSV) sweep from −50 mV to +50 mV, the voltage step was set to 0.5 mV, the sweep rate to 50 mV/s and the gate-to-source voltage was set to 0 V. In the gate-to-source voltage (GSV) sweep from −50 V to +50 V, the voltage step was set to 0.1 V, the sweep rate to 10 V/s and the drain-to-source voltage (DSV) was set to 50 mV. The internal averaging was set to 20 ms for all the measurements. Only the backward sweep traces were considered for the analysis, refer to the The devices were electrically characterized under vacuum  in tapping mode. The AFM was equipped with a TESPA-V2 cantilever with a tip apex radius of 7 nm, with a resonant frequency of 320 kHz and a spring constant of 37 N/m. The AFM data were processed with Gwyddion (version 2.56) which was used to extract the single and average height profiles and export the images.−1) equipped with a TE-cooled charge-coupled device . One Raman spectrum for each device was collected (150 devices per chip). The laser power was set to 0.1 mW and 0.5 mW for the Gr/C60 and Gr/Pentacene heterostructures, respectively. In both cases, the integration time was set to 30 s. The average Raman spectra of graphene, Gr/C60 and Gr/Pentacene were obtained using all the Raman spectra acquired on each set of devices. Refer to Raman spectra were acquired in ambient conditions using a 532 nm excitation wavelength with a WITec Alpha 300R confocal Raman microscope mounting an 100× objective  and a 300 mm lens-based spectrometer (grating: 600 g mmL. The weights were set to Python scripts were used for the data selection, analysis and visualization. Raman spectra averaging and polynomial background subtraction was done using numpy . The resng scipy . The RC dent TLM .


Saline stress is a major abiotic stress that inhibits plant growth and yields worldwide. The plant transcription factor (TF) family plays an important role in converting abiotic stress signals into gene expression changes. In this study, a transcriptome-based comparative analysis was performed to investigate the global gene expression of all the TFs in diploid and autotetraploid rice during the early stage of NaCl stress and recovery period. The phenotypic data indicated that the tetraploid rice exhibited a superior salt-tolerant ability compared to the diploid rice. A total of 55 TF families were co-expressed in the tetraploid and diploid rice, and the cumulative number of TF-expressed genes was relatively higher in the diploid rice than in the tetraploid rice at all time points. Unlike the diploid rice, the overall gene expression levels of the tetraploid rice were comparable to the control during recovery. The number of differentially expressed TFs (DE-TFs) in the tetraploid rice decreased after recovery, whereas it increased to a large extent in the diploid rice. GO and KEGG pathway enrichment analysis of the DE-TFs discovered the early switching of the ABA-activated signaling pathway and specific circadian rhythm in the tetraploid rice. Combining the PPI network and heatmap analysis, some core DE-TFs were found that may have potential roles to play in tetraploid salt tolerance. This study will pave the way for elucidating the complex network regulatory mechanisms of salt tolerance in tetraploid rice. Oryza sativa L.) is the primary source of energy for over half of the world’s population. Unfortunately, rice yields and the grain quality are deeply affected by unfavorable environmental factors, such as drought, salinity, or extreme temperatures [Rice  has been recognized as one of the widespread tools for measuring gene transcriptional expression profiles, and it can provide a superior platform for a comprehensive understanding of the contribution of TFs to plant regulatory systems ,27. To dThe differences between the GFD–T and GFD–D seedling responses to saline stress were evaluated via the phenotypic changes at different time points of stress reactivity and recovery. Previous studies have shown that rice is relatively tolerant to salt stress during the germination, tillering, and maturity stages but is sensitive during the seedling (50–120 mM NaCl) and reproductive (30–100 mM NaCl) stages ,32,33. SR2 = 0.815) with the qRT-PCR results, indicating the reliability of the RNA-seq data  of saline stress and recovery, a comparative transcriptomic analysis was carried out. We sequenced twenty-four cDNA libraries of GFD–T and GFD–D and obtained equivalent reads and coverage depths between replicates for each sample by comparing FPKM boxplots of the gene expression levels among all the genes under different experimental conditions A, which seq data . Furtherseq data B. At allseq data . Among tseq data . And theseq data . Furtherseq data . And oneseq data . Interesseq data .LOC_Os02g32590 (HSF)) co-expressed among T1 vs. T0/D1 vs. D0_common, T2 vs. T0/D2 vs. D0_common, and RT vs. T0/RD vs. D0_common, suggesting that this gene may play a key role in both GFD–T and GFD–D in the response to saline stress (LOC_Os01g51690 (WRKY) and LOC_Os01g10580 (DBB)) were involved in the “response to stimulus” and “photomorphogenesis” processes in the intra-group comparisons of the GFD–T (LOC_Os02g52670 (ERF), LOC_Os08g36920 (ERF), LOC_Os01g58420 (ERF), LOC_Os01g60640 (WRKY), LOC_Os02g26430 (WRKY), LOC_Os11g02520 (WRKY), LOC_Os09g28310 (bZIP), LOC_Os02g52780 (bZIP), and LOC_Os01g53220 (HSF)) were involved in the “ET signaling pathway”, “response to stimulus”, “ABA signaling pathway”, “response to salt stress”, “response to water deprivation”, and “response to ABA” processes in the intra-group comparisons of the GFD–D  and diploid  rice, we compared the gene expression values of the intra-group pairwise comparisons between the GFD–T  and GFD–D . The Venn diagrams showed that there were 18, 47, and 34 unique differentially expressed TFs (DE-TFs) from 9, 23, and 16 TF families in T1 vs. T0, T2 vs. T0, and RT vs. T0, respectively. In contrast, D1 vs. D0, D2 vs. D0, and RD vs. D0 contained 124, 144, and 298 unique DE-TFs from 30, 30, and 38 TF families, respectively . These de stress B. The upe stress . Among the GFD–T E,F. Addihe GFD–D G–I. The he GFD–D D. As thehe GFD–D E. And thhe GFD–D F. In conhe GFD–D G. In D2 he GFD–D H. Howevehe GFD–D I. All biLOC_Os08g42440, LOC_Os004g31804, LOC_Os03g26130, and LOC_Os05g03800) were shared among three comparison groups, and two genes (LOC_Os055g39790 and LOC_Os11g47910) were shared among four comparison groups (LOC_Os04g35010 and LOC_Os06g06750) shared among three comparison groups (LOC_Os06g06750 (MIKC_MADS), LOC_Os11g02520 (WRKY), LOC_Os01g58420 (ERF), and LOC_Os05g36100 (ERF)) were involved in “cell differentiation”, “response to stimulus”, “response to ET”, “response to salicylic acid (SA)”, “response to water deprivation”, and “ET signaling pathway”, respectively (LOC_Os03g32230 (C2H2) was upregulated in T0 vs. D0, while it was downregulated in RT vs. RD (LOC_Os09g25060) belonging to the WRKY family was upregulated in T1 vs. D1, while it was downregulated in RT vs. RD  via volcano plots. There were 12 upregulated DE-TFs and 10 downregulated DE-TFs in the GFD–T compared to the GFD–D at 0 hpt (T0 vs. D0); 9 upregulated and 18 downregulated DE-TFs in the GFD–T compared to the GFD–D at 3 hpt (T1 vs. D1); 13 upregulated and 12 downregulated DE-TFs in the GFD–T compared to the GFD–D at 24 hpt (T2 vs. D2); as well as 37 upregulated and 194 downregulated DE-TFs in the GFD–T compared to the GFD–D at 24 hpr (RT vs. RD) . To expln groups B. In addn groups C. Among ectively D–G. The ectively D–G. Inteectively D–G. NotaT vs. RD D,G. And T vs. RD E,G.LOC_Os02g10860 and LOC_Os08g06110) derived from “circadian rhythm” were specifically downregulated in T2 vs. T0 . However, these three identified genes derived from the MAPK signaling pathway were upregulated in the GFD–D intra-group comparisons , and most of the 6 genes in C2, 5 genes in C1, 9 genes in C4, and 39 genes in C5–C7 were highly expressed at D0, D1, D2, and RD, respectively. Five (C3–C7) out of seven clusters exhibited an increased trend, especially as RD was significantly higher than D0 A. By conOsWRKY24, OsWRKY53, OsbZIP23, OsbZIP46, OsbHLH148) and 22 predicted binding genes. Among them, bHLH148 TFs were found to be actively interacting with TIFY TFs; WRKY53 interacted largely with MPK5, WRKY24, Q6ER79, Q8H540, Q8H542, and A3BGT3; bZIP mainly interacted with SAPK and indirectly interacted with other proteins, such as PP2C, VP1, Q6K3D4, etc. We further found six genes  that play crucial roles in the regulation of salt or drought tolerance, some of which through the JA signaling or ABA-dependent regulation pathway  showed significant downregulation trends during the saline stress and recovery periods in the GFD–T versus the GFD–D. Different from the GFD–D, OsSAPK1, OsSAPK2, OsSAPK10, and OsVQ8 were significantly upregulated in T2 vs. T0 of the GFD–T. Strikingly, the vacuolar H+–pyrophosphatase  OsVP1 was obviously upregulated in T0 vs. D0. Moreover, it was downregulated during the saline stress and recovery periods in the GFD–T, whereas it was upregulated in the GFD–D at 24 h of the saline stress and recovery period. In addition, OsbHLH148 was significantly upregulated in T0 vs. D0, and it was upregulated in the GFD–D at 24 h of the recovery period, but not in the GFD–T. OsPP2C30 was also significantly upregulated in T0 vs. D0, but there was no significant change in the RT vs. T0 of the GFD–T, unlike in the GFD–D. Taken together, the expression levels of the TFs in the diploid and tetraploid rice at different stages of stress were significantly different, and it was especially during the recovery period in the diploid rice that the expression levels of most of the TFs significantly changed, which implies that diploid rice needs to activate considerably more TFs than tetraploid rice to rescue it from salt poisoning.To elucidate the expression levels of the DE-TFs involved in the network in different comparisons, a heatmap with significantly expressed genes was generated B. The reIn agricultural production, it is well known that abiotic stresses, such as saline stress, are major disturbances that negatively affect crop performances. Plant defense mechanisms against stress have been studied at the molecular level for many years to identify the main factors that enhance plant tolerance . Noticea+-specific responses have been reported to occur in roots during the early stages (<24 h) of saline stress exposure [LOC_Os02g32590, OsHsfA3) that belongs to the HSF family was co-expressed among T1 vs. T0/D1 vs. D0_common, T2 vs. T0/D2 vs. D0_common, and RT vs. T0/RD vs. D0_common  during the early stages of saline stress ) that negatively controls the UV–B tolerance in rice [UVR8-overexpressing plants showed more severe growth defects under osmotic and salt stress, while the inactivation of UVR8 expression enhanced the shoot growth under high-salt stress [OsbZIP18 in the GFD–T may have endowed it with stronger salt tolerance in our study . In contrast, the above three identical genes derived from the MAPK signaling pathway were upregulated in the GFD–D intra-group comparisons  promoted salt tolerance [OsABI5 was significantly downregulated in the GFD–T versus the GFD–D at 0 hpt, and it showed a significant downregulation trend during the saline stress and recovery periods in the GFD–T versus the GFD–D, suggesting that the low expression level of OsABI5 in tetraploid rice may play an important role in its salt tolerance. OsSAPK10 and OsVQ8 were significantly upregulated in T2 vs. T0 of the GFD–T.In common cells or organisms, physiological and biological processes, including cellular interactions, as well as developmental control and metabolic processes, are regulated by PPIs between different TFs . To explenotypes . The “SA in rice . The oveand cold . And thestresses . Howeverstresses  and mainstresses . In this vacuole . In addi vacuole . The WRKugarcane ,63. Baseat 0 hpt B. Becausolerance , these dectively ,64. In aectively . Previouolerance . In thisOryza sativa L. ssp. japonica were used in the present study. The GFD–T was a natural mutant autotetraploid rice derived from GFD–D screened in the field, and self-pollinated for six generations. Two weeks after seed germination, seedlings at the trefoil stage were treated with a series of 0 mM, 50 mM, 100 mM, 125 mM, 150 mM, and 200 mM saline solutions 2SO4; 0.09 mM KNO3; 0.18 mM Ca(NO3)2; 3 μM H3BO3; 0.5 μM MnCl2; 1 μM (NH4)6Mo7O24; 0.4 μM ZnSO4; 0.2 μM CuSO4; and 20 μM Fe (III)-EDTA, and it was adjusted to pH 4.5–5.6 for the preparation of the saline solution and recovery culture. The experimental progress was conducted in a growth chamber with 16 h light  and 8 h darkness (25 °C) with 70% relative humidity at the College of Agronomy of Jilin Agricultural University . Ninety GFD–D and ninety GFD–T seedlings with representative and vigorous conditions from three replicates were selected and exposed to the stress for phenotyping. Among them, three individual plants each from the GFD–D and GFD–T for RNA-seq analysis were selected for collecting samples at 0 h, 3 h, and 24 h after stress and 24 h after recovery, labeled as T0, T1, T2, and RT in the GFD–T, and D0, D1, D2, and RT in the GFD–D. Therefore, a total of twenty-four RNA samples for RNA sequencing (RNA-seq) were collected, which were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.Diploid (GFD–D) and tetraploid rice (GFD–T) plants of solution ) for 24 Oryza sativa Nipponbare reference genome  [https://github.com/DaehwanKimLab/hisat2 (accessed on 29 July 2023).Total RNA of rice seedlings was isolated using the TRIzol method  according to the manufacturer’s protocol. The RNA purity, concentration, and integrity were assessed with NanoDrop 2000 , a Qubit 2.0 Fluorometer , and the Agilent 2100 Bioanalyzer system, respectively. Qualified RNA was processed for RNA-seq to construct the RNA-seq library, which was performed on the Illumina sequencing platform by Biomarker Technologies Co., Ltd. . Raw image data generated via sequencing were transformed from base-calling to sequence data, which are called raw data/raw reads, and were stored in fastq format. Clean reads were obtained by removing low-quality sequences from raw data. High-quality reads were mapped to the er 2022) , using Her 2022) , which ihttps://qprimerdb.biodb.org/, accessed on 4 January 2023) was used for the primer design . Each PCR cycle was conducted as follows: 94 °C for 10 s, 55 °C for 10 s, and 72 °C for 10 s, followed by 40 cycles. All reactions were performed in triplicate, and the relative gene expression levels were calculated using the 2−ΔΔCt method [The qRT-PCR-based expression analysis was used to perform validation for seven genes. The qPCR Primer Database . The functions of the differentially expressed TFs were examined via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) [p-values  with q < 0.05 were treated as significant.De novo assembly was performed in StringTie . Unigene (DAVID) . All falq-values < 0.05 and |log2 (fold changes)| ≥ 1, and plotted using TBtools (version 1.09866). The Venn diagrams were generated via a web-based tool (Venny 2.1 at https://bioinfogp.cnb.csic.es/tools/venny/ (accessed on 10 July 2023)), and the DiVenn analysis was performed online at https://divenn.tch.harvard.edu/ (accessed on 15 July 2023). The volcano plots were generated using TBtools. The upset plots were built with the R package UpSetR . A protein–protein interaction (PPI) analysis was performed with the STRING database  as the background interactions. The parameter-of-confidence score was set to 0.9 for the filter of the network. In the STRING network, nodes represented the proteins generated by a single protein-coding gene locus, and edges represented PPIs containing known and predicted interactions. The k-means algorithm clustering analysis was conducted using the factoextra R package and online platform for data analysis and visualization .Gene expression levels or transcripts were measured according to FPKM (fragments per kilobase of transcript per million fragments mapped) in StringTie using the maximum-flow algorithm. The genes with count values > 30 were detected for differentially expressed genes (DEGs) with https://cran.r–project.org/web/packages/pheatmap/index.html, accessed on 5 May 2023), respectively. Other data visualization was carried out in Microsoft Office Excel Version 2307 (Build 16827.20166).The heatmaps for the gene expression levels of the global TFs and log2 (fold-change) values of differentially expressed TFs derived from different comparisons were visualized using the TBtools and pheatmap package in R   were found that may have potential roles to play in tetraploid salt tolerance. The systematic identification and classification of the TF genes in this study are essential for understanding the evolutionary directions, biological roles, and regulatory networks of polyploid rice.This study performed a transcriptome-based comparative analysis of the TF families in salt-tolerant GFD–T and salt-sensitive GFD–D during NaCl stress and recovery periods. Our results showed that the GFD–T had superior phenotypic characteristics and an intrinsic transcriptional regulation ability compared with the GFD–D during the early stage of saline stress and recovery period, mainly manifested in the fact that its overall gene expression levels were comparable to those of the control during the recovery period, which may have been due to its rapid activation of the ABA signaling pathway and the initiation of a specific circadian rhythm process, resulting in a unique gene regulatory network for GFD–T to resist saline stress. However, further work is necessary to verify these new findings. In addition, some core DE-TFs (such as


Glycyrrhiza uralensis extract exhibited ~50% inhibition of sEH at 100 μg/mL, and column chromatography yielded compounds 1–11. Inhibitors 1, 4–6, 9, and 11 were non-competitive; inhibitors 3, 7, 8, and 10 were competitive. The IC50 value of inhibitor 10 was below 2 μM. Molecular simulation was used to identify the sEH binding site. Glycycoumarin (10) requires further evaluation in cells and animals.Soluble epoxide hydrolase (sEH) is a target enzyme for the treatment of inflammation and cardiovascular disease. A  Epoxyeicosatrienoic acids (EETs) are derived from arachidonic acid, which is metabolized by cytochrome P450 (CYP) epoxygenase enzymes . The CYPIn particular, sEH is an enzyme mainly expressed in the liver, and alcohol-induced inflammation, injury, and steatosis were reduced in hepatic sEH-knockout mice . MoreoveGancao) prepared from the roots of Glycyrrhiza species (Fabaceae) is widely used to treat various human ailments -benzamide were excluded from the A-chain. The revised A-chain was hydrogenated using AutoDockTools ; the Gasteiger charge model was then applied. Flexible ligand docking was evaluated using a torsion tree that detected torsion roots and rotatable bonds. The grid box sizes were 126 × 126 × 126 at 0.375 Å and 60 × 60 × 60  for blinded docking and ligand docking to the active site, respectively. Molecular docking was evaluated using a Lamarckian algorithm and the maximum possible evaluation number. The resulting values were calculated and visualized using AutoDockTools, Chimera ver. 1.14 , and LIGPLOT .3D ligand structures were built using the GlycoBioChem server. The sEH Gro was developed using the GROMOS96 45a3 force field. All complexes were surrounded by water molecules (cubic size 12 × 12 × 12) and six Cl anions. The energy level attained 10.0 kJ/mol via steepest descent minimization. Each inhibitor–sEH complex was sequentially subjected to NVT equilibration at 300 K, NPT and Particle Mesh Ewald evaluation of long-range electrostatics at 1 bar, and MD simulation for 30 ns.All measurements were performed in triplicate (three independent experiments) and the results are means ± standard errors of the means (SEMs). The results were compared using Sigma Plot 10.0 .G. uralensis roots inhibited sEH, but the main compound, glycyrrhizin, lacked such an effect. In this study, eleven polyphenol compounds, 1–11, were isolated from G. uralensis. Among them, compounds 1, 3–9, and 11 were found to exert moderate inhibitory effects, and inhibitor 10 showed a potent effect, with an IC50 value of 1.9 ± 0.2 μM. Molecular simulation was used to study the binding of 1, and 3–11 to sEH and it was found that the potential inhibitor 10 maintained a stable fluidic bond with the enzyme. Therefore, of the polyphenols, compound 10 was the strongest sEH inhibitor.Bioactivity screening revealed that an ethanol extract of


Portunus trituberculatus) stored at 20 and 4 °C were investigated. It showed that samples stored at 4 °C had a longer shelf life, lower total viable count (TVC), pH, and total volatile base nitrogen (TVB-N) contents than those stored at 20 °C. The biogenic amine (BA) results demonstrated that tyramine (tyr), putrescine (put), and cadaverine (cad) were the dominant amines in all samples, and samples stored at 4 °C had lower BA contents. A microbiome analysis indicated that a salt–alcohol water mixture significantly inhibited the growth of Tenericutes. Firmicutes, Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, and Cyanobacteria were the dominant bacteria of stored pickled crabs, and storage at 4 °C significantly inhibited the growth of dominant bacteria, more than that of 20 °C. In conclusion, 4 °C storage guaranteed the quality of samples by inhibiting changes in biochemical properties and the growth of dominant bacteria, thereby prolonging its shelf life.The storage quality and microbiome analysis of pickled swimming crabs ( Portunus trituberculatus) is a highly rated seawater crustacean native to East China and other East Asian countries. In China, swimming crabs are a major aquacultural fish species, with approximately 5.3 million tons produced in 2020 [The swimming crab  through analysis of sensory scores and biochemical properties . Furthermore, the dominant microorganisms were also evaluated using 16S rRNA gene high-throughput sequencing. This study was anticipated to provide useful information to help consumers understand pickled crab characteristics.Sensory evaluation is a technique used to analyze quality changes in pickled crab, and it is generally highly correlated with other quality indices. As shown in In high-temperature storage (20 °C), the initial quality characteristics changed from a bright appearance, firm meat, and muscles strongly attached to the shell to a dark exterior and loose meat, leading to HQL being reached after storage for 24 h, and sensory scores over two (SL) after 48 h of storage. This might be due to the enzymatic activity from the hepatopancreas and mirobial breeding [Microbial activity is one of the major factors responsible for seafood spoilage; microbiological analysis can help to elucidate quality and safety properties during storage . ChangesArgyrosomus regius) fillet. Moreover, in this study, microbiological analysis results correlated well with sensory scores.In samples stored at 20 °C A, TVC sipH can be used as an indicator of the extent of proteolysis. As shown in p < 0.05). Briefly, the initial TVB-N value was approximately 8.16 ± 0.47 mg/100 g. At 20 °C, TVB-N significantly increased to 24.94 ± 0.11 mg/100 g at 48 h, which is close to the unacceptable limit (25 mg/100 g), and then increased to 37.48 ± 0.95 mg/100 g after 60 h. Meanwhile, TVB-N increased slowly over 8 days of storage at 4 °C, reaching 19.67 ± 0.58 mg/100 g after 10 days (p < 0.05). Similarly, Xu, Xia, and Kim [TVB-N can be used as an indicator of a food’s fitness for consumption . TVB-N o and Kim  reported and Kim . Further and Kim ,15. TherThe average BA values during storage at 20 and 4 °C are presented in Compared with other three BAs, all samples had lower try, spd, and hist contents, which were <3.0. In fact, neither try, spd, nor hist could be detected at first. Try and spd were first detected at 48 h and 10 d of storage at 20 and 4 °C, respectively. Hist was detected at 72 h and 14 d of storage at 20 and 4 °C, respectively. Among the BAs evaluated, histamine is of particular concern because of its toxicity and allergenic properties , who fouFirmicutes (27.19%), Proteobacteria (26.63%), Bacteroidetes (15.73%), Tenericutes (13.71%), Acidobacteria (3.02%), Actinobacteria (2.86%), and Cyanobacteria (2.11%)  and Proteobacteria (Gram-negative bacteria) can negatively affect the safety and quality of food [Actinobacterias, a probiotic in humans and animals, was significantly higher in pickled swimming crabs than in fresh crabs [Based on the sequencing results, 23,050 operational taxonomic units (OTUs) of all samples were generated under 97% similarity. The coverage was ≥95%, meaning most microbial phylotypes were detected. OTUs of the same kind were analyzed at the phylum and genus levels, and the distributions of dominant bacteria are shown in  (2.11%) . In the  of food . Actinobsh crabs . MoreoveCandidatus_Bacilloplasma (13.41%), Lactobacillus (9.21%), Psychrobacter (6.26%), uncultured_bacterium_f_Muribaculaceae (3.45%), and Bacteroides (2.52%). In the samples stored at 4 °C, Lactobacillus, Psychrobacter, Bacteroides, and Escherichia-Shigella were the predominant bacteria after 8 days of storage. Meanwhile, in the samples stored at 20 °C, Lactobacillus, uncultured_bacterium_f_Muribaculaceae, and Bacteroides were the predominant bacteria after 48 h of storage. During storage, Lactobacillus accounted for the highest proportion of bacteria, and it first increased then decreased for pickled crab samples stored at both 4 and 20 °C. Lactobacillus is an essential lactic acid bacteria strain used in traditional fermentation, and it has been found previously in marine products [Psychrobacter slightly decreased from 25.10 to 3.58% from 4 to 8 d, and then increased from 3.58 to 7.24% from 8 to 16 d. Psychrobacter, a novel marine probiotic, played an important role in the degradation of organics based on unique lipase activity. Psychrobacter sp. also exhibited strong potential for producing aroma compounds with pronounced “cheese” notes, and improving the flavor qualities of cheese [Psychrobacter significantly increased the total soluble nitrogen content and amino acid nitrogen content, and promoted the umami taste and meaty aroma of crab paste [Candidatus_Bacilloplasma, which belongs to the phylum Tenericutes, was exclusively dominant in fresh samples. Some previous studies have reported that the Candidatus_Bacilloplasma exhibited is normally present in healthy crustaceans [Candidatus_Bacilloplasma was not detected in crabs stored at either temperature. This indicates that a salt–alcohol water mixture can significantly inhibit Candidatus_Bacilloplasma growth, which is consistent with the finding of Liu [At the genus level, the initial dominant bacteria (relative abundance > 2%) in the fresh crab were f cheese . The higab paste . Candidastaceans ,24, and bacter 6.%, uncultSpecies richness was assessed using the Chao and ACE indices . Among aBeta diversity analysis was used to compare the degree of similarity in species diversity among the different samples. PCA could reflect the differences and distances between samples by analyzing their feature compositions. The PCA results A revealef_Planococcaceae, f_Staphylococcaceae, g_Candidatus_Bacilloplasma, g_Planococcu, and g_Staphylococcus were significantly higher in the fresh, 12 d, and 16 d groups.To search for a specific bacterial genus of crabs during storage, we identified groups using an LDA effect size (LEfSe) analysis of significantly different species. The LDA values of the microbial groups with significant effects in different groups were calculated using the LDA analysis. Bacterial communities with significant differences were identified during sample storage using LEfSe analysis . The lenPortunus trituberculatus) weighing 212.36 ± 10.85 g were purchased from the local aquatic market  and immediately thoroughly scrubbed under running water. Two hundred fresh crabs were placed in a glass jar containing saturated salt water and alcohol (5%) at a volume ratio of 10:1 and refrigerated at 4 °C for 12 h. Subsequently, the pickled crabs were drained for 10 min and packed into polyethylene containers. One hundred pickled crabs were stored in a 20 °C incubator for 96 h, and the remaining one hundred were stored in a 4 °C of refrigerator for 16 d. The inedible parts were removed, and swimming crab abdominal muscles were obtained for various indicators tests. All chemicals used were of analytical grade.Fresh swimming crabs  with twenty volunteer trained panelists (ten men and ten women) . All pan2CO3, and was absorbed by a boric acid solution. Titration was carried out with 0.02 N HCl. The TVB-N content is expressed as mg N/100 g ± standard deviation of three replicates.TVB-N value was determined using a steam-distillation procedure, according to Parlapani et al. . Two gra® 400 Circulator  for 1 min, followed by 10-fold dilutions using sterile 0.85% NaCl solution. Next, 0.1 mL of the mixture was spread on aerobic plate count agar to enumerate TVC. The plates were incubated at 30 °C for 72 h, then bacterial colonies were counted. TVC values were reported as logarithmic average values of the parallels and expressed as log (cfu/g).Microbiological analysis was carried out, according to Lorentzen et al. . The picThe crab meat was homogenized with 100 mL deionized water then filtered using Whatman No. 1 filter paper. The pH of the mixture solution was measured using a pH meter .4, 0.4 mol/L) in a 50 mL polypropylene conical tube, followed by centrifuging for 10 min at 3000 rpm, after which the supernatant was filtered through a 0.45 μm pore Millipore filter. The precipitate was repeatedly washed with 10 mL HClO4 and centrifuged as above. Then, 100 μL NaOH (2 mol/L), 300 μL saturated NaHCO3, 2 mL dansyl chloride solution (10 mg/mL acetone) were added to 1 mL of the prepared filtrate solution. The mixture was incubated in a water bath at 40 °C for 45 min in the dark. After this reaction, 100 μL ammonia (25%) was added to the solution, which was stored at room temperature (25 ± 0.5 °C) for 30 min. Then, dansylated extract solution was adjusted to 5 mL using 0.1 mol/L ammonium acetate to acetonitrile at a 1:1 ratio, followed by filtering through a 0.45 μL pore Millipore filter. Ten microliters of solution were injected for HPLC analysis. The HPLC was equipped with a diode array detector  and C18 column . A mixture of methanol and water (V:V 70:30) was used as the isocratic mobile phase, with a flow rate of 1 mL/min at room temperature. The dansyl derivatives of the BAs were quantified by measuring the UV-absorption at 254 nm.BAs of samples were performed, as described by Qian et al. . BrieflyThe pickled crabs were placed on an anatomical disk, the body surface was disinfected with alcohol, the abdominal muscle was removed in a sterile environment, and the sample was placed in a sterile centrifuge tube. After homogenization of muscle samples, DNA was extracted using a TGuide S96 Magnetic DNA Kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The 338F: 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′ universal primer set was used to amplify the V3-V4 region of 16s RNA gene from the extracted genomic DNA. Both forward and reverse 16s primers were tailored to sample-specific Illumina index sequences for deep sequencing. For the constructed library, Illumina NovaSeq 6000  was used for sequencing analysis. All samples were analyzed by the Biomarker Biological Testing Company. The data were analyzed using the BMK Cloud . The bacp < 0.05. Operational taxonomic units (OTUs) were obtained using an Illumina NovaSeq 6000 platform  for high-throughput sequencing data. The heatmap of species abundance was plotted using the R project pheatmap package (version 1.0.12). The Alpha diversity was evaluate by QIIME (version1.9.1) and Beta diversity was calculated by GuniFrac package (version 1.0) in R project. Furthermore, online analysis tools  were used for LEfSe analysis, and a logarithmic LDA score of 3.0 was set as the threshold for discriminative features.All experiments were replicated in triplicate (n = 3) for each group, and the average values are expressed as the mean ± standard deviation. Duncan’s analysis of variance (ANOVA) was performed using SPSS statistical software , and statistical significance was set at Tenericutes; in contrast, Firmicutes, Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria and Cyanobacteria were the dominant bacteria in the stored samples. Compared with 20 °C, storage at 4 °C significantly protected the quality and food safety of pickled swimming crabs by inhibiting the growth of dominant bacteria. Overall, the present study provides some basic yet valuable information on pickled swimming crabs, which could enhance customers’ understanding of instant food items.In this study, the sensory scores and biochemical properties of pickled swimming crabs were investigated to study how storage conditions influence their quality and safety. According to the sensory analysis, TVC, TVB-N, pH and BAs, the shelf life of pickled crabs was 36 h and 6 days for samples stored at 20 and 4 °C, respectively. BAs data revealed that tyr, put, and cad were the major amines present during storage. Compared with another three BAs, all samples had the lower try, spd and hist contents, and these three BAs only could be detected during the product’s shelf life. The high-throughput sequencing results showed that the microbial complexity gradually decreased during cold storage. The salt–alcohol water mixture could significantly inhibited the growth of